Cento De Investigación Y Asistencia En Tecnología Y

Home , Puerarin

CENTO DE INVESTIGACIÓN Y ASISTENCIA EN TECNOL OGÍA Y DISEÑO DEL ESTADO DE JALISCO, A. C

ENZYMATIC FRUCTOSYLATION OF FLAVONOIDS WITH β-FRUCTOSIDASES FROM DIFFERENT

GLYCOSIDE HYDROLASE FAMILIES TESIS

QUE PARA OBTENER EL GRADO

ACADÉMICO DE

DOCTOR EN CIENCIA Y TECNOLOGÍA EN LA ESPECIALIDAD DE BIOTECNOLOGÍA PRODUCTIVA

PRESENTA

M.C.Q. MARÍA AZUCENA HERRERA GONZÁLEZ

GUADALAJARA, JAL. FEBERO 2019

Publications

Functionalization of natural compounds by enzymatic fructosylation. Herrera-González A, Núñez- lópez G, Morel S, Amya-Delgado L, Sandoval G, Gschaedler A, Remaud-Simeon M, Arrizon J (2017) Appl Microbiol Biotechnol 101:5223–5234. doi: 10.1007/s00253-017-8359-5

Fructosylation of phenolic compounds by levansucrase from Gluconacetobacter diazotrophicus. Núñez-López G, Herrera-González A, Hernández L, Amaya-Delgado A, Sandoval G, Gschaedler A, Arrizon J, Remaud-Simon M, Morel S (2019) Enzyme Microb Technol 122:19–25. doi: 10.1016/J.ENZMICTEC.2018.12.004

Poster communications

Comparing two GH32 enzymes for fructosylation of phenolic compounds. Herrera-González A, Hernández L, Morel S, Amaya-Delgado L, Sandoval G, Gschaedler A, Remaud-Simeon M, Arrizon J. Symposium Enzyme Technology, Congress Biotechnology Havana (BH2017). Varadero (Cuba). 3th- 6th December 2017.

Fructosylation of phenolic compounds by levansucrase from Gluconacetobacter diazotrophicus Núñez-López G, Herrera-González A, Hernández L, Amaya-Delgado L, Gschaedler A, Arrizon J, Remaund-Simeon M, Morel S. Symposium Enzyme Technology, Congress Biotechnology Havana (BH2017). Varadero (Cuba). 3th-6th December 2017. This work was awared as the Best Poster.

Enzymatic fructosylation of phenolics compounds by a plant sucrose:sucrose 1- fructosyltransferase. . Herrera-González A, Hernández L, Morel S, Amya-Delgado L, Sandoval G, Gschaedler A, Remaud-Simeon M, Arrizon J. 13th International Symposium on Biocatalysis and Biotransfromation (BIOTRANS) Budapest (Hungary). 9th-13th July, 2017.

Fructosyltransferase Activity of non-Saccharomyces Yeasts from Mezcal Fermentation for Fructosylation of Polyphenols. Herrera-González A, Hernández L, Morel S, Amaya-Delgado L, Sandoval G, Gschaedler A, Remaud-Simeon M, Arrizon J 8th International Fructan Symposium (IFS2016). Oaxaca (Mexico), June 26th to July 1, 2016.

Acknowledgments

Thank God for giving me the inspiration, strength, energy and optimism to achieve this goal, thanks for guiding me in this chapter of my life.

Thanks to my mother and my father for their love and unconditional support, for always believing in me, for trust in me and for motiving me. Thanks to my sister, for all her support, trust and help. Also, I would like to thank you for carrying me to the laboratory in the early morning and in the weekends during these four years. Thanks also for taking care of me and for all their support that helped to me to achieve this project.

Thanks to Dr. Javier Arrizon, for his unconditional support, for being a great supervisor, a guide throughout all this time on research, for his advices, his teachings and for giving to me the opportunity of sharing an international project with France. Thanks for all the moments shared at Budapest and Cuba.

Thanks to Dr. Sandrine Morel, for the time-shared during this PhD thesis, for all her teachings in the laboratory, for all the scientific discussions for let me practice my French. Thanks also for sharing with me the sparkle of passion to science. Thanks for the time-shared in France, in Mexico and Cuba. Thanks also for all her help while finishing the thesis.

Thanks to Dr. Magali Remaud-Simeon, for all her unconditional support during this project, for sharing the passion of science, for all her teaching and all her patience and for all the scientific discussions. Thanks also for changing my life through all the learning experiences in the field of enzymatic glycosylation. Thanks for giving to me the wonderful opportunity to work in the group of Catalysis and Enzyme Molecular Engineering (CIMEs) at LISPB (Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés) at INSA Toulouse, France.

Thanks to Dr. Lázaro Hernández for all his support, for all his teaching in the field of Cloning and expression of heterologous genes in Pichia pastoris. Thanks for allow us to test his interesting enzymes, for all the scientific discussions. Thanks also for give me inspiration and for all the good moments shared in CIATEJ and Cuba.

Thanks to Dr. Lorena Amaya-Delgado, Dr. Anne Gschaedler and Dr. Georgina Sandoval thanks for all the teachings in the different science fields, thanks for all your support during my PhD thesis. Thanks for tour valuable support and advices in my seminars.

Thanks to everyone in the Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés. Thanks to Dr. Yoann Brison, Dr. Yannike Malbert, for all valuable help while performing the experiments, for all their teachings in the field of enzymatic glycosylation. Thanks to Nelly Monties for training me in the LC-MS and Dr. Gianluca Cioci for teaching me the methodology for enzyme purification. Thanks to Nathalie Caron for all her valuable technical support in the laboratory. Thanks to the group composed by Emna Bouhajja, Haiyang Wu, Jiao Zhao, Eleni Ioannou, Manon Molina, Marie Guicherd, Carlos Robles, Sayani Ray, Mounir, Alvaro Lafraya, Etienne Séverac, Laure de la Satte, Pauline Bondy, Marion Clavalier, Pablo Alvira, Lisa Ufarte, Lousie Badruna, thanks also for all the good moments spent each day in lab and thanks also for all the good talks shared in the cafeteria.

To my friends at France Esmeralda Cuevas, Emna Bouhajja, Haiyang Wu, Jiao Zhao, Eleni Ioannou, Jesus Rodriguez and Nelson Nuñez thanks for the lunches shared with me and for all the fantastic trips and dinners. Thanks for all the discussions about music, dance, French culture. Thanks also for being thanks for being such a good friends and thanks for sharing all this great and funny moments at Toulouse.

Thanks to Marianne Oltrogge for receiving in her house. Thanks for being my friend, for showing me the French culture, for all her care and support, even in hard moments of the stay in France. Thanks for the trips, French dinners and for all the beautiful moments that we spent together at Toulouse. Thanks also to my roommates Fatma, Hazard, Mongo, Jesus, Nelson, Gallina and Marwa, for your friendship, for sharing with me so many talks. Thanks for all the good moments shared at Marianne´s house.

Thanks to all the friends and colleagues from the laboratory of Biotegnología Industrial in the CIATEJ, for sharing such good moments and for their unconditional help and support. Thanks to Anahi Martinez, Mariana Pineda, Gema Nuñez, Ming, Kelly Reis, Monique Silva, Jorge Gomez, Luis Muñoz, Juan Carlos Leyva, Flor García, Nilda Flores, Jesus Rodriguez, Josue Nova, Araceli Hernandez, Felipe Bonilla and Laura Iñiguez for all the moments shared during this amazing PhD period.

Thanks to all my friends in Mexico, for believing on me, for waiting for me and for giving to me all their support even in the distance. Thanks to Anahi Martinez, Mariana Pineada, Dulce Carolina Mendez, Priscilla Muñiz and Pilar Haro for your unconditional help. Thanks for being such a good friend for those talks that can only be shared with you.

Finally, I would like to thanks to CONACYT for the scholarship for the development of my PhD thesis (237785). Thanks to the bilateral project CONACYT-ANUIES/ECOS-NORD M14A01 for the financial support. Thanks to CIATEJ in particular to the Laboratorio de Bioetcnología Industrial. Thanks to Metasys, the Metabolomics and Fluxomics Center at the Laboratory for Engineering of Biological Systems and Processes (Toulouse, France), for the NMR experiments. Thank to the ICEO facility dedicated to enzyme screening and discovery, part of the Integrated Screening Platform of Toulouse (PICT, IBiSA), for providing the HPLC equipment and protein purification system.

This thesis is dedicated to my parents (Angelica González and Humberto Herrera), to my sister (Angelica Herrera)

“Look deep into nature, and then you will understand everything better”

Albert Einstein

Summary

Flavonoids constitute an important source of bioactive molecules. They have some health benefits such as anti-inflammatory, antiviral, anti-tumor and anti-oxidant activities; however most of them show a rather low bioavailability due to their hydrophobic nature. Enzymatic fructosylation is an interesting approach to overcome these limitations. In this thesis work, the focus was to study the capacity for the enzymatic fructosylation of flavonoids using β-fructosidases from the families GH32 and GH68 in order to produce new fructosides that do not exist in nature. Thus, we performed a bibliographic revision of the enzymatic fructosylation of different molecules, in order to chose the reaction conditions for the fructosylation of flavonoids. Next, new β-fructosidases from non- saccharomyces yeast isolated from fermenting must of Mezcal were tested for the enzymatic fructosylation of flavonoids. Consequently, the gene of a new β-fructosidase from Rhodotorula mucilaginosa was cloned and expressed in Pichia pastoris; the produced enzyme was tested for the fructosylation of different class of flavonoids. The results showed that it was possible to fructosylate acceptors other than sucrose in absence of co-solvents and the yields were around 20%. The best yield was obtained in the case of coniferyl alcohol and only the presence of monofructosyl coniferyl alcohol was detected. Thus, it was the first time that the fructosylation of puerarin by a β-fructosidases from non-saccharomyces yeasts was described. Moreover, the enzymatic fructosylation of phlorizin was compared by β-fructosidases from different sources and specificities belonging to GH32 and GH68 families, the new β-fructosidase from R. mucilaginosa, a plant fructosyltransferase (sucrose:sucrose 1- fructosyltransferase) and a levansucrase. The best results were obtained with levansucrase from Gluconacetobacter diazotrophicus (LsdA), which is able to synthetize β-D-fructofuranosyl–(26)- phlorizin as a new substance from phlorizin by enzymatic fructosylation in 79.1 % of conversion in absence of co-solvents. The mono-fructosylated product was 15.9 fold (30.5 g L-1 at 25°C) more soluble in water than the original substrate phlorizin (1.93 g L-1 at 25°C), although exhibited a 1.4-fold reduction in the antioxidant capacity.

GENERAL INTRODUCTION ...... 10 General introduction ...... 11 CHAPTER I: LITERATURE REVIEW ...... 14 A. Generalities on flavonoids ...... 15 A.1. Structure of flavonoids ...... 16 A.2. Classification of flavonoids ...... 16 A.3. Solubility of flavonoids ...... 19 A.4. Benefits of flavonoids for health ...... 19 A.5. Bioavailability of flavonoids ...... 20 B. Glycosylation of flavonoids ...... 22 B.1. Chemical glycosylation ...... 22 B.2. Enzymatic glycosylation ...... 24 C. Glycoside-Hydrolases (GHs) ...... 30 C.1. Glycoside-Hydrolases overview ...... 30 C.2. Enzymes from GH32 family ...... 34 C.3. Enzymes from GH68 family ...... 36 C.4. Catalytic mechanism ...... 38 D. Aims of the thesis ...... 40 E. Hypothesis ...... 40 F. References ...... 41 CHAPTER II: FUNCTIONALIZATION OF NATURAL COMPOUNDS BY ENZYMATIC FRUCTOSYLATION ...... 46 A. Summary ...... 48 B. Introduction ...... 49 C. Fructosylation of different acceptors ...... 51 C.2. Fructosylation of alkyl alcohol ...... 51 C.3. Fructosylation of aromatic alcohols ...... 56 C.4. Fructosylation of alkaloids ...... 59 C.5. Fructosylation of flavonoids and xanthonoids ...... 61 D. Physical-chemical properties of β-D-fructofuranosides ...... 65 E. Pharmacokinetics parameters ...... 67 F. Conclusions and perspectives ...... 69 G. References ...... 70 CHAPTER III: ENZYMATIC FRUCTOSYLATION OF FLAVONOIDS USING β- FRUCTOSIDASES FROM NON-SACCHAROMYCES YEASTS ISOLATED FROM FERMENTING MUST OF MEZCAL ...... 74 A. Introduction ...... 76 B. Results and discussion ...... 78 B.1. Screening of transfructosylation activity by β-fructosidases from non-saccharomyces yeasts isolated from fermenting must of Mezcal ...... 78 B.2. Screening of transfructosylation activity on different class of flavonoids ...... 79 B.3. Gene search in silico from yeast genomes for new β-fructosidases ...... 83 B.4. Cloning and expression of the new β-fructosidase from Rhodotorula mucilaginosa for enzymatic fructosylation of flavonoids ...... 91 B.5. Enzymatic fructosylation of flavonoids by a recombinant β-fructosidase from Rhodotorula mucilaginosa (RhInv) ...... 97

C. Experimental methods…………………………………………………………………………………………103 C.1. Yeast propagation ...... 103 C.2. Production of β-fructosidases ...... 103 C.3. Activity assay ...... 103 C.4. Screening of enzymatic fructosylation of different flavonoids by enzymatic extracts from non-Saccharomyces yeasts ...... 104 C.5. Quantification of percentage of conversion of flavonoids by Liquid Chromatography- Mass Spectrometry (LC-MS) ...... 104 C.6. Search of a new β-fructosidase gene sequence in silico from available genomes ..... 105 C.7. Cloning and expression of β-fructosidase from Rhodotorula mucilaginosa in Pichia pastoris X-33 ...... 107 C.8. Enzymatic fructosylation of flavonoids by a recombinant β-fructosidase (RhInv) from Rhodotorula mucilaginosa ...... 108 D. Conclusions ...... 109 E. References ...... 110 CHAPTER IV: ENZYMATIC FRUCTOSYLATION OF PHLORIZIN BY LEVANSUCRASE FROM Gluconacetobacter diazotrophicus ...... 113 A. Summary ...... 115 B. Introduction ...... 116 C. Results and discussion ...... 118 C.1. Enzyme selection for phlorizin fructoside production ...... 118 C.2. Effect of sucrose, phlorizin and enzyme concentration on phlorizin conversion ..... 121 C.3. Structural characterization of phlorizin mono-fructoside ...... 124 C.4. Water solubility and antioxidant activity of mono fructosyl phlorizin ...... 126 D. Experimental methods ...... 127 D.1. Chemical materials ...... 127 D.2. Enzyme production ...... 127 D.3. Enzyme activity assays...... 127 D.4. Enzyme selection for phlorizin fructoside production ...... 128 D.5. Effect of sucrose, phlorizin and enzyme concentration on the percentage of conversion of phlorizin with LsdA ...... 128 D.6. HPLC-MS analysis of fructosylation reaction mixture ...... 128 D.7. Large-scale production of fructosyl phlorizin ...... 129 D.8. Structural analysis of phlorizin mono-fructoside ...... 129 D.9. Solubility determination of mono-fructosyl phlorizin ...... 130 D.10. Antioxidant activity of mono-fructosyl phlorizin ...... 130 D. Conclusions ...... 131 E. Supplementary data ...... 132 F. References ...... 134 CONCLUSIONS AND PERSPECTIVES ...... 137 Conclusions and perspectives ...... 138 FIGURE AND TABLE CONTENTS ...... 141 Figure and Table contents ...... 142 Figure contents...... 142 Table contents ...... 144 ABBREVIATIONS ...... 145 Abbreviations...... 146

GENERAL INTRODUCTION

General introduction

Flavonoids are secondary metabolites of plants. They have as main functions the protection of plants against UV radiation, oxidation and pathogen agents. Nowadays, flavonoids and their glycosides are getting attention due to their physicochemical and biological properties such as antioxidant, antiviral, anti-inflammatory, anti-tumor, etc. Thus, the effects of these properties in human health are more and more studied in order to develop new active molecules of interest for the pharmaceutical, cosmetic and food industry.

Nevertheless, absorption of these compounds in their aglycone form is limited due to their high hydrophobicity. When flavonoids are not absorbed in the gut, microbiota metabolizes them, leading to major changes in their structure and altering their beneficial properties. Thus, glycosylation of flavonoids is considered as an efficient approach to protect the aglycone structure and to increase their solubility, stability and bioavailability. In addition, glycosylation opens access to a large structural diversity of compounds with new pharmacological properties. Two approaches are commonly used for glycosylation: the chemical and the enzymatic method. The chemical method is quite difficult due to the complexity of sugar molecules, in particular the presence of multiple alcohol functions showing similar reactivities. In addition, glycosylation reactions often use heavy metals, which limit such developments. Moreover, enzymatic glycosylation presents the advantage of being regio- and stereoselective while using mild conditions.

In the last two decades, several types of carbohydrate-active enzymes (CAZy) have been used in glycosylation reactions, each with specific characteristics, such as Glycosyltransferases (GT) and Glycoside Hydrolases (GHs). According to the literature, glycosylation of flavonoids has been explored using Leloir and non-Leloir type Glycosyltransferases, and Glycoside Hydrolases. Consequently, several new glycosides were successfully obtained. However, enzymatic fructosylation of flavonoids using enzymes from GH32 and GH68 families has been little explored although it could open access to added-value and new-to-nature compounds. This was the main objective of our work, which aimed at characterizing new transfructolysases of interest for flavonoid glucosylation and was performed within the frame of a collaboration supported by CONACYT with the group of Catalysis and Enzyme Molecular Engineering (CIMEs) in the Laboratoire d’ingénierie des Systèmes Biologiques et des Procédés (LISPB) at INSA Toulouse, France.

11 General introduction

The thesis manuscript is developed as follows:

In the Chapter I, the thesis topic is placed in context. The chapter is divided in three parts. The first one presents an overview of flavonoids (structure, classification, solubility, biological activity, and bioavailability). The second part describes the different strategies used for glycosylation of flavonoids to increase their solubility and bioavailability. The last part is dedicated to the glycoside hydrolases (from GH32 and GH68 families) reported as able to catalyze flavonoid fructosylation and describes their catalytic mechanism for fructoside linkage hydrolysis or transfructosylation.

The second chapter is focused on the fructosylation of natural compounds and is presented as scientific literature review that was published in 2017. It compiles the tested enzymes and acceptors, the reaction conditions and the obtained yields. It also describes the physico-chemical and bioactive properties of the fructosides. This review allowed us to confirm that enzymatic fructosylation of polyphenolic compounds was little explored and that this reaction could lead to new molecules that do not exist in nature.

The Chapter III presents the enzymatic fructosylation of flavonoids by β-fructosidases from different non-saccharomyces yeasts isolated from fermenting must of mezcal. A screening of fructosyltransferase activities found in the extracellular enzymatic extract of yeasts was first performed and the positive strains were tested for the enzymatic fructosylation of a set of flavonoids belonging to different classes. From this screening, the enzymatic extract of Rhodotorula mucilaginosa MB4 was retained as the most promising one, as it contained an activity leading to the fructosylation of the isoflavone puerarin. Subsequently, the analysis of R. mucilaginosa genome enabled the identification of a gene sequence encoding a new β-fructosidase from the GH32 family putatively responsible for puerarin fructosylation. The corresponding gene was cloned and expressed in Pichia pastoris X-33 in order to explore its capacity for the enzymatic fructosylation of flavonoids.

The last chapter (Chapter IV) is focused on the fructosylation of phlorizin using three different enzymes belonging to the clan GH-J. The enzymes employed were a levansucrase from Gluconacetobacter diazotrophicus (LsdA, 2.4.1.10), a sucrose:sucrose 1-fructosyltransferase from Schedonorus arundimaceus (Sa1-SSTrec, EC 2.4.1.99) and a β-fructofuranosidase from Rhodotorula mucilaginosa (RhInv, EC 3.2.1.26). The best enzyme able to fructosylate the acceptor was selected in order to optimize the reaction conditions.

12 General introduction

The second part of the chapter presents the purification and the structure characterization of the new fructoside. Finally, the solubility and the antioxidant activity of the new fructo-conjugate were evaluated and compared to the properties of the aglycone.

This chapter presents an overview of flavonoid glycosylation. The first part of this chapter is focused on the chemical features of flavonoids and provides a description of their physicochemical properties and their potential uses as bioactive compounds. The second part presents different strategies for glycosylation of flavonoids, which aimed to increase their solubility and bioavailability. Finally, the final part is dedicated to the glycoside hydrolases reported to catalyze flavonoid fructosylation, ie the enzymes from the GH32 and GH68 families, and describes the catalytic mechanism adopted for fructoside hydrolysis and transfructosylation.

CHAPTER I: LITERATURE REVIEW

Chapter I: Literature review

A. Generalities on flavonoids

Flavonoids (from the latin word flavus meaning yellow, their color in nature) are one of the largest families of natural compounds present in the kingdom of plants. They were first isolated in 1930 by Albert Szent-György (Quideau et al. 2011). At the beginning, flavonoids were called “vitamin P”, but this term was rapidly replaced by “bioflavonoids” as it was revealed that this isolated product was a mixture of various molecules. Subsequently, over the years it was possible to identify many individual polyphenols compounds in these mixtures, which finally classified under the general term “flavonoids” (Quideau et al. 2011).

Flavonoids are natural compounds that belong to the general class of polyphenol molecules. These are secondary metabolites and their main functions are pigmentation, protection against ultraviolet radiation and pathogen agents. In nature more than 8,000 flavonoids have been found distributed in plants, algae, fungi, flowers, fruits, vegetables and cereals (Crozier et al. 2009; Pandey et al. 2009; Quideau et al. 2011). They are synthesized in the plants through the phenylpropanoid pathway (Falcone Ferreyra et al. 2012; Kumar and Pandey, 2013).

Nowadays, flavonoids are widely studied due to their physicochemical and biological properties that make them very promising candidates to develop new active compounds of interest for the pharmaceutical, cosmetic and food industry (Di Carlo et al. 2009; Panche et al. 2016).

15 Chapter I: Literature review

A.1. Structure of flavonoids

Chemically, flavonoids are classified in different polyphenol families. Structurally, they have the

C6C3C6 carbon formula. The general structure of the flavonoids is shown in Figure 1, where it can be observed that they are composed of two aromatic rings (A and B) and one heterocyclic ring (C). The basic flavonoid skeleton can have numerous substituents. Hydroxyl groups are usually present at the 4’, 5’ and 7 positions. Moreover sugars such as glucose, galactose and rhamnose are very common substituents, thus the majority of flavonoids exists naturally as glycosides in the position C7 or C3 (Crozier et al. 2009; Pandey et al. 2016).

3' 4' 2' 1 B 8 O 5' 2 1' 7 A C 6' 6 3 5 4

Figure 1. General structure of flavonoids (Crozier et al. 2009).

A.2. Classification of flavonoids

Flavonoids are classified into different groups depending on the substitution in the ring C, the degree of unsaturation and their level of oxidation. Mainly, they are grouped into several classes and table 1 shows the classification, the structure and examples of each class of flavonoids (Di Carlo et al. 1999; Crozier et al. 2009; Pache et al. 2016):

 Flavonols are flavonoids with a ketone group in position C4 and a hydroxyl group in position C3 of the ring C.  Flavones show a double bond between position C2 and C3 and a ketone in position C4 of the ring C. Also, most of the flavones have a hydroxyl group in position C5 and C7 of the ring A and sometimes they are hydroxylated also in ring B at C3’ and C4’.

16 Chapter I: Literature review

 Flavan-3-ols have the hydroxyl group always bound to position C3 of the ring C.  Anthocyanines are constituted by the flavylium ion at the ring C. In addition, they are characterized by the presence of a cationic charge and they are glycosylated in position C3, C5, C7, C3’ and C7’, but mostly in position C3  Flavanones display a saturated C ring, the double bond is found between positions C2 and C3. They are non-planar and have a chiral center at C2.  Dihydroflavonols are flavanones substituted at C3 with a hydroxyl group. They exhibit two asymmetric carbons, C2 and C3, thus giving two couples of enantiomers.  Isoflavones have the B ring attached at C3 rather than C2. Also, they have hydroxyl groups at C7 and C4.  Chalcones display two aromatic rings (ring A and B) linked by three carbon atoms with a ketoethylenic group (-CO-CH=CH-).  Aurones constitute a smaller class of flavonoids showing a benzofuran linked to a benzylidene.

Individual differences within each group arise from the variation in number and arrangement of the hydroxyl groups and their degree of alkylation and or glycosylation. Quercetin, myricetin, catechins etc., are some of the most common flavonoids.

17 Chapter I: Literature review

Table 1. Flavonoids classification: structure and examples.

Group Structure Example OH

HO O Flavones R1 Apigenin (R1=H), Luteolin (R1=OH)

OH O R1 OH Quercetin (R1=OH, R2=H), Kaempferol (R1=H, R2=H), Myricetin (R1=OH, R2=OH), Flavonols HO O R2 Isorhametin (R1=OMe, R2=H)

OH OH O R1 R2 Hespertin (R1=OH, R2= OMe), Flavanones HO O Naringenin (R1=H, R2=OH)

OH O R1 O Genistein (R1=OH, R2=OH), Isoflavones Daidzein (R1=OH, R2=H) R2 O OH R1 OH

Flavan-3-ols HO O Catechin (R1=OH)

OH OH R1 OH

HO O Dihydrokaempferol (R1=H, R2=H), Dihydroflavonol R2 Dihydroquercetin (R1=H, R2=OH) OH OH O R1 OH

HO O+ Cyanidin (R1=OH, R2=H), Delphidin Anthocyanidin R2 (R1=OH, R2=OH), OH OH OH O R1 Chalcone Butein (R1=OH), Isoliquiritigenin (R1=H)

HO OH O

Sulfuretin (R1=OH) Aurone O Hispidol (R1=H)

18 Chapter I: Literature review

A.3. Solubility of flavonoids

Solubility is one of the most important physicochemical properties of bioactive compounds. Many natural flavonoids have extremely low solubility in aqueous media due to their structural characteristics (Plaza et al. 2016; Zhang et al. 2017). For example, solubility in water for rutin, naringin, quercetin and neohesperidine ranges around 0.12, 0.5, 0.01 and 0.4 g L-1 at 20 ºC, respectively (Chebil et al. 2007). Thus, the best solvents for flavonoid solubilization are polar, such as methanol and ethanol, and aprotic, such as acetone and DMSO (Razmara et al. 2010). However, the presence of a sugar moiety usually increases the solubility of the flavonoids in water solutions. For example, glycosylquercetin, as well as other glycosylated flavonols, were reported to have considerably enhanced water solubility compared to quercetin and its aglycone analogues (Plaza et al. 2014).

A.4. Benefits of flavonoids for health

According to the literature, the biological studies about flavonoids are associated with a broad diversity of health-promoting effects such as antioxidative, anti-inflammatory, cardio-protective and anti- carcinogenic properties, thus they are an indispensable component in a variety of nutraceutical, pharmaceutical, medicinal and cosmetic applications (Lu et al. 2013; Pache et al. 2016). In the following section some of their benefits for health are described:

 Antioxidant activity: Flavonoids possess many biological properties, however the most described one is their capacity to act as antioxidants. The antioxidant activity depends on the arrangement of the functional groups on the basic structure. The configuration, substitution, and total number of hydroxyl groups influence several mechanisms of antioxidant activity such as radical scavenging (Kumar and Pandey, 2013). The B ring is the most significant determinant of scavenging activity (Cao et al. 1997).  Anti-inflammatory effect: Inflammation is a normal biological process in response to tissue injury, microbial pathogen infection, and chemical irritation. Several studies indicate that flavonoids can reduce neuro-inflammation acting mainly through the regulation of microglial cells. In particular, the modulatory effects induced by these compounds are mediated by their impact on important signaling pathway. For example, quercetin at 0.1 mM concentration was able to reduce neuroinflammation induced by the parkinsonian toxin (Kumar and Pandey, 2013; Spagnuolo et al. 2018).

19 Chapter I: Literature review

 Cardio-protective effect: One of the most important functions of flavonoids is their potential role in the prevention of cardiovascular diseases. Several studies have demonstrated that polyphenols are potent inhibitors of Low Density Lipoprotein (LDL) and this type of oxidation is considered to be a key mechanism in the development of atherosclerosis (Pandey and Rizvi, 2009). Another study demonstrated that flavones and flavanols protect against hypertension (Kozłowska and Szostak-Wegierek, 2014).  Anti-carcinogenic effect: Numerous studies have suggested that flavonoids exhibit a growth inhibitory effect on different kinds of cancer cells acting through diverse metabolic pathways. Many polyphenols, such as quercetin, catechin, isoflavones, flavanones, resveratrol and curcumin have been tested; all of them showed protective effects in some models and their mechanisms of action were found to be different (Sak, 2014).

Epidemiologic evidences showed that flavonoids are correlated with a low incidence of chronic diseases, such as cardiovascular diseases, neurodegenerative diseases, and cancers. However, for most of them the molecular mechanism in the cells is unknown (Kozłowska and Szostak-Wegierek, 2014).

A.5. Bioavailability of flavonoids

Bioavailability is the proportion of the nutrient that is digested, absorbed and assimilated through metabolism (Pandey and Rizvi, 2009; Quideau et al. 2011). Absorption of flavonoids from food to the intestine depends on their physicochemical properties, size of molecules, lipophilicity and solubility (Sordon et al. 2016).

The flavonoids can be absorbed from the small intestine or continue to the colon before absorption. This may dependent on their structures and in particular their level of glycosylation (Kumar and Pandey, 2013). Nevertheless, absorption of these compounds in their aglycone form is limited due to their high hydrophobicity. When flavonoids are not absorbed in the gut, the microbiota metabolizes them, leading to major modifications in their structures and altering their beneficial properties (D’Archivio et al. 2007).

Furthermore, aglycones of flavonoids can be easily absorbed by the small intestine, while flavonoid glycosides have to be converted into aglycone form in order to able to be absorbed by passive diffusion (Kumar and Pandey, 2013).

20 Chapter I: Literature review

Flavonoid glycosides retain their structures even after cooking processing. They are also resistant to low pH in stomach and to digestive enzymes found there and they may be actively transported by Na+/glucose transporters from intestinal lumen to enterocytes, where they are subsequently cleaved to aglycones by cytosolic glucosidases. Since the absorption of flavonoid glycosides in small intestine is weak, it is suggested that flavonoid glycosides are hydrolyzed by β-glucosidase, secreted by the brush border of human small intestine epithelial cells, known as LPH (lactase phloridzin hydrolase) (Kumar and Pandey, 2013). LPH is characterized by substrate specificity and the glycosides are not substrates for this enzyme, thus they are transported to the large intestine, where they are hydrolyzed by intestinal bacteria enzymes such as α-arabinofuranosidase, α-fructosidase, α-rhamnosidase, β-fructosidase, β- glucosidase and β-glucuronidase. The aglycones are absorbed in the colon (Kumar and Pandey, 2013; Xiao et al. 2014).

From intestine, flavonoids are transported in blood to liver, where they may be deconjugated and again conjugated with either glucuronic acid or sulfate with the help of enzymes, such as β-glucuronidase and sulphatase, or they may be metabolized (Perez-Vizcaino et al. 2012). Next, the flavonoid glucuronates or sulfates formed in liver are delivered with blood to tissues of the whole organism (Desmet et al. 2012). The excess of flavonoids delivered to the body is not accumulated. There are two ways of elimination of flavonoids by the organism: flavonoids absorbed in intestine are eliminated with urine, whereas flavonoids not absorbed are eliminated with faeces in the form of glycosides (Perez- Vizcaino et al. 2012).

In the case of quercetin glucoside, it was observed that after oral administration, the maximal concentration of quercetin in plasma was 20 fold higher than after the intake of quercetin rutoside. Therefore, it seems that quercetin glucoside is absorbed from small intestine in unchanged form, whereas absorption of quercetin rutoside takes place after its deglycosylation (Gee et al. 1998; Gee et al. 2000). In conclusion, it is possible to imply that glycosylated flavonoids tend to be better absorbed in the small intestine than aglycone form.

21 Chapter I: Literature review

B. Glycosylation of flavonoids

Glycosylation of flavonoids is considered as an efficient approach to increase their solubility, stability and bioavailability, which lead to improvement of their pharmacological properties (Slámová et al. 2018). In this section, the chemical and enzymatic approaches will be discussed in order to perform the synthesis of glycosylated flavonoids.

B.1. Chemical glycosylation

Glycosylation reactions by chemical synthesis are used intensively in the field of glycochemistry. Nowadays, a variety of glycosylation protocols was developed and four types of glycosyl donors used for chemical synthesis (Desmet et al. 2012).

According to the literature, figure 2 presents the most used glycosyl donors and activation agents in chemical synthesis for glycosylation. Glycosyl halides are used as glycosyl donors, which are activated with silver salts (Wallace and Schroeder, 1977). Then, the glycosyl trichloroacetamidates have showed to be powerful donor substrates with an excellent leaving group (Schmidt and Michel, 1980). Alternatively, more stable glycosides, such as thioglycosides and n-pentenyl glycosides, can be used when activated by electrophilic reagents such as iodonium dicolidine satls, N-bromosuccinimide (NBS) and triflic acid (Zu and Schmidt 2009). However, two main problems are present in these reactions, the regioselectivity and the configuration of the glycosidic linkages. The regioselectvity can be resolved by appropriate protection/deprotection strategies, although the configuration of the glycosidic linkage is strongly dependent on the adjacent group (participation of the C2 substituent). Moreover, solvents and catalysts have an important effect on the anomeric outcome during glycosylation reactions. Finally, despite all these efforts in the improvement of reaction conditions, the recognition of a single general procedure to describe chemical glycosylation in its totality is yet to be accomplished (Crich, 2010; Das and Mukhopadhyay, 2016).

Finally, as it has been reported in the literature, chemical approaches have several drawbacks, such as activation and protection of hydroxyl groups, multistep synthetic routes with low overall yields, the use of toxic catalysts and solvents and the amount of waste (Desmet et al. 2012). Therefore, in order to overcome these limitations, specific enzymes may be used for the synthesis of glycosides.

22 Chapter I: Literature review

Glycosyl donor Activation Agents

OAc O AcO Ag salts AcO AcO Br OAc O AcO O CCl 3 Lewis acids (e.g. BF3) AcO AcO NH OAc O + AcO SR Electrophilic promotors (e.g. I , iodonium dicolidine AcO salts) AcO R= alkyl OAc O Electrophilic promotors AcO O AcO (e.g. N-bromosuccinimide(NBS) and triflic acid) AcO

Figure 2. Prominent glycosyl donors used in chemical synthesis (Desmet et al. 2012).

23 Chapter I: Literature review

B.2. Enzymatic glycosylation

In the last two decades, enzymatic synthesis of glycosides has been explored due to their advantages compared with chemical synthesis. Consequently, enzymatic catalysis provides some benefits such as the control of regioselectivity and stereoselectivity and the use of mild conditions. According to the literature, several types of carbohydrate-active enzymes (CAZy) can be used in glycosylation reactions, each with specific characteristics, such as Glycosyltransferases (GTs) and Glycoside hydrolases (GHs) (Desmet et al. 2012).

Figure 3 illustrates the main mechanism for the synthesis of glycosidic bond by different enzymes, wherein glycosyltransferases (GTs) catalyze the formation of glycosidic bond by transferring sugar moieties from donor molecules to a specific aglycon with strict stereo or regioselectivities. The acceptors can be either sugars or other molecules such as proteins, lipids, nucleic acids, steroids, and polyphenols (Xu et al. 2016). Therefore, based on catalytic properties, glycosyltransferases are divided into two categories: Leloir and non-Leloir enzymes. The Leloir glycosyltransferases are mainly responsible for the synthesis and modification of glycan in vivo, requiring activated glycosyl donors such as uridine diphosphate glucose (UDP-glucose) (Hoffer, 2016). Moreover, non-Leloir glycosyltransferases can use non-activated sugars for glycosyl transfer such as sucrose. In comparison, Leloir glycosyltransferases catalyze glycosyl transfer with strict specificity and high efficiency, but require expensive glycosyl donors and generally tolerate natural acceptors. Instead, non-Leloir glycosyltransferases are compatible with low-cost donors and a wide range of acceptors, but lead to relatively low product yields (Xu et al. 2016). Glycosidases or transglycosylases (GHs), are responsible for the cleavage of glycosidic linkages in vivo. GHs catalyze the hydrolysis of non- reducing terminal or internal glycosidic bonds. In addition, some of the glycosidases can catalyze glycoside synthesis by transglycosylation under suitable reaction conditions, using monosaccharides, oligosaccharides, or glycosides as glycosyl donors and a variety of compounds containing hydroxyl groups as glycosyl acceptors (Xu et al. 2016).

24 Chapter I: Literature review

Glycosyltransferases (GTs) a) Leloir glycosyltransferase

b) Non-Leloir glycosyltransferase: glycoside hydrolase and transglycosylase

Glycoside Hydrolase (GHs)

Transglycosylase (GHs)

Figure 3. General mechanism for the synthesis of glycosidic bond by different enzymes (adapted from Xu et al. 2016).

Tables 2, 3 and 4 show an overview of the enzymatic glycosylation of flavonoids using Leloir, non- Leloir glycosyltransferases and glycoside hydrolases, respectively. A wide variety of flavonoids have been glycosylated using Leloir-GTs, non-Leloir GTs and GHs. However, each enzyme has its advantages and drawbacks. In the case of Leloir glycosyltransferases (table 2), an important advantage is the possibility to obtain values of conversion up to 100% for the glycosylation of flavonoids. However, these reactions have two major drawbacks, first the reactions use low acceptor concentrations, typically around 0.1 mM and second the reactions need UDP-glucose as sugar donor, which is expensive. These drawbacks render difficult their use for biotechnological applications (Xu et al. 2016).

Alternatively, glycosylation of flavonoids catalyzed by non-Leloir glycosyltransferases, which are members of glycoside hydrolase families but naturally display a high transglycosylase activity, has been attempted. As seen in table 3, several glycosides were successfully obtained with cyclodextrin glycosyltransferases, maltogenic amylases and also glucansucrases. Hence, an advantage of non- Leloir GTs is that they are able to use cheap sugar donors such as starch, sucrose and maltodextrin, etc.

25 Chapter I: Literature review

Moreover, non-Leloir GTs (transglycosylases) are usually natural promiscuous towards various types of acceptor molecules such as saccharides, alcohols and flavonoids (Hofer, 2016). Due to their promiscuity, various types of flavonoids such as (+)-catechin, quercetin, myricetin, naringenin, daidzein, neohesperidin and luteolin have been successfully glycosylated. However, some drawbacks are reported for non-Leloir GT, such as low yields and the necessity to use of co-solvent to increase the solubility of the flavonoid before glycosylation.

Finally, glycoside hydrolases showing a high hydrolytic activity have also been tested in order to find new classes of enzymes prone to glycosylate flavonoids as acceptor (table 4). In order to limit the hydrolytic activity of GHs, high concentrations (100 mM and above) of the donor (sucrose or maltose) were employed and flavonoid concentrations typically in the range of 10–25 mM were used. In these conditions the percentage of conversion usually vary between 20–90% (Hoffer, 2016).

Moreover, for the aims of this thesis, it is worth mentioning that Wu et al. (2013) reported the synthesis of new puerarin fructosides, using a β-fructosidase from Arthrobacter nicotianae XM6 CCTCC M2010164. They obtained 90 % of conversion with the addition of 20 % of dimethyl sulfoxide (DMSO) as co-solvent, an exceptionally high acceptor concentration of 265 mM was used (Wu et al. 2013).

In conclusion, GHs appears interesting candidates for flavonoid glycosylation, and for the production of a broad range of new fructosylated derivatives. Moreover, GHs are able to glycosylate flavonoids by using cheap and abundant glycosyl donors such as sucrose. Thus, GHs constitute the focus of the next part of this literature review, in particular GHs that belong to the GH-J clan (GH32 and GH68 families). These enzymes are able to transfer the fructosyl moiety of sucrose onto hydroxylated acceptors; they are also named fructosyltransferases or transfructosylases.

26 Chapter I: Literature review

Table 2. Enzymatic glycosylation of flavonoids by Leloir glycosyltransferases.

Flavonoid acceptor Enzyme Sugar donor Flavonoid glycosides Reference UDP- Quercetin OH glycosyltransferase quercetin 3-O-β- Ohgami et UDP-Glucose HO O from Camellia glucopyranoside al. 2014 OH sinensis Glycosyltrasferasa OH quercetin 3-O-glucoside Hyung Ko from Bacillus UDP-Glucose OH O quercetin 7-O-glucoside et al. 2006 cereus 10987 Naringenin OH UDP – glucose naringenin 7-O-glucoside Singh et UDP-glucose from W. somnifera naringenin 4’-O-glucoside al. 2013 HO O Glycosyltrasferasa Hyung et from Bacillus UDP-glucose naringenin 4’-O-glucoside al. 2006 cereus 10987 OH O Betadin 5-O- Kaempferol OH glucoayltransferase Das et al. UDP-Glucose kaempferol 3-O-glucoside HO O from Amaranthus 2003 tricolor Glycosyltrasferasa OH Hyung Ko from Bacillus UDP-glucose kaempferol 7-O-glycosyde OH O et al. 2006 cereus 10987 OH Apigenin Glycosyltrasferasa HO O apigenin 7-O-glucoside Hyung Ko from Bacillus UDP-glucose apigenin 4’-O-glucoside et al. 2006 cereus 10987

OH O OH Luteolin HO O Glycosyltrasferasa luteolin 7-O-glucoside Hyung et OH from Bacillus UDP-glucose luteolin 4’-O-glucoside al. 2006 cereus 10987

OH O phloretin-4’-O-glucoside phloretin 2’-O-glucoside UDP- phloretin 4’,4-O- Glucosyltransferase Pandey et Phlor etin UDP-Glucose diglucoside O OH from Bacillus al. 2013 phloretin6’-4-O-diglucoside lichenifromis phloretin 2’,4,4-O- triglucoside HO HO OH UDP- Glucosyltransferase Gosch et UDP-glucose phoretin 2’-O-glucoside from Malus al. 2010 domestica

27 Chapter I: Literature review

Table 3. Enzymatic glycosylation of flavonoids by non-Leloir glycosyltransferases.

Sugar Flavonoid acceptor Enzyme Flavonoid glycosides Reference donor (+)-Catechin-4’-O-α-D- glucopyranoside α-Glycosyltransferase from Meulenbeld OH Sucrose (+)-Catechin-4’,7-O-α-di-D- (+)-Catechin Streptococcus mutans GS-5 et al. 1999 glucopyranoside HO O OH α-Amylosucrase from (+)-Cathechin-3’-O-α-glucoside Cho et al. Deinococcus geothermalis Sucrose OH (+)-Catechin-3’-O-α-maltoside 2011 DSM 113000 OH α-Cyclodextrin (+)-Cathechin-3’-O-α- Funayama glucanotrasferase from Starch glucopyranoside et al. 2014 Bacillus macerans O Neohesperidin

O O O HO OH Cyclodextrin Starch, HO O Neohesperidin-3’’-α-(13)-D- Kometani OH OH O glucanotransferase from cyclodext HO glucopyranose et al. 1996 O Alkalophlic Bacillus rin

HO OH

Daidzin OH Maltosyltransferase from OH O Thermotoga maritime Maltotrio Li et al. HO OH Daidzein 7-O-triglucoside se 2004 HO O O O

OH Naringin

O O O HO Cyclodextrin Starch, HO O Naringin-3’’-α-(16)-D- Kometani OH OH O glucanotransferase from cyclodext HO glucopyranose et al. 1996 O Alkalophlic Bacillus rin

HO OH luteolin-4’-O- α -D- glucopyranoside, OH Glucansacrase from luteolin-4’-O-α-D- glucopyranosyl- Luteolin Malbert et Neisseria polysaccharea Sucrose α-(14)-D-glucopyranoside and HO O al. 2014 OH luteolin-4’-O-α-D-glucopyranosyl- α-(14)-D-glucopyranosyl-α- (16)-D- glucopyranoside Dextransucrase from OH O luteolin-3’-O-α-D-glucopyranoside Betrand et Leuconostoc mesenteroides Sucrose luteolin-4’-O-α-D-glucopyranoside al. 2006 NRRL B-512F Quercetin OH quercetin-4’-O-α-D-di- glucopyranoside Alternansucrase from HO O quercetin-4’-O-α-D- Betrand et OH Leuconostoc mesenteroides Sucrose glucopyranoside al. 2006 NRRL B-23192 OH quercetin-3’-O-α-D- OH O glucopyranoside OH

OH Myricetin myricetin-3’-O-α-D- Alternansucrase from glucopyranoside Betrand et HO O Leuconostoc mesenteroides Sucrose OH myricetin-4’-O-α-D- al. 2006 NRRL B-23192 glucopyranoside OH OH O

28 Chapter I: Literature review

Table 4. Enzymatic glycosylation of flavonoids by glycoside hydrolases (GHs).

Flavonoid acceptor Enzyme Sugar donor Flavonoid glycosides Reference p-Nitrophenyl-β- Celullase from (+)catechin 4’-β-D- OH D- (+)-Catechin Aspergillus niger fucopyranoside fucopyranoside HO O α-glucosidase Gao et al. OH (+)-catechin 5-α-D- from Bacillus Maltose 2000 glucopyranoside OH steatothermophilus OH (+)-catechin 7-α-D-maltoside α-amylase Dextrin (+)-catechin 5-α-D-maltoside Maltogeninic puerarin-α-(16)-D- amylase from Puerarin glucopyraoside Choi et al. OH Bacillus Maltodextrin O puerarin-α-(16)-D- 2010 steatothermophilsu glucopyraoside

HO O β-Fructosidase from OH β-D-fructofuranosyl-(26)- O Arthrobacter puerarin Wu et al. HO nicotianae XM6 Sucrose OH β-D-di-fructofuranosyl-(26)- 2013 CCTCC OH puerarin M2010164

Kaempferol OH

HO O Cellulase from Chen et al. Maltose Kaempferol monoglucoside P. decumbens 2011 OH OH O Quercetin OH

HO O Cellulase from Chen et al. OH Maltose Quercetin monoglucoside P. decumbens 2011 OH OH O

29 Chapter I: Literature review

C. Glycoside-Hydrolases (GHs)

This section will present an overview of the enzymes belonging to the Glycoside-Hydrolase families (GHs), their classification, their mechanism and the reactions catalyzed. Moreover, in the second part, the focus will be placed on the clan GH-J with GH32 and GH68 families to which belong the enzymes at the core of this thesis. The main features of these enzymes including their catalytic mechanisms and structures will be first described. Finally, the last part will be devoted to the hydrolysis and transfructosylation reactions catalyzed by enzymes from GH32 and GH68 families.

C.1. Glycoside-Hydrolases overview

The IUBMB (International Union of Biochemistry and Molecular Biology) enzyme nomenclature is based on their substrate specificity and on the reaction catalyzed. In this classification, glycoside Hydrolases (GHs, EC 3.4…) and glycosyltransferases (GT EC 2.4..) belong to difference classes. However, in the classification based on sequence alignment and molecular mechanism initially proposed by B. Henrissat and at the origin of the CAZy database (Carbohydrate Active Enzymes, http://www.cazy.org/), glycoside hydrolases (EC 3.2.) and some transferases (EC2.4..) showing a strong ability to catalyse glycosyl transfer were shown to be homologous, thus sharing related sequences and similar mechanism of glycosidic bond cleavage. Consequently, they were all classified in glycoside hydrolase family. In the CAZy classification, GHs are structurally divided into over 156 families (February 2019). In addition, these families are grouped in clans according to similarity in structure-function; this classification is showed in table 5 (Desmet et al. 2012; Cantarel et al. 2009).

Moreover, GHs have two different main mechanisms for the cleavage of the glycosidic bond. In 1953, Koshland for the first time described these mechanisms as retaining or inverting, which depends on the variation of the anomeric carbon configuration during the reaction (Koshland, 1953). Figure 4 shows the general mechanism of GHs. In the inverting mechanism, the catalytic acid residue donates a proton to the anomeric carbon while the catalytic base residue removes a proton from a water molecule, increasing its nucleophilicity, facilitating its attack on the anomeric center.

30 Chapter I: Literature review

Table 5. The established Glycoside Hydrolases clans of related families in the CAZy database (adapted from http://www.cazy.org/).

GH clans Shared features Related GH Families 1, 2, 5, 10, 17, 26, 30, 35, 39, 42, 50,51, 53, 59, 72, 79, 86, GH-A (β/α)8 113, 128, 147, 148 GH-B β-jelly roll 7, 16 GH-C β-jelly roll 11, 12 GH-D (β/α)8 27, 31, 36 GH-E 6-fold β-propeller 33,34 83,93 GH-F 5-fold β-propeller 43,62 GH-G (α/α)6 37, 63, 100, 125 GH-H (β/α)8 13, 70, 77 GH-I β+α 24, 80 GH-J 5-fold β-propeller 32, 68 GH-K (β/α)8 18, 20, 85 GH-L (α/α)6 15, 65 GH-M (α/α)6 8, 48 GH-N β-helix 28, 49 GH-O (α/α)6 52, 116 GH-P (α/α)6 127, 146 GH-Q (α/α)6 94, 149 GH-R (β/α)8 29,107

In the retaining mechanism, a general acid/base catalyst works first as an acid and then as a base in two steps: glycosylation and deglycosylation, respectively. In the first step, it facilitates departure of the leaving group by donating a proton to the glycosyl oxygen atom while the nucleophile forms a glycosyl-enzyme intermediate of opposite anomeric configuration. In the second step, the deprotonated acid/base acts as a general base to activate a water molecule that carries out a nucleophilic attack on the glycosyl-enzyme intermediate; these two inversion steps resulted in the stereochemistry retention at the anomeric center (Davies and Henrissat, 1995; Vuong and Wilson, 2010).

31 Chapter I: Literature review

a) Inverting mechanism

Catalytic acid

A A H

O O O R

O HOR OH H H

B BH Catalytic base b) Retaining mechanism

Acid/base A A AH H H O O O O O R O R* R* HOR Nuc Nuc Nuc Nucleophile Intermediate

Figure 4. General glycoside hydrolases a) inverting and b) retaining mechanisms. AH: a catalytic acid residue, B-: a catalytic base residue, Nuc: a nucleophile, and R: a carbohydrate derivative. HOR: an exogenous nucleophile, often a water molecule (adapted from Vuong and Wilson, 2010).

In addition, GHs are able to perform hydrolysis and synthesis reactions (figure 5). The hydrolysis is the main reaction that these enzymes typically perform; thus they hydrolyze the glycosidic bond by transferring the cleaved glycosyl moiety to water as an acceptor substrate. For some enzymes, the glycosyl moiety can however be transferred on to a free hydroxylated group of an acceptor molecule different from water (e.g. simple sugar, oligosaccharides or aglycon). This process is called transglycosylation and allows the formation of a new glycosidic bond, instead of hydrolysis reaction. Thus, transglycosylation is kinetically controlled, and during the reaction, it is assumed that there is a competition between the nucleophilic water and the acceptor substrate at the glycosyl-enzyme intermediate (Desmet et al. 2012). Moreover, when enzymatic synthesis is thermodynamically controlled this process is called reverse hydrolysis (van Rantwijk et al. 1999).

32 Chapter I: Literature review

R2OH R1OH

Glycosyl-O-R1 Glycosyl-O-R2 transglycosylation

s h i y s H2O d ly r o o r l d H2O y y s i h s e s r e v 1 e 2 R OH r R OH

Glycosyl-O-OH

Figure 5. Synthetic and hydrolytic reactions catalyzed by Glycoside Hydrolases (Desmet et al. 2012).

Indeed, many GHs can perform hydrolysis or synthesis reactions using different kinds of acceptors such as glucose, fructose, lactose, xylose, sucrose, etc. The GHs that transfer fructosyl units are classified into clan GH-J according to the CAZy database (Carbohydrate Active Enzymes, http:// www.cazy.org/). The clan GH-J contains the GH32 and GH68 families; they share some similarities such as a retaining mechanism, a 5-fold β-propeller tertiary structure and conserved aspartate and glutamate residues that act as nucleophile and acid/base catalyst, respectively. Structurally, this clan has a common β-propeller catalytic domain with three conserved amino acids, located in the deep axial pocket of the active site. The propeller has a 5-fold repeat of blades, each consisting of four antiparallel β-strands with the classical ‘W’ topology around the central axis, enclosing the negatively charged cavity of the active site (Lammens et al. 2009).

Although, enzymes from GH-J clan have structural similarities, especially in the amino acid residues near to the active site, they have a broad variation in substrate specificities (Yuan et al. 2012). Mainly, these enzymes use sucrose or fructans as donor substrates and sucrose, fructans or water as acceptor substrates. Some examples are levansucrases and inulosucrases from the GH68 family, plant and microbial invertases, microbial endo- and exo-type inulinases as well as levanases and a wide variety of plant fructosyltransferases plant fructan and exo-hydrolases all of them from the GH32 family (Lammens et al. 2009). The next section is entirely devoted to the description of the enzymes belonging to the GH32 and GH68 families.

33 Chapter I: Literature review

C.2. Enzymes from GH32 family

GH 32 family enzymes are present in fungi, microbial and plant organism. The members of this family are shown in table 6, some examples of these enzymes are acid-type invertases (cell wall and vacuolar type in plants), fungal and bacterial endo and exo-inulinases, levanases, plant fructan exohydrolases and plant fructan biosynthetic enzymes (Carbohydrate Active Enzymes, http://www.cazy.org/).

Table 6. Enzyme members and some features of GH32 family (adapted form CAZy, http:// www.cazy.org/).

invertase or β-fructosidase(EC 3.2.1.26), endo-inulinase (EC 3.2.1.7), β-2,6 fructan 6-levanbiohydrolase (EC 3.2.1.64), endo-levanase (EC 3.2.1.65), exo-inulinase (EC 3.2.1.80), fructan β-(2,1)-fructosidase/1- exohydrolase (EC 3.2.1.153), fructans β-(2,6)-fructosidase/6- Activities in exohydrolase (EC 3.2.1.154), sucrose:sucrose 1-fructosyltransferase Family (EC 2.4.1.99), fructans:fructans 1-fructosyltransferase (EC2.4.1.100), sucrose:fructans 6-fructosyltransferase (EC 2.4.1.10), fructans:fructans 6G-fructosyltransferase (EC 2.4.1.243), levan fructosyltransferase (EC 2.4.1.-), sucrose:sucrose 6-fructosyltransferase (6-SST) (EC 2.4.1.-), cycloinulo-oligosaccharide fructanotransferase (EC 2.4.1.-) Structure 5-fold-β-propeller Catalytic Asp Nucleophile/Base Catalytic Proton Glu donor

Structurally, the GH32 family possesses a five bladed β-propeller fold and they are characterized by an additional β-sandwich in the C-terminal region, this domain is absent in GH68 family (Lammens et al. 2009). In addition, the active site is composed of a WMNDPNG motif as the nucleophile and the EC motif as the acid/base catalyst (Reddy and Maley, 1996; Pons et al. 2004). The aspartate in the RDP motif could not to be directly implicated in the catalytic mechanism and apparently plays a role in substrate recognition and stabilization of the transition-state (Meng and Fütterer, 2003). Figure 6 illustrates the three-dimensional structure of the invertase from Thermotoga maritime, where it can be observed the propeller module, the five blades and β-sandwich module (Alberto et al. 2004).

34 Chapter I: Literature review

Figure 6. Three-dimensional structure of invertase from Thermotoga maritime. The N-terminal β-propeller module, the five blades (numbered I–V), and the C-terminal β-sandwich module (dark red) were highlighted in colors (adapted from Alberto et al. 2004).

Finally, the most well-known enzyme in this family in order to give an example of the GH32 family, is the invertase, β-fructofuranosidase or β-fructosidase (EC 3.2.1.26). Invertases occur mostly in microorganisms. Among microbial strains, for many decades yeast species have been extensively studied for invertase production, characterization, and applications in industries. The main reaction catalyzes by an invertase is sucrose hydrolysis; the hydrolysis of sucrose liberates D-glucose and D- fructose (Lincoln and More, 2017). The monosaccharide units formed are equimolar and the mixture is referred to as invert sugar. However, at high sucrose concentrations the enzyme exhibits a transferase activity and the enzyme is designated as fructosyltransferase (EC 2.4.1.9). The nomenclature of the enzyme has been in dispute as some authors have reported the transfructosylating property as β-D- fructofuranosidases (Warchol et al. 2002), while other researchers have referred this action as fructosyltransferase or transfructosylating enzymes (Kim et al. 2000). Some microbial strains exhibit both activities by efficiently hydrolyzing sucrose (invertase activity, U ), as well as significantly h transferring fructosyl moieties (transfructosylating activity, U ). However, the ratio U /U (Tranfer t t h versus Hydrolysis) varies considerably from one enzyme to another (Linconln and More, 2017).

35 Chapter I: Literature review

C.3. Enzymes from GH68 family

The enzymes that belong to GH68 family are shown in table 7 according to CAZy database. Three types of activity have been described in this family: levansucrase (EC 2.4.1.10), β-fructofuranosidase (EC 3.2.1.26) and inulosucrase (EC 2.4.1.9). These enzymes are present in bacteria and archeas (Cantarel et al. 2009). For example, levansucrases are widespread in gram-positive and gram-negative bacteria, inulosucrases have been reported only in gram-positive bacteria, specifically in species from three genera (Streptococcus, Leuconostoc and Lactobacillus) and β-fructofuranosidases are found in Arthrobacter genera (Öner et al. 2016; Cantarel et al. 2009).

Table 7. Enzymes members and some features of GH68 family (adapted form CAZy, http:// www.cazy.org/).

levansucrases (EC 2.4.1.10), β- Activities in Family fructofuranosidase (EC 3.2.1.26), inulosucrase (EC 2.4.1.9) Structure 5-fold-β-propeller Catalytic Nucleophile/Base Asp Catalytic Proton donnor Glu

The cleavage of the glycosidic bond is carried out by three fully conserved carboxylic residues forming the catalytic triad, which is located at the bottom of the active site pocket. The catalytic triad is composed by one aspartic acid (Asp, D) functioning as the nucleophile, a glutamic acid as acid/base catalyst (Glu, E) and an aspartic acid that acts as the transition-state stabilizer (Asp, D) (Meng and Fütterer, 2003; Pons et al. 2004). According to the literature, the highly conserved motifs related to the catalytic triad are VWD, EWSG and RDP (Meng and Fütterer, 2003).

Moreover, GH68 family does not contain an additional C-terminal β-sheet domain; they only have a 5- fold-β-propeller, with the classical ‘W’ topology around the central axis, enclosing the negatively charged cavity of the active site (Lammens et al. 2009). In addition, in figure 7 the three-dimensional structure of Levansucrase from Gluconacetobacter diazotrophicus (PDB 1W18) is presented, the five- bladed β-propeller fold is observed. Also, the catalytic residues Asp135, Asp309 and Glu401 form the catalytic triad (Martínez-Fleites et al. 2005).

36 Chapter I: Literature review

Figure 7. Three-dimensional structure of levansucrase from Gluconacetobacter diazotrophicus (adapted from Martínez-Fleites et al. 2005)

Finally, the bacterial levansucrase (EC 2.4.1.10) is chosen as a representative of the GH68 family. Mainly, levansucrases use sucrose as substrate and catalyze the synthesis of levan, which is a class of fructan composed by β-(26)-linked fructosyl units. According to the literature, levansucrases are able to form β-(21)-fructosyl-fructose linkages either to synthesize inulin-type fructooligosaccharides

(FOS) or to create branching points that connect the β-(26) linked chains of the polymer. They also possess a ratio of hydrolytic activity at low concentration of sucrose (≤0.146 M) (Raga-Carbajal et al. 2018; Öner et al. 2016; Hernández et al. 1995). The ratio of each activity is highly dependent on the enzyme’s origin and selectivity towards the various natural acceptors produced during the reaction. For instance, levansucrase from the Gram-positive bacterium Bacillus subtilis mainly converts sucrose into levan, whereas levansucrase from the Gram-negative species Gluconacetobacter diazotrophicus catalyzes the formation of relatively high levels of 1-kestotriose and 1-1-kestotetraose, and in consequence, lower levels of levan, under the same reaction conditions (Hernández et al. 1995).

37 Chapter I: Literature review

C.4. Catalytic mechanism

Enzymes from GH32 and GH68 family possess the same retaining mechanism (figure 9). This mechanism operates via a double displacement mechanism, using a fructosyl-enzyme covalent intermediate. Generally, they cleave the glycosidic bond between two monosaccharides or between a carbohydrate and an aglycon moiety. For the consecutive binding sites, Davies et al. (1997) proposed the -n to +n subsite nomenclature. This nomenclature is represented in figure 8, where, by convention the point of cleavage is indicated by an arrow with the non-reducing end of the substrates on the left and its reducing one on the right. For example, hydrolysis of disaccharides takes place between the –1 and +1 subsite (Davies et al. 1997). In addition, it was proposed for retaining enzymes that the different binding positions of the sugars in the –1 subsite are more crucial, than the distance between the catalytic residues (Davies et al. 1997).

Non-reducing end Reducing end

Figure 8. Schematic drawing of the sugar-binding subsites from –n to +n nomenclature (adapted from Davies et al. 1997).

Figure 9 illustrates the catalytic mechanism in GH32 and GH68 families, which involves a two-step reaction in which a covalent fructosyl-enzyme intermediate is formed. In the first step (fructosylation) a nucleophilic attack is performed on the anomeric carbon of the sugar substrate by the carboxylate of the nucleophile, forming a covalent fructosyl–enzyme intermediate. The acid/base catalyst acts as a general acid donating a proton to the glycosyl-leaving group. In the second step (defructosylation) the acid/base catalyst acts as a general base, removing a proton from the incoming fructosyl acceptor (water or an appropriate sugar acceptor), which cleaves the fructosyl–enzyme intermediate (Lammens et al. 2009). Therefore, the outcome of the reaction is dependent on the acceptor. In the case of water, the reaction results in hydrolysis, whereas transfructosylation is performed when the fructosyl moiety is transferred to a sugar acceptor (or hydroxylated molecule).

38 Chapter I: Literature review

Figure 9. Retaining mechanism form GH32 and GH68 families (adapted from Lammes et al. 2009).

39 Chapter I: Literature review

D. Aims of the thesis

Flavonoids belong to a class of important molecules showing a high potential for improving health due to their biological properties such as antioxidant, anti-inflammatory or anti-tumor properties. However due to their hydrophobic nature, they show a low solubility, sometimes a poor stability and a limited bioavailability. In order to improve these properties, enzymatic fructosylation of flavonoids is a promising biotechnological alternative to produce new bioactive molecules with a higher specificity and regioselectivity than chemical methods.

To further investigate the potential of β-fructosidases from the families GH32 and GH68 to fructosylate flavonoids, we proposed to screen enzymes secreted from Mezcal and test them together with other fructosyltransferases generously provided by Dr. Lázaro Hernández from the Centro de Ingeniería Genética y Biotecnología, La Habana, Cuba. The thesis work encompassed the following approaches:

 Screening of non-saccharomyces yeasts isolated from Mezcal for their ability to catalyze transfructosylation from sucrose to flavonoids

 In silico gene search encoding new β-fructosidases in selected yeast genomes; cloning and expression a selected gene and testing of the produced enzyme for flavonoid fructosylation

 Comparison of the enzymatic fructosylation of phlorizin by β-fructosidases from different sources, belonging to GH32 and GH68 families and showing different specificities.

E. Hypothesis

The enzymatic fructosylation of acceptors different from sugars such as flavonoids can be efficiently performed through the use of β-fructosidases with different specificities and can lead to novel derivatives of high potential for food and health industries.

40 Chapter I: Literature review

F. References

Bertrand A, Morel S, Lefoulon F, Rollan Yves, Monsam P, Remaud-Simeon M (2006) Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous- organic solvents. Carbohydr Res 341:855–863. doi: 10.1016/j.carres.2006.02.008

Cantarel BI, Coutinho PM, Rancurel C, Bernard T, Lombard V, HenriSSAT B (2009) The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Res 37:233–238. doi: 10.1093/nar/gkn663

Cao G, Sofic E, Prior RL (1997) Antioxidant and prooxidant behavior of flavonoids: Structure-activity relationships. Free Radic Biol Med 22:749–760. doi: 10.1016/S0891-5849(96)00351-6

Chebil L, Humeau C, Anthoni J, Dehez F, Engasser JM, Ghoul M (2007) Solubility of flavonoids in organic solvents. J Chem Eng Data 52:1552–1556. doi: 10.1021/je7001094

Chen S, Xing X-H, Huang J-J, Xu M-S (2011) Enzyme-assisted extraction of flavonoids from Ginkgo biloba leaves: Improvement effect of flavonol transglycosylation catalyzed by Penicillium decumbens cellulase. Enzyme Microb Technol 48:100–105. doi: 10.1016/j.enzmictec.2010.09.017

Choi C-H, Kim J-W, Park C-S, park K-H, Kyung M-O, Park J-T, Lee C-K, Kim Y-W, lee J-H, park S- H, Shim J-H, Li X-H (2010) Enzymatic biosynthesis of a puerarin–cycloamylose inclusion complex by 4-α-glucanotransferase and maltogenic amylase. Biocatal Biotransformation 28:209– 214. doi: 10.3109/10242421003754538

Cho H-K, Kim H-H, Seo D-H, Jung J-H, park J-H, Baek N-I, Kim M-J, Yoo S-H, Cha J, Kim Y-R, park C-S (2011) Biosynthesis of (+)-catechin glycosides using recombinant amylosucrase from Deinococcus geothermalis DSM 11300. Enzyme Microb Technol 49:246–253. doi: 10.1016/j.enzmictec.2011.05.007

Crich D (2010) Mechanism of a chemical glycosylation reaction. Acc Chem Res 43:1144–1153. doi: 10.1021/ar100035r

Crozier A, Jaganath IB, Clifford MN (2009) Dietary phenolics: chemistry, bioavailability and effects on health. Nat Prod Rep 26:1001–1043. doi: 10.1039/b802662a

Das R, Mukhopadhyay B (2016) Chemical O-Glycosylations: An Overview. ChemistryOpen 5:401– 433. doi: 10.1002/open.201600043

Das SS, Gauri SS, Misra BB, et al (2013) Purification and characterization of a betanidin glucosyltransferase from Amaranthus tricolor L catalyzing non-specific biotransformation of flavonoids. Plant Sci 211:61–69. doi: 10.1016/j.plantsci.2013.07.003

Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859. doi: 10.1016/S0969-2126(01)00220-9

Davies GJ, Wilson KS, Henrissat B (1997) Nomenclature for sugar-binding subsites in glycosyl hydrolases. Biochem J 321,Pt 2:557–9.

41 Chapter I: Literature review

Desmet T, Soetaert W, Bojarová P, et al (2012) Enzymatic glycosylation of small molecules: Challenging substrates require tailored catalysts. Chem-A Eur J 18:10786–10801. doi: 10.1002/chem.201103069

Di Carlo G, Mascolo N, Izzo A, Capasso F (1999) Flavovoids: old and new aspects of a class of natural therapeutic drugs. Life Sci 65:337–353. doi: 10.1016/S0024-3205(99)00120-4

Falcone Ferreyra ML, Rius SP, Casati P (2012) Flavonoids: biosynthesis, biological functions, and biotechnological applications. Front Plant Sci 3:1–15. doi: 10.3389/fpls.2012.00222

Funayama M, Nishino T, Hirota A, Murao S, Takenishi S, nakano H (1993) Enzymatic synthesis of (+)-catechin-α-glucoside and its effect on tyrosinase activity. Biosci Biotechnol Biochem 57:1666–1669. doi: 10.1080/bbb.57.1666

Gao C, Mayon P, MacManus DA, Vulfson EN (2000) Novel enzymatic approach to the synthesis of flavonoid glycosides and their esters. Biotechnol Bioeng 71:235–243. doi: 10.1002/1097- 0290(2000)71:3<235::AID-BIT1013>3.0.CO;2-M

Gee JM, DuPont MS, Rhodes MJC, Johnson IT (1998) Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic Biol Med 25:19-25. doi.org/10.1016/S0891- 5849(98)00020-3

Gee JM, DuPont MS, Day AJ, Plumb GW, Williamson G, Johnson IT (2000) Intestinal transport of quercetin glycosides in rats involves both deglycosylation and interaction with the hexose transport pathway. J Nutr 130: 2765–27771.

Gosch C, Halbwirth H, Schneider B, Hölscher D, Stich K (2010) Cloning and heterologous expression of glycosyltransferases from Malus x domestica and Pyrus communis, which convert phloretin to phloretin 2 ′ -O-glucoside (phloridzin). Plant Sci 178:299–306. doi: 10.1016/j.plantsci.2009.12.009

Hernández L, Arrieta J, Menendez C, Vazquez R, Coego A, Suarez V, Selman G, Petit-Glatron MF, Chambert R (1995) Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane. Biochem J 309:113–118. doi: 10.1042/bj3090113

Hofer B (2016) Recent developments in the enzymatic O-glycosylation of flavonoids Biochem Biotechnol 100:4269–4281. doi: 10.1007/s00253-016-7465-0

Hyung Ko J, Gyu Kim B, Joong-Hoon A (2006) Glycosylation of flavonoids with a glycosyltransferase from Bacillus cereus. FEMS Microbiol Lett 258:263–268. doi: 10.1111/j.1574- 6968.2006.00226.x

Kim BW, Kwon HJ, Park HY, Nam SW (2000) Production of a novel transfructosylating enzyme from Bacillus macerans EG-6. Bio-process Eng 23:11–16. doi: 10.1007/s004499900078

Kometani T, Nishimura T, Nakae T, et al (1996) Synthesis of Neohesperidin Glycosides and Naringin Glycosides by Cyclodextrin Glucano-transferase from an Alkalophilic Bacillus Species. Biosci Biotechnol Biochem 60:645–649. doi: 10.1271/bbb.60.645

42 Chapter I: Literature review

Koshland DEe (1953) stereochemistry and the mechanism of enzymatic reactions. Biol Rev 28:416– 436. doi: 10.1111/j.1469-185X.1953.tb01386.x

Kozłowska A and Szostak-Wegierek D (2014) Flavonoids, Food sources and health benefits. Rocz Państwowego Zakładu Hig 65:79–85.

Kumar S, Pandey AK (2013) Chemistry and Biological Activities of Flavonoids : An Overview Chemistry and Biological Activities of Flavonoids : An Overview. doi: 10.1155/2013/162750

Martínez-Fleites C, Ortíz-Lombardía M, Pons T, Tarbouriech N, Taylor E, Arrieta J, Hernández L, Davies G (2005) Crystal structure of levansucrase from the Gram-negative bacterium Gluconacetobacter diazotrophicus. Biochem J 390:19–27. doi: 10.1042/BJ20050324

Lammens W, Le Roy K, Schroeven L, et al (2009) Structural insights into glycoside hydrolase family 32 and 68 enzymes: Functional implications. J Exp Bot 60:727–740. doi: 10.1093/jxb/ern333

Li D, Park J-H, Park J-T, Oark CS, Park K-H (2004) Biotechnological Production of Highly Soluble Daidzein Glycosides Using Thermotoga maritima Maltosyltransferase. J Agric Food Chem 52:2561–2567. doi: 10.1021/jf035109f

Lincoln L, More SS (2017) Bacterial invertases: Occurrence, production, biochemical characterization, and significance of transfructosylation. J Basic Microbiol 57:803–813. doi: 10.1002/jobm.201700269

Lu MF, Xiao ZT, Zhang HY (2013) Where do health benefits of flavonoids come from? Insights from flavonoid targets and their evolutionary history. Biochem Biophys Res Commun 434:701–704. doi: 10.1016/j.bbrc.2013.04.035

Malbert Y, Pizzut-Serin S, Massou S, Cambon E, Laguerre S, Monsan P, Lefoulon F, Morel S, André I, Remaud-Simeon M (2014) Extending the Structural Diversity of α-Flavonoid Glycosides with Engineered Glucansucrases. ChemCatChem 6:2282–2291. doi: 10.1002/cctc.201402144

Meng G, Fütterer K (2003) Structural framework of fructosyl transfer in Bacillus subtilis levansucrase. Nat Struct Biol 10:935.

Meulenbeld GH, Zuilhof H, van Veldhuizen A, van den Heuvel R, Hartmans S (1999) Enhanced (+)- catechin transglucosylating activity of Streptococcus mutans GS-5 glucosyltransferase-D due to fructose removal. Appl Environ Microbiol 65:4141–7.

Ohgami S, Ono E, Toyonaga H, et al (2014) Identification and characterization of Camellia sinensis glucosyltransferase, UGT73A17: A possible role in flavonol glucosylation. Plant Biotechnol 31:573–578. doi: 10.5511/plantbiotechnology.14.1027a

Öner ET, Hernández L, Combie J (2016) Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnol Adv. doi: 10.1016/j.biotechadv.2016.05.002

43 Chapter I: Literature review

Panche AN, Diwan AD, Chandra SR (2016) Flavonoids: An overview. J Nutr Sci. doi: 10.1017/jns.2016.41

Pandey, KB and Rizvi, SI (2009) Plant polyphenols as dietary antioxidants in human health and disease. Oxidative Medicine and Cellular Longevity, 2(5), 270–8.

JS, Sohng JK (2013) Enzymatic synthesis of novel phloretin glucosides. Appl Environ Microbiol 79:3516–3521. doi: 10.1128/AEM.00409-13

Pandey RP, Parajuli P, Koffas MAG, Sohng JK (2016) Microbial production of natural and non-natural flavonoids: Pathway engineering, directed evolution and systems/synthetic biology. Biotechnol Adv 34:634–662. doi: 10.1016/j.biotechadv.2016.02.012

Perez-Vizcaino F, Duarte J, Santos-Buelga C (2012) The flavonoid paradox: conjugation and deconjugation as key steps for the biological activity of flavonoids. J Sci Food Agric 92:1822– 1825. doi: 10.1002/jsfa.5697

Plaza M, Pozzo T, Liu J, Ara GZK, Turner C, Karlsson EN (2014) Substituent effects on in vitro antioxidizing properties, stability, and solubility in flavonoids. J Agric Food Chem 62:3321– 3333. doi: 10.1021/jf405570u

Pons T, Naumoff DG, Martínez-Fleites C, Hernández L (2004) Three acidic residues are at the active site of a β-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68. Proteins Struct Funct Bioinforma 54:424–432. doi: 10.1002/prot.10604

Quideau S, Deffieux D, Douat-Casassus C, Pouységu L (2011) Plant polyphenols: Chemical properties, biological activities, and synthesis. Angew Chemie - Int Ed 50:586–621. doi: 10.1002/anie.201000044

Raga-Carbajal E, López-Munguía A, Alvarez L, Olvera C (2018) Understanding the transfer reaction network behind the non-processive synthesis of low molecular weight levan catalyzed by Bacillus subtilis levansucrase. Sci Rep 8:15035. doi: 10.1038/s41598-018-32872-7

Razmara RS, Daneshfar A, Sahraei R (2010) Solubility of Quercetin in Water + Methanol and Water + Ethanol from (292.8 to 333.8) K. J Chem Eng Data 55:3934–3936. doi: 10.1021/je9010757

Reddy A, Maley F (1996) Studies on Identifying the Catalytic Role of Glu-204 in the Active Site of Yeast Invertase. J Biol Chem 271:13953–13958. doi: 10.1074/jbc.271.24.13953

Sak K (2014) Cytotoxicity of dietary flavonoids on different human cancer types. Pharmacogn Rev 8:122. doi: 10.4103/0973-7847.134247

Schmidt RR, Michel J (1980) Facile Synthesis of α- and β-O-Glycosyl Imidates; Preparation of Glycosides and Disaccharides. Angew Chemie Int Ed English 19:731–732. doi: 10.1002/anie.198007311

Singh S, Vishwakarma R, Kumar RJS, Sonawane PD KB (2013) Functional Characterization of a Glucosyltransferase Specific to Flavonoid 7-O-Glucosides from Withania somnifera. Plant Mol Biol Report 31:1100–1108. doi: 10.1007/s11105-013-0573-4

44 Chapter I: Literature review

Slámová K, Kapešová J, Valentová K (2018) “Sweet Flavonoids”: Glycosidase-Catalyzed Modifications. Int J Mol Sci 19:1–19. doi: 10.3390/ijms19072126

Sordon S, Popłoński J, Huszcza E (2016) Microbial Glycosylation of Flavonoids. Polish J Microbiol 65:137–51.

Spagnuolo C, Moccia S, Russo GL (2018) Anti-inflammatory effects of flavonoids in neurodegenerative disorders. Eur J Med Chem 153:105–115. doi: 10.1016/j.ejmech.2017.09.001 van Rantwijk F, Woudenberg-van Oosterom M, Sheldon RA (1999) Glycosidase-catalysed synthesis of alkyl glycosides. J Mol Catal B Enzym 6:511–532. doi: 10.1016/S1381-1177(99)00042-9

Vuong T V., Wilson DB (2010) Glycoside hydrolases: Catalytic base/nucleophile diversity. Biotechnol Bioeng 107:195–205. doi: 10.1002/bit.22838

Wallace JE, Schroeder LR (1977) Koenigs–Knorr reactions. Part 3. Mechanistic study of mercury( II) cyanide promoted reactions of 2-O-acetyl-3,4,6-tri-O-methyl-α- D - glucopyranosyl bromide with cyclohexanol in benzene–nitromethane. J Chem Soc, Perkin Trans 2 2:795–802. doi: 10.1039/P29770000795

Warchol M, Perrin S, Grill JP, Schneider F (2002) Characterization of a purified β-fructofuranosidase from Bifidobacterium infantis ATCC 15697. Lett Appl Microbiol 35(6):462–467. doi: 10.1046/j.1472-765X.2002.01224.x

Wu X, Chu J, Wu B, Zhang S, He B (2013) An efficient novel glycosylation of flavonoid by β- fructosidase resistant to hydrophilic organic solvents. Bioresour Technol 129:659–62. doi: 10.1016/j.biortech.2012.12.041

Xiao J, Muzashvili TS, Georgiev MI (2014) Advances in the biotechnological glycosylation of valuable flavonoids. Biotechnol Adv 32:1145–1156. doi: 10.1016/j.biotechadv.2014.04.006

Xu L, Qi T, Xu L, Lu L, Xiao M (2016) Recent progress in the enzymatic glycosylation of phenolic compounds. J Carbohydr Chem 35:1–23. doi: 10.1080/07328303.2015.1137580

Yuan S, Le Roy K, Venken T, Lammes W, Van den Ende W, De Maeyer M (2012) pKa Modulation of the Acid/Base Catalyst within GH32 and GH68: A Role in Substrate/Inhibitor Specificity? PLoS One 7:e37453. doi: 10.1371/journal.pone.0037453

Zhang H, Wang M, Chen L, Liu Y, Liu H, Huo H, Sun L, Ren X,Deng Y, Qi A (2017) Structure- solubility relationships and thermodynamic aspects of solubility of some flavonoids in the solvents modeling biological media. J Mol Liq 225:439–445. doi: 10.1016/j.molliq.2016.11.036

Zhu X, Schmidt RR (2009) New Principles for Glycoside-Bond Formation. Angew Chemie Int Ed 48:1900–1934. doi: 10.1002/anie.200802036

CHAPTER II: FUNCTIONALIZATION OF NATURAL COMPOUNDS BY ENZYMATIC FRUCTOSYLATION

This chapter was published in Applied Microbiology and Biotechnology.

Functionalization of natural compounds by enzymatic fructosylation

Herrera-González A, Núñez-lópez G, Morel S, Amaya-Delgado L, Sandoval G, Gschaedler A, Remaud-Simeon M, Arrizon J, (2017). Appl Microbiol Biotechnol 101:5223–5234.

Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Chapter II of this thesis work is devoted to a bibliographic study on the enzymatic fructosylation of natural compounds using glycoside hydrolases from GH32 and GH68 families. Thus, in the first part of this chapter, general consideration on enzyme belonging to glycoside hydrolase family GH 32 and GH68 are presented such as their mechanism, reactions, products and main features. The second part focuses on the enzymatic fructosylation of different acceptors such as aliphatic alcohols, aromatic alcohols, alkaloids, flavonoids and xanthonoids. In addition, in these sections, the microorganisms, the catalysts, the reaction condition (acceptor, donor, solvent, pH, temperature) and the yield of the fructosylation were reviewed. Moreover, in the third part, the solubility and antioxidant activity of these new fructo-conjugates are presented and compared to their aglycone. Finally, an analysis of the pharmacokinetic properties of the new fructo-conjugates is showed.

This review has highlighted the interest to synthetize these new fructo-conjugates and it allowed us to know what kind of molecules; reactions conditions and enzymes have been reported in the literature. In addition, this chapter is part of the first publication of this thesis.

47 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Mini review

Functionalization of natural compounds by enzymatic fructosylation

Azucena Herrera-Gonzáleza,b, Gema Núñez-Lópeza,b, Sandrine Morelb, Lorena Amaya- Delgadoa, Georgina Sandovala, Anne Gschaedlera, Magali Remaud-Simeonb, Javier Arrizona aCentro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.- Unidad Zapopan. Camino Arenero 1227, El Bajio del Arenal, 4519, Zapopan, Jal. México b LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France LISBP-INSA Toulouse 135 Avenue de Rangueil, 31077 Toulouse, France

A. Summary

Enzymatic fructosylation of organic acceptors other than sugar opens access to the production of new molecules that do not exist in nature. These new glycoconjugates may have improved physical- chemical and bioactive properties like solubility, stability, bioavailability and bioactivity. This review focuses on different classes of acceptors including alkyl alcohols, aromatic alcohols, alkaloids, flavonoids, xanthonoids, which were tested for the production of fructoderivatives using enzymes from the glycoside hydrolase (GH) families 32 and 68 that use sucrose as donor substrate. The enzymatic strategies and the reaction conditions required for the achievement of these complex reactions is discussed, in particular with regard to the type of acceptors. The solubility, pharmacokinetic and antioxidant activity of some of these new -D-fructofuranosides in comparison is reviewed and compared with their glucoside analogs to highlight the differences between these molecules for technological applications.

48 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

B. Introduction

In the last two decades, the functionalization of natural organic acceptors such as aliphatic and aromatic alcohols, alkaloids, xanthonoids and flavonoids with fructosyl moiety has been investigated to improve their physical-chemical and biological properties. These new compounds can be obtained chemically or by enzymatic synthesis (Lee et al. 2003; Du et al. 2004; van Rantwijk et al. 1999; Wu et al. 2013a; Xiao et al. 2014). The chemical process has some drawbacks as it involves many steps to protect and deprotect hydroxyl groups and the use of heavy metals. In addition, it may produce large quantities of toxic wastes (Lee et al. 2003; Du et al. 2004). An alternative way to reduce the impact on the environment is the use of enzymes that catalyze reactions under mild conditions, consume less energy and generate less waste as the reactions can usually be performed in water. In addition they have the major advantage of being regio- and stereoselective.

In particular, various glycoside hydrolases (GH) belonging to the families 32 and 68 and which use sucrose as substrate were reported to perform fructosylation of organic compounds (Cantarel et al. 2009) and, among them, -fructofuranosidases (EC 3.2.1.26) belong to the GH 32 and 68 family, levansucrases (EC 2.4.1.10) and inulosucrases (2.4.1.9) belong to the family GH 68. The main reaction catalyzed by the -fructofuranosidases of GH32 and GH68 families is sucrose hydrolysis. These enzymes can also transfer fructosyl units from sucrose to a suitable acceptor (glucose, fructose or sucrose), but to a much lesser extent, as the transfructosylation versus hydrolysis ratio (T/H ratio) is mainly in favor of hydrolysis. In contrast, levansucrases and inulosucrases are fructosyltransferases. They present a much higher T/H ratio and mainly catalyse fructosyl transfer (2) to a growing fructan chain to produce levan, consisting of -(2®6)-linked fructosyl residues, or inulin consisting of -(2 ®1)-linked fructosyl residues through a polymerization reaction, (1) to water (hydrolysis reaction), or (3) to acceptor molecules (acceptor reaction) (figure 10).

GH 32 and 68 enzymes share the same 5-fold -propeller structure and double displacement retaining mechanism and mainly differ by an additional -sandwich domain appended to the C-terminal domain found in GH32 family (Lammens et al. 2009) Due to these characteristics, they have been classified as belonging to the GH-J clan. The retaining mechanism operates through, first, the nucleophilic attack of an Asp residue to the anomeric carbon of the fructosyl moiety of sucrose, which is assisted by the protonation of the leaving group (glucosyl) involving a Glu residue playing the role of the acid/base catalyst. A fructosyl-enzyme type covalent intermediate is formed which, in the second step, undergoes a nucleophilic attack by an acceptor molecule deprotonated with the assistance of the acid-base

49 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation catalyst. The acceptor is a water molecule in the case of hydrolytic reactions or another hydroxylated molecule (sucrose or growing fructan chain) in the case of transfructosylation. Transfructosylation may result in the formation of different types of -fructosidic linkages depending on the enzyme linkage specificity (Lafraya et al. 2011; Lammens et al. 2009).

Certain sucrose active enzymes of GH family 32 and 68 have been also reported as being able to fructosylate different types of organic acceptors other than sugars to generate new fructo-conjugates. The aim of this paper is thus to provide for the first time an overview of all published knowledge in this field and describe the enzymatic reactions as well as the biological and physical-chemical properties of the new-to-nature fructo-conjugates.

Figure 10. Schematic diagram of hydrolysis and transfructosylation reactions catalyzed by enzymes from GH32 and GH68 families.

50 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

C. Fructosylation of different acceptors

C.2. Fructosylation of alkyl alcohol

Alkyl alcohols were the first non-sugar molecules that were tested as acceptors for GH32 or GH38 enzymes. Table 8 makes a summary of the conditions used and the fructosylation yields obtained. Most of the studies were performed with GH32 invertase and GH68 levansucrase. The first attempt of alcohol fructosylation was reported by Breuer and Bacon (1957). The reaction was carried out in the presence of methanol and sucrose with an invertase from Saccharomyces cerevisiae. Methanol fructosylation was observed in parallel with sucrose hydrolysis (Breuer and Bacon 1957). Since then, invertase from Saccharomyces cerevisiae has been widely used to study the production of alkyl-β-D- fructofuranosides under different conditions. Straaforth et al. (1988) performed the fructosylation of different alcohols including methanol, ethanol, n-propanol, butanol, allyl alcohol and tert-butanol in similar experimental conditions to those used by Breuer and Bacon (1957) (25 ºC, pH 4.8, 239 mM sucrose, 40 % (w/v) methanol). Methyl-β-D-fructoside, ethyl-β-D-fructoside, propyl-β-D-fructoside and butyl-β-D-fructoside were obtained with a yield of 40, 18, 12, and 9% respectively. Fructosylation decreased with the increase of the carbon chain length. The authors suggested that the aglycone binding site of invertase has many non-polar residues that reduce the affinity of long carbon same authors did not succeed in fructosylating secondary or tertiary alcohols and this was attributed to a possible steric hindrance. Invertase activity in anhydrous solvents was measured and no enzymatic activity was detected in absence of water and presence of alcohols (Straathof et al. 1988). Selisko et al. (1990) found that the increase in the yield of alkyl-β-D-fructofuranosides depended on the amount of enzyme. At high enzyme concentrations (24 U mL-1), the yield of alkyl-fructosides decreased, possibly due to hydrolysis of the synthesized alkyl-fructosides. Rodriguez et al. (1996) studied the effect of temperature, pH, the amount of enzyme and acceptor concentrations in order to avoid the hydrolysis of methyl-β-D-fructoside. These authors found that the main factors that improved synthesis yield were pH and temperature (4 ºC, pH 6, 12 U mL-1, yield 35%). The temperature effect in the production of methyl-fructoside showed that (40, 24.5 and 4 ºC, 12 U mL-1, pH 6, 150 mM sucrose, 20 % (v/v) methanol) at 40 ºC and 24.5 ºC the hydrolysis of methyl--D-fructofuranosides was observed, and at 4 ºC, hydrolysis decreased by a factor 5, which was correlated with the activation energy of alkyl--D- fructoside hydrolysis (Rodriguez et al. 1996). The concentration of the acceptor has been another interesting parameter to study; Straaforth et al. (1988) reported that the reaction is possible with a high percentage of methanol up to 50-60%, v/v. However, Rodriguez et al. (1996) showed that high concentrations of alcohol caused enzyme inhibition due to the loss of enzyme stability in long duration reactions. Indeed, using more than 20% methanol (v/v) caused irreversible changes in the enzyme

51 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation conformational structure and led to protein precipitation (Rodriguez et al. 1997; Straaforth et al. 1988). When alcohols are used, it is consequently important to consider the limit of the acceptor concentration. More recently, Dudíková et al. (2007) adopted a new approach for alcohol fructosylation by using cell wall fragments of Cryptococcus laurentii CCY 17-3-6 containing - fructosidases from GH32 family. They propose to use the acceptor such as co-solvent and an acceptor at the same time. The results showed that the use of 90% (v/v) of the acceptor as a co-solvent allowed reaching up to 38% and 26% fructosylation yield for butanol and pentanol, respectively. However, in these conditions, alcohols with double or triple bonds, such as allyl alcohol and propargyl alcohol were fructosylated with very low yield of only 3% or 4%, respectively (Table 8) (Dudíková et al. 2007).

Levansucrases from GH family 68, which naturally catalyze levan synthesis, have also been tested for the fructosylation of alkyl alcohols (Li et al. 2015). Perez Oseguera et al. (1996) performed fructosylation of methanol, sorbitol and glycerol using a levansucrase from Bacillus circulans. A higher yield was obtained for methyl fructoside (50%), than for glycerol and sorbitol fructoside (30%). This was attributed to the competition between hydrolysis and transfructosylation, which varies with the acceptor (Perez Oseguera et al. 1996). Kim et al. (1998) used solvents including methanol, ethanol, ethylene glycol and propylene glycol at 20% (v/v) as a strategy to increase the yield of levan synthesis with a levansucrase from Rahnella aquatilis ATCC 33071. Interestingly they observed residual fructosylation of the solvents on the available hydroxyl group and consequently investigated the production of fructosides with these solvents. The highest yield (70%) was obtained at 10 ºC, 60% (w/v) of methanol and 146 mM sucrose (Kim et al. 2000). Gonzalez-Muñoz et al. (1999) investigated the fructosylation of glycerol and observed the formation of a mixture of glycerol fructosides with -(2 ®2’) and -(2 1’) fructopyranosyl glycerol linkages, indicating that the fructosyl transfer occurred on two different positions. The highest yield (78 %) was obtained at 37° C, 500 g L-1 of glycerol and 818 mM sucrose (Gonzalez-Muñoz et al. 1999). Butanol at different concentrations (400, 500 and 850 mM butanol, at 30 ºC, pH 6, 400 mM sucrose, 5 U mL-1) was also more recently tested as acceptor with a levansucrase from Bacillus subtilis Marburg 168 in presence of 400 mM sucrose (Mena-Arizmendi et al. 2011). The best fructosylation yield (1.91 %) was obtained with 850 mM butanol. These authors suggested that as a primary alcohol, butanol is not a good acceptor due to the difficulty of the deprotonation of the hydroxyl group, and the presence of high concentrations of butanol increased levan synthesis due to low water activity (Mena-Arizmendi et al. 2011).

52 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

According to the comparison of the different strategies, the enzymatic synthesis of alkyl--D- fructofuranosides is highly dependent on the type of enzyme. In the case of GH family 32 enzymes, the invertase from S. cerevisiae has some drawbacks including low yield and hydrolysis of the fructoside product with a rapid loss of the enzyme activity in presence of organic solvents. However, – fructosidases produced by Cryptococcus laurenti CCY-17-3-6 cell walls work more efficiently than the S. cerevisiae invertase with a higher affinity for different acceptors and a higher solvent robustness, possibly due to the natural entrapment of the enzyme in the cell wall that can be seen as a kind of enzyme immobilization. In contrast, the synthesis of alkyl--D-fructofuranosides using levansucrases from different sources (GH family 68 enzymes) generally gives higher synthesis yields, high versatility for acceptor affinity, which, according to Mena-Arizmendi et al. (2011), is possibly due to better reactivity of the nucleophile.

53 Table 8. Fructosylation of alkyl alcohols from sucrose by microbial enzymes. Conditions Microorganism Biocatalyst Acceptor Donor Solvent Temperature Time Yield Reference pH (mM) (ºC) (h)

Saccharomyces Invertase Methanol Sucrose 50 mM Breuer and 4.8 Room temperature 5 NR cerevisiae GH 32 40% (w/v) (239) Sodium Bacon 1957 acetate buffer Methanol 40a (40% v/v)

Ethanol 18a (50% v/v)

n-Propanol Invertase 80 mM 12a Saccharomyces (55% v/v) Sucrose Straathof et (240 U mg-1) Sodium 4.8 25 NR cerevisiae Butanol (438) al.1988 GH 32 acetate buffer 8a (50% v/v)

Allyl alcohol 7a (75% v/v)

tert-Butanol ND (10% v/v) Methanol, Invertase 25 mM Saccharomyces Ethanol, Sucrose 11.7b Selisko et al. (0.6 U mL-1) sodium 5 25 30 cerevisiae n-Propanol (146) 4.6b 1990 GH 32 acetate buffer 50% (v/v) 0b

Invertase Methanol Sucrose Rodriguez et Saccharomyces 50 mM 6 4 3 35c (3 U mL-1) 20% (v/v) (146) al. 1996 cerevisiae acetate buffer GH 32 Methanol* 4 17c 30 %(v/v)

Ethanol* 4 19c 30 %(v/v)

Propanol* 14 28c 90 %(v/v)

Butanol* 14 38c 90 %(v/v) Cell walls 100 mM Pentanol* Cryptococcus laurenti β-fructosidase Sucrose McIlvaine 20 26c Dudíková et al. 90 %(v/v) 4.8 37 CCY 17-3-6 (0.15 U mg-1) GH (146) buffer 2007 Hexanol* 32 20 12c 90 %(v/v)

Heptanol* 20 6c 90 %(v/v)

Allyl alcohol * 3 4c 30 %(v/v)

Propargyl alcohol* 3.5 3c 30 %(v/v)

Methanol +++d Perez Bacillus Levansucrase Sucrose Glycerol Water NR NR NR ++d Oseguera et al. circulans GH 68 (156) Sorbitol ++d 1996

Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Methanol 20% (v/v) 4.4c Ethanol Levansucrase 20% (v/v) 0.7c Rahnella aquatilis -1 Sucrose 50 mM Kim et al. a (1 U mL ) Ethylene glycol 6 17 72 ATCC 33071 (584) acetate buffer c 1998 GH 68 20% (v/v) 19 Propylene glycol 20% (v/v) 19c

Levansucrase 50 mM Gonzalez- Bacillus Glycerol Sucrose (0.7 U mL-1) GH phosphate 6 37 40 78 Muñoz et al. circulans (500 g L-1) (818) 68 buffer 1999 Levansucrase Methanol Sucrose Kim et al. Rahnella aquatilis (1 U mL-1) GH68 MES buffer 6 10 48 70c 60 % (v/v) (146) 2000 ATCC 33071 50 mM Levansucrase Mena- Bacillus subtilis Butanol Sucrose phosphate (5 U mL-1) GH 68 6 30 20 1.91c Arizmendi et Marburg 168 (500 mM) (400) buffer al. 2011

a Yields are calculated from sucrose consumption b Yields are calculated from the peak area of fructoside was referred to the sum of all peak area are of product mixture c Yields are calculated from alkyl-fructoside production d Yields are expressed as the fructosyl product area/sucrose area determined by thin-layer chromatography densitometry. +++: more than 50%, ++: between 30 and 50%, +: less than 30% * Used as co-solvent NR: no reported ND: no detected

55 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

C.3. Fructosylation of aromatic alcohols

Fructosylation of aromatic compounds is attractive mainly due to their antioxidant capacity, which could be useful in the pharmaceutical and cosmetics industries. Chemical synthesis to produce them is quite challenging (van Der Heijden et al. 1999), so biotechnological synthesis has industrial potential. Table 9 summarizes all the reactions reported in the literature for the fructosylation of different aromatic alcohols as acceptors.

Dudíková et al. (2007) investigated the fructosylation of aromatic compounds in presence of different water-solvent mixtures using cell walls from Cryptococcus laurentii CCY 17-3-6 with fructosyltransferase activity. They propose to add an aprotic co-solvent (acetone) to increase the solubility of the acceptor in the reaction mixture. A 70:30 (v/v) mixture of McIlvaine buffer and acetone was successful for the fructosylation of furfuryl alcohol, 4-hydroxybenzyl alcohol, vanillyl alcohol and coniferyl alcohol, the yield was 12 % for furfuryl fructoside and 4 % for the others aromatic fructosides, probably because conditions were not optimal and due to product hydrolysis. One advantage of using cell walls as biocatalysts is their recycling capacity, which ranged between 1 and 20 work cycles depending on the nature of the acceptor (Dudíková et al. 2007).

Aromatic compounds have also been fructosylated by enzymes from GH family 68. Kang et al. (2009) studied the fructosylation of hydroquinone using a levansucrase from Leuconostoc mesenteroides B- 512FMC to avoid the side effects of hydroquinone used in dermatological skin whitening treatments. The best fructosylation yield reported by Kang et al. (2009) was 14% (0.7 U mL-1, 350 mM hydroquinone, 115 mM sucrose, 28 ºC, pH 5.2). Fructosylated hydroquinone exhibited a lower antioxidant capacity and inhibition of melanin synthesis compared to aglycone hydroquinone, but a higher antioxidant capacity than -arbutin, a glycosylated hydroquinone. This work shows that the fructosylation of aromatic compounds has advantages for their formulation in cosmetics (Kang et al. 2009).

In order to improve the yield of fructosylated hydroquinone, Mena-Arizmendi et al. (2011) used a B. subtilis Marburg 168 levansucrase and investigated the influence of the sucrose, acceptor and enzyme concentrations and that of the use of a co-solvent such as 2-methy-2-propanol (2M2P). A high concentration of sucrose (400 mM) favored the reaction displacement towards transfructosylation and 30 mM of fructosyl hydroquinone. The effect of enzyme concentration was studied using 1, 5, 10 and 20 U mL-1 at 30 ºC 400 mM sucrose and 500 mM hydroquinone. It was achieved an increase in the

56 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation production of fructosyl hydroquinone (30 mM) when the reaction was performed at 5 U mL-1. Nevertheless, at higher concentrations of between 10 and 20 U mL-1 of the enzyme, the same yield was obtained in a shorter time (2 h), after which rapid product hydrolysis was observed. It is thus important to take the enzyme concentration and reaction time into account to avoid loss of the desired product. In addition, the presence of 50% (v/v) of 2-methyl-2-propanol reduced the formation of fructosyl- hydroquinone but increased levan synthesis. The authors explained that in presence of the mixture of 2- methyl-2-propanol-water the solubility of hydroquinone reduce (Mena-Arizmendi et al. 2011; Castillo and López-Munguía, 2004). These optimal conditions established for hydroquinone fructosylation were tested for the fructosylation of other acceptors including benzyl alcohol, 4-methoxyphenol, catechol and resorcinol, and yields were shown to vary depending on the nature of the acceptor. It is important to note that best yields of fructosides were obtained with low nucleophilic molecules such as resorcinol, hydroquinone and catechol with 11.17, 9.51 and 6.86 % respectively. The authors explained the difference in acceptor reactivity by the facility of deprotonation of the acceptor reactive hydroxyl group, which could also be correlated with the pKa of the acceptor (Mena-Arizmendi et al. 2011). Thus, deprotonation of the hydroxyl group is necessary for the formation of a glycoside bond between the fructose and the acceptor. For this reason, the aromatic hydroxyl group could be better acceptor molecules than alkyl alcohols (Mena-Arizmendi et al. 2011; Lammens et al. 2009).

There is only one report (Mena-Arizmendi et al. 2011) describing the use of inulosucrase for the fructosylation of aromatic alcohol. The authors have obtained a low amount of fructosyl-hydroquinone in presence of sucrose (292 mM), hydroquinone (200 mM), inulosucrase (1 U mL-1), phosphate buffer 50 mM, pH 6, 30°C.

The main factors involved in fructosylation of aromatic compounds were the concentration of sucrose, acceptor concentration, the nature and the percentage of co-solvent, which are important to avoid the product hydrolysis and increase the acceptor solubility, and a low concentration of the enzyme.

57 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Table 9. Fructosylation of aromatic alcohols from sucrose by microbial enzymes.

Conditions Microorganism Biocatalyst Acceptor Donor Temperature Time Yield Reference Solvent pH (mM) (mM) (ºC) (h) Furfuryl alcohol 100 mM 12 12a 90 % (v/v) McIlvaine buffer Cell wall 4-Hydroxybenzyl 3.5 4a Cryptococcus Fructosidase alcohol Sucrose Dudíková et al. laurenti 4.8 37 (150 U mL-1) GH (200) (500) 30 % Acetone 2007 CCY 17-3-6 32 Vanillyl alcohol (v/v) 2 4a (300) Coniferyl alcohol 3.5 4a (200) 50 mM Leuconostoc Levansucrase Hydroquinone Sucrose Sodium Kang et al. mesenteroides (0.7 U mL-1) 5.2 28 6 14a (350) (115) acetate 2009 B-512FMC GH 68 buffer

Hydroquinone 9.51b (500) Resorcinol 11.70b (500) Levansucrase Catechol 50 mM 6.86b Mena- Bacillus subtilis (5 U mL-1) Sucrose 6 30 20 (500) Phosphate Arizmendi et Marburg 168 GH 68 (400) 4-Methoxyphenol Buffer 6.25b al. 2011

(500) Benzyl alcohol 0.81b (500) Hydroquinone 20 % 2M2P 3.48b (500) (v/v)

50 mM Inulosucrase Mena- Leuconostoc Hydroquinone Sucrose Phosphate (1 U mL-1) 6 30 NR NR Arizmendi et citreum (200) (292) Buffer GH 68 al. 2011

a Yields are calculated from aromatic-fructoside production b Yields are calculated from sucrose consumption NR: no reported

58 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

C.4. Fructosylation of alkaloids

Alkaloids including morphine and nicotine have psychoactive effects, which is why they are used for the treatment of mental illness and pain. It is known that a change in the structure of a molecule may modify its biochemical properties and new molecules could be a great interest to the pharmaceutical industry (Křen et al. 1991).

There are only two reports in the literature on alkaloid fructosylation (Table 10). Flieger et al. (1990) was the first to report on fructosyl alkaloids. These authors studied the fructosylation of chanoclavine via cell biotransformation using a strain of Claviceps fusiformis W1 for the fructosylation reaction. They described the formation of two elymoclavine fructosides and other products including chanoclavine I aldehyde, elymoclavine and agroclavine. Mass spectra, 1H and 13C NMR confirmed the structure of chanoclavine 1 O-β-D-fructofuranoside and chanoclavine I O-β-D-fructofuranosyl-(2® 1)-O-β-D-fructofuranoside. The authors proposed that the reaction was catalyzed by the β-fructosidase produced by C. fusiformis W1 in the periplasmic space. The 10-day biotransformation only produced a yield of 6.8%. Unfortunately, using a fungus, the production of these compounds takes a long time, so obtaining a purified enzyme to optimize the process would be a major advantage. The physical- chemical properties of these new fructosides were not studied (Flieger et al. 1990).

Like Flieger et al. (1990), Křen (1990, 1992) investigated enzymatic fructosylation of chanoclavine, lysergol, elymoclavine, 9,10-dihydrolysegol and ergometrine maleate using a commercial yeast invertase and obtained lysergol-O-β-D-fructoside, elymoclavin-O-β-D-fructoside, chanoclavine-O-β- D-fructoside, 9,10-dihydrolysegol-O-β-D-fructoside with low yields of between 6.7 and 8.7%, and in the case of ergometrine maleate, no fructosides were detected. Křen (1990) showed that the concentration of sucrose, pH, and temperature were the most important parameters to control the synthesis yield. The same author suggested that a low pH favored sucrose hydrolysis, whereas a pH between 5 and 7 enhanced transfructosylation (Křen, 1992).

59 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Table 10. Fructosylation of alkaloids from sucrose by microbial enzymes. Conditions

Microorganism Biocatalyst Acceptor Donor Temperature Time Yield Reference Solvent pH (mM) (mM) (ºC) (h)

Chanoclavine a 14 Flieger et al. (5.8) Biotransfor Lysergol 1990 Claviceps Sucrose mation (0.79) Water 5.5 27 240 6.8a fusiformis W1 (292) Elymoclavine a (0.79) 8.1

Chanoclavine a (0.79) 8.7

9,10- Commercial β- McIlvain Dihydrolysegol Sucrose a invertase from fructosidase buffer 50 5.6 33 17 6.1 Křen 1992 (0.79) (1286) yeast GH 32 mM

Ergometrine maleate ND (0.79) a Yields are expressed as a decrease in the concentration of alkaloids at the end of the reaction ND: no detected

60 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

C.5. Fructosylation of flavonoids and xanthonoids

Flavonoids and their glycosides are widely distributed in vascular plants and are very attractive as they play an important role in the prevention, treatment and cure of different chronic degenerative diseases (Xiao et al. 2014; Yao et al. 2004; Heim et al. 2002). Xanthonoids, secondary metabolites present in a few higher plants (Cardona et al. 1990), have pharmacological properties including antioxidant, antitumor, antidiabetic and immunomodulatory effects (Muruganandan et al. 2005), specifically mangiferin (Sanchez et al. 2000; Rouillard et al. 1998). However, the absorption of these compounds in their aglycone form is limited due to their high hydrophobicity. When flavonoids are not absorbed in the gut, microbiota metabolize them, leading to major changes in their structure and altering their beneficial properties (D'Archivio et al. 2007). However, glycosylated flavonoids have been found to be better absorbed in the small intestine than aglycone (Gee et al. 1998; Gee et al. 2000). Hence, glycosylation of flavonoids and xanthonoids increases their solubility and bioavailability, which increases their value as an ingredient in the pharmaceutical industry (Gill and Valivety, 2000; Plaza et al. 2014). So far there are several reports on the glycosylation of phenolic compounds catalyzed by various glycosyltansferases (Xiao et al. 2014). However, only few of them describe the use of fructosyltransferases from GH32 and 68 families (Table 11).

Table 11 lists existing reports on the fructosylation of flavonoids and xanthonoids. According to the literature, β-fructosides were synthetized by GH family 32 enzymes two methods: (i) enzymatic fructosylation with a β-fructofuranosidase or (ii) bioconversion with whole cells in the presence of the substrates and a co-solvent.

As can be seen in Table 11, the reaction conditions varied in the substrate concentrations and solvents, such as DMSO or ethanol, used to increase the flavonoid solubility. Such solvents affect the catalytic activity of GH enzymes as they are hydrophilic, but some can function under certain concentrations (Table 11) (Castillo and López-Munguía, 2004).

The first study describing the fructosylation of a flavonoid was by Yu et al. (2010) with the production of puerarin fructoside by permeabilized cells of Microbacterium oxylans CGMCC 1788 in presence of 40% of ethanol, puerarin-7-O-glucoside and puerarin-7-O-fructoside were the main products. The same authors also reported that after ten cycles of reused permeabilized cells a molar conversion rate of 72% was obtained for puerarin-7-O-fructoside, in the first cycle they obtained 4.5 % of molar conversion,

61 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation thus it was possible shift the production of puerarin-7-O-glucoside to puerarin-7-fructoside (Yu et al. 2010).

The fructosylation of flavonoids using bacteria was also taken up by Wu et al. (2013a), who isolated different species from contaminated soil including Arthrobacter nicotianae XM6 CCTCC M2010164, Microbacterium testaseum W12, Arthrobacter arilaitensis NJM01 CCTCC M2012155 and Acinetobacter johnsonii G2; which contain -fructosidases capable of achieving fructosylation of puerarin in presence of 10 to 25% (v/v) of DMSO, with yields higher than 70% after 36-72 h reaction time. A purified -fructosidase from Arthrobacter nicotianae XM6 CCTCC M2010164 was selected to further optimize the puerarin-O-fructoside production. The highest yield (91%) was obtained with 25% of DMSO. Product purification and structural characterization showed that two different products were obtained in different proportions (75:15). The major product was -D-fructofuranosyl-(2®6)-puerarin and the minor one was -D-difructofuranosyl-(2®6)-puerarin (Wu et al. 2013a). It is important to mention that this reaction was performed using a -fructosidase from GH family 32, the amino acid sequence and the crystal structure of this enzyme have not yet been reported. The same authors reported the synthesis of other fructosides with an enzymatic extract from A. arilaitensis NJMO1, which displayed high activity and stability in solvents including DMSO, methanol and ethanol (15- 20% v/v). The synthesis of -D-monofructoside and -D-difructoside was carried out with mangiferin as acceptor (90% yield), both with fructosyl residue linked to the C6 of the glucose, as well as the synthesis of vitexin-O-fructoside with vitexin as acceptor (35% yield) (Wu et al. 2013c).

In 2014 Wang et al. reported the fructosylation of puerarin by microbial biotransformation with Lysinibacillus fusiformis CGMCC 4913 obtaining a yield of 97% after 48 h of reaction. It is important to note that the solvent used is a key factor for such biotransformation, as it can affect the microbial viability. First, the authors tested different organic solvents including toluene, methanol, cyclohexane, chloroform, DMSO, and ethanol to find the most suitable one for microbial viability. Their results showed that ethanol was the best solvent. Subsequently, they varied the ethanol concentration from 0 to 20% (v / v) and the best performance was observed with 10% ethanol (v/v) (Wang et al. 2014).

Generally, the position of the fructosylation is dependent on the biocatalyst specificity. Depending on the enzymatic preparation (cells or enzymes), fructosylation occurred on the 7-OH position of puerarin aglycone or 6-OH position of glucose moiety.

62 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

All these studies revealed that fructosylation of natural organic molecules can be performed with fructosyltransferases from GH family 32 or 68. Some questions remain concerning the benefit of fructosylation compared to other types of glycosylation. Some answers will be given in the next section dedicated to the description of the physicochemical properties of the fructo-conjugates.

63 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Table 11. Fructosylation of flavonoids from sucrose by microbial enzymes.

Conditions Microorganism Biocatalyst Yield Reference Acceptor Donor Temperature(º Time Solvent pH (mM) (mM) C) (h) Microbacterium Permeabilized Puerarin Sucrose PBS buffer oxylans CGMCC cells 8.0 30 48 72a Yu et al. 2010 (0.48) (58) 66 mM 1788 10 cycles

Arthrobacter 6.47 35 72 a Puerarin DMSO 90.6 nicotianae XM6 (265) 25% (v/v) CCTCC M2010164 a 6.47 35 48 71 Puerarin DMSO Microbacterium (48) 15% (v/v) testaseum W12 β-Fructosidase Sucrose Wu et al. (0.24 U mL-1) (584) DMSO 2013a Arthorobacter GH 32 a Puerarin 20% (v/v) 6.47 35 72 81 Arilaitensis (108) NJEM01

CCTCC M2012155

DMSO Acinetobacter Puerarin 10% (v/v) 6.47 37 36 62a johnsonii G2 (24)

Lysinibacillus Biotransformati Puerarin Sucrose Ethanol Wang et al. 8 30 48 97a fusiformis on (9.6) (60) 10% (v/v) 2014 CGMCC 4913

Mangiferin 91a

Arthrobacter (120) β-Fructosidase DMSO arilaitensis -1 Sucrose Wu et al. (0.24 U mL ) Puerarin 25% 6.47 35 24 b NJEMO1 (20 w/v) 90.5 2013c GH32 (NR) (v/v) CCTCC M2012155 Vitexin 35b (NR) a Yields calculated based on the amount of the products b Yields calculated based on remaining acceptor NR: no reported

64 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

D. Physical-chemical properties of β-D-fructofuranosides

In this section, we present the physical-chemical properties of -D-fructofuranosides produced from alkyl alcohols, aromatic alcohols and flavonoids. Little information is available in the literature about the solubility, pharmacokinetic and scavenging activity of these new compounds. These properties are important in order to address a suitable industrial application. Water solubility is one of the most important parameters due to its effect on biological absorption; for all these compounds the solubility was determined at 25 ºC (Table 12). Puerarin-7-O-fructoside is five times more soluble than puerarin, (Wang et al. 2014). When the solubility of -D-glucosyl-(1®6)-puerarin and puerarin-7-O-fructoside was compared, solubility was seen to be higher when the glucose is linked at the 6-OH position of glucose. When two molecules of glucose was added to puerarin (-maltosyl-(1 ® 6)-puerarin), solubility was 297 times higher than puerarin (Li et al. 2004). Thus, the number of sugars linked by glycosidic bonds has an effect in the solubility. In the case of -D-fructofuranosyl-(2®6)-mangiferin and -D-difructofuranosyl-(2®6)-mangiferin the same phenomena was observed, the solubility was increased by a factor of 646 and 2182 respectively. The water solubility of the 4-Hydroxyphenyl--D- fructofuranoside was not evaluated, although its glucoside analogous -Arbutin (4-Hydroxyphenyl-- D-glucopyranoside) was shown to be 1.8 times more soluble than hydroquinone (Mathew and Adlercreutz, 2013).

Another important property of these compounds is the antioxidant activity (DPPH scavenging activity) as it is correlated with biological activities (Table 13). Wu et al. (2013b) measured the antioxidant activity of mangiferin, -D-fructofuranosyl-(2 ® 6)-mangiferin, -D-difructofuranosyl-(2 ® 6)- mangiferin in a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. These authors found that di and mono-fructosyl mangiferin had higher antioxidant activity than mangiferin, possibly due to the position of the linked fructose on the 6-OH position of the linked glucose (Wu et al. 2013c). Conversely, when the hydroquinone was glycosylated, the antioxidant activity of -arbutin (4- Hydroxyphenyl--D-glucopyranoside) and 4-Hydroxyphenyl--D-fructofuranoside decreased (Kang et al. 2009). Hydroquinone has two hydroxyl groups available to catch free radicals. When a glucose or fructose is attached to hydroquinone, its antioxidant activity decreases but is still sufficient for use as a skin whitening agent (Kang et al. 2009).

65 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Table 12. Solubility of glucosylated and fructosylated compounds Solubility in water g L-1 Molecule Reference at 25 ºC Puerarin 5.2 ± 0.6 Wang et al. 2014 Puerarin-7-O-fructoside 28.9 ± 0.6 β-D-fructofuranosyl-(2 ®6)-puerarin NR Wu et al. 2013a α-D-Glucosyl-(1 ®6)-puerarin 103.22 ± 2.48 Li et al. 2004 α-Maltosyl-(1 ®6-puerarin) 1549.97 ±21.77 Mangiferin 0.464 β-D-Fructofuranosyl-(2 ®6)-mangiferin 299.89 Wu et al. 2013c β-D-Difructofuranosyl-(2 ®6)-mangiferin 1013.76 Hydroquinone 70 Mathew and Adlercreutz, 2013 β –Arbutin 128 ± 0.6 4-hydroxyphenyl-β-D-fructofuranoside NR Mena-Arizmendi et al. 2011 NR: no reported

Table 13. Antioxidant activity of fructoside and glucoside compounds determined by DPPH (2, 2-diphenyl-1- picrylhydrazyl) radical scavenging activity IC50

DPPH radical scavenging Molecule Reference activity IC50 (mM) Puerarin NR Wu et al. 2013a Puerarin-7-O-fructoside NR Wang et al. 2014 β-D-Fructofuranosyl-(2 ®6)-puerarin NR Wu et al. 2013a Puerarin-7-O-glucoside NR Jiang et al. 2008 α-D-Glucosyl-(1 ®6)-puerarin NR Li et al. 2004 α-Maltosyl-(1 ®6)-puerarin NR Mangiferin 0.0335 β-D-Fructofuranosyl-(2 ®6)-mangiferin 0.0311 Wu et al. 2013c β-D-Difructofuranosyl-(2 ®6)-mangiferin 0.0298 Hydroquinone 0.33 ± 0.02 β-Arbutin 6.04 ± 0.02 Kang et al. 2009 4-Hydroxyphenyl-β-D-fructofuranoside 5.83 ±0.03 NR: no reported

66 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

E. Pharmacokinetics parameters

The enhancement of bioavailability of fructosides is related to pharmacokinetics parameters. As can be seen in table 14, little information is available about the pharmacokinetics parameters of fructoside and glucoside molecules. These parameters have been evaluated intravenously in rats for glycoside and fructoside flavonoids. The half-time elimination (t1/2) of -D-fructofuranosyl-(2 ® 6)-puerarin and puerarin-7-O-glucoside was respectively, 4.2 and 2.85 times higher than puerarin. The peak serum concentration (Cmax) suggested that puerarin-7-O-glucoside is 1.57 times higher than puerarin, in the case of puerarin-7-O-fructoside- this value was not reported by the authors concerned (Jiang et al. 2008; Wang et al. 2014). The mean residence time revealed better retention in the plasma for puerarin- 7-O-glucoside and fructofuranosyl-(2 ®6)-puerarin than for puerarin (Jiang et al. 2008; Wu et al.

2013b). The parameters of area under the time concentration curve (AUC(0-t)) showed that puerarin-7-

O-glucoside was 3.18 times more retained in the plasma than puerarin, AUC(0-t) for fructofuranosyl-(2 6)-puerarin was 11.52 times more retained than puerarin and 5 times more than puerarin-7-O- glucoside (Jiang et al. 2008; Wu et al. 2013b). In addition the extrapolation to the infinity time of area under the time concentration curve (AUC(0-∞)) present the same trend as that observed for AUC(0-t). According to the pharmacokinetics parameters reported, one can assume that the bioavailability of puerarin-7-O-glucoside and fructofuranosyl-(2®6)-puerarin is better than that of puerarin (Jiang et al. 2008; Wu et al. 2013b). Nevertheless, it is important to note that the bioavailability of fructofuranosyl- (2®6)-puerarin is better than that of puerarin-7-O-glucoside. Finally, as little information is available on these properties, more studies are needed to establish their potential for the pharmaceutical treatment of some diseases.

67 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Table 14. Pharmacokinetics properties of glucosyl and fructosyl compounds

at bC cAUC dAUC eMRT Molecule ½ max (0-t) (0-∞) Reference (min) (μg mL-1) (μg min L-1) (μg min L-1) (min)

Jiang et al. Puerarin 4.70 ± 1.13 60.16±14.80 484.19±97.44 486.25±97.40 16.42±2.37 2008 Jiang et al. Puerarin-7-O-glucoside 13.40 ± 2.70 94.65±5.02 1,529.45±116.19 1,549.51±119.23 31.50±3.60 2008 Wang et al. Puerarin-7-O-fructoside NR NR NR NR NR 2014 β-D-Fructofuranosyl-(2 ®6)- Wu et al. 19.74 ± 2.34 NR 5,523.44±688.10 5,605.211±723.96 25.707±2.32 puerarin 2013b α-D-Glucosyl-(1 ®6)-puerarin NR NR NR NR NR Li et al. α-Maltosyl-(1 ®6)-puerarin NR NR NR NR NR 2004 Mangiferin NR NR NR NR NR β-D-Fructofuranosyl-(2 ®6)- NR NR NR NR NR Wu et al. mangiferin 2013c β-D-Difructofuranosyl-(2 ®6)- NR NR NR NR NR mangiferin Hydroquinone NR NR NR NR NR β -Arbutin NR NR NR NR NR Kang et al. 4-Hydroxyphenyl-β-D- 2009 NR NR NR NR NR fructofuranoside a Elimination half-time b Peak serum concentration c Area under the concentration time curve d Area under the time concentration curve extrapolated to infinity e Mean residence time NR: No reported

68 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

F. Conclusions and perspectives

As it can be seen, the enzymatic fructosylation of different organic acceptors produced new molecules with increased water solubility, modified pharmacokinetics properties and antioxidant activity, making them of great potential interest to the pharmaceutical and cosmetics industries. According to the literature, the most important factors to take into account for the enzymatic production of fructosides are the concentration of the donor and acceptor, pH, the presence or absence of co-solvents to avoid hydrolysis of the product and to increase the solubility of hydrophobic acceptors, the concentration of the enzyme and the nucleophilicity of the organic acceptors.

It could be interesting to understand why -fructosidase from Arthrobacter nicotianae XM6 and Lysinibacillus fusiformis were the most efficient for the fructosylation of puerarin and mangiferin. The amino acid sequence and the crystal structure need to be known to understand the catalytic mechanism behind their high catalytic efficiency. This information would be useful to achieve changes by directed mutagenesis to improve the catalytic performance in presence of hydrophobic acceptors and organic solvents, which in turn, would help understand why they can accept longer and more hydrophobic acceptors. Moreover, since fructosylation in flavonoids on the linked glucose to the aglycone has already been achieved, why is it not possible to link them to aglycone flavonoids. Knowledge is also lacking on the physical-chemical properties of the new -D-fructofuranosides, and on how these new molecules could be absorbed for their use in different treatments. -fructosidases belonging to GH family 32 have been widely used to produce a wide range of -D-fructofuranosides, but their yields were low due to their high hydrolytic activity as well as to steric hindrance in the catalytic pocket that prevent the voluminous acceptors from reaching the active site. The highest yields for fructoside synthesis were obtained with bacterial -fructosidases from GH family 32 and levansucrases from B. subtilis Marburg 168 (GH family 68). Therefore, it could be interesting to understand the differences in the catalytic mechanisms between both types of enzymes.

69 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

G. References

Breuer HJ and Bacon JSD (1957) Sucrose Formation by Taka-diastase : Action of the Enzyme on Methyl- -Fructofuranoside and Raffinose. Biochem J 66:462–468. doi.org/10.1042/bj0660462

Cardona ML, Fernandez MI, Pedro JR, Serrano A (1990) Xanthones from Hypericum reflexum. Phytochem 29:3003-3006. doi: 10.1016/0031-9422(90)87123-C

Castillo E, López-Munguía A (2004) Synthesis of levan in water-miscible organic solvents. J Biotechnol 114:209–217. doi:10.1016/j.jbiotec.2004.06.003

D’Archivio M, Filesi C, Di Benedetto R, Gargiulo R, Giovannini C, Masella R (2007) Polyphenols, dietary sources and bioavailability. Ann Ist Super Sanita, 43:348–361.

Du Y, Wei G, Linhardt RJ (2004) Total Synthesis of Quercetin 3-Sophorotrioside. J Org Chem 69:2206–2209. doi: 10.1021/jo035722y

Dudíková J, Mastihubová M, Mastihuba V, Kolarova N (2007) Exploration of transfructosylation activity in cell walls from Cryptococcus laurentii for production of functionalised -D- fructofuranosides. J Mol Catal B Enzym 45:27–33. doi:10.1016/j.molcatb.2006.11.003

Flieger M, Křen NF, Zelenkova P, Sedmera J, Novák, Sajdl P (1990) Ergot alkaloid glycosides from saprophytic cultures of Claviceps, II. Chanoclavine I fructosides. J Nat Prod 53:171–175. doi: 10.1021/np50067a025

Gee JM, DuPont MS, Rhodes MJC, Johnson IT (1998) Quercetin glucosides interact with the intestinal glucose transport pathway. Free Radic Biol Med 25:19-25. doi.org/10.1016/S0891- 5849(98)00020-3

Gill I, Valivety R (2000) Enzymatic glycosylation in plasticized glass phases: A novel and efficient route to O-glycosides. Angew Chem Int Ed 39:3804-3808. doi: 10.1002/1521- 3773(20001103)39:21<3804::AID-ANIE3804>3.0.CO;2-

Gonzalez-Munoz F, Perez-Oseguera A, Cassani J, Jiménez-Estrada M, Vazquez-Duhalt R, López- Munguía A (1999) Enzymatic synthesis of fructosyl glycerol. J Carbohydr Chem 18:275–283. doi: 10.1080/07328309908543995

Heim KE, Tagliaferro AR, Bobilya DJ (2002) Flavonoid antioxidants: Chemistry, metabolism and structure-activity relationships. J Nutr Biochem 13:572–584. doi: 10.1016/S0955- 2863(02)00208-5

Jiang J-R, Yuan S, Ding J-F, Zhu S-C, Xu H-D, Chen T, Cong X-D, Xu W-P, Ye H, Dai Y-J (2008) Conversion of puerarin into its 7-O-glycoside derivatives by Microbacterium oxydans (CGMCC 1788) to improve its water solubility and pharmacokinetic properties. Appl Microbiol Biotechnol 81:647–657. doi: 10.1007/s00253-008-1683-z

70 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Kang J, Kim Y-M, Kim N, Kim D-W, Nam S-H, Kim D (2009) Synthesis and characterization of hydroquinone fructoside using Leuconostoc mesenteroides levansucrase. Appl Microbiol Biotechnol 83:1009–16. doi: 10.1007/s00253-009-1936-5

Kim MG, Seo JW, Song K-BW, Kim C-H, Chung BH, Rhee S-K (1998) Levan and fructosyl derivatives formation by a recombinant levansucrase from Rahnella aquatilis. Biotechnol Lett 20:333–336. doi: 10.1023/A:1005310926399

Kim MG, Kim CH, Lee JS, Song KB, Rhee SK (2000) Synthesis of methyl -D-fructoside catalyzed by levansucrase from Rahnella aquatilis. Enzyme Microb Technol 27:646–651. doi: 10.1016/S0141-0229(00)00267-2

Křen V (1991) Bioconversions of ergot alkaloids. In: Fiechter A (ed) Advances in biochemical engineering /biotechnology volume 44. Springer Berlin, Heidelberg, pp 123–144

Křen V (1992) Fructosylation of ergot alkaloids by yeast invertase. Biotechnol Lett 14:769–772. doi:10.1007/BF01029136.

Křen V, Flieger M, Sajdl P (1990) Glycosylation of ergot alkaloids by free and immobilized cells of Claviceps purpera. Appl Microbiol Biotechnol 32: 645-650. doi:10.1007/BF00164733

Lafraya A, Sanz-Aparicio J, Polaina J, Marín-Navarro J (2011) Fructo-oligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase. Appl Environ Microbiol 77:6148–6157. doi: 10.1128/AEM.05032-11

Lammens W, Le Roy K, Schroeven L, van Laere A, Rabijns A, van den Ende W (2009) Structural insights into glycoside hydrolase family 32 and 68 enzymes: Functional implications. J Exp Bot 60:727–740. doi: 10.1093/jxb/ern333

Lee DYW, Zhang WY, Karnati VVR (2003) Total synthesis of puerarin, an isoflavone C-glycoside. Tetrahedron Lett 44:6857–6859. doi: 10.1016/S0040-4039(03)01715-5

Li D, Park SH, Shim JH, Lee HS, Tang SY, Park CS, Park KH (2004) In vitro enzymatic modification of puerarin to puerarin glycosides by maltogenic amylase. Carbohydr Res 339:2789–2797. doi: 10.1016/j.carres.2004.09.017Li W, Yu S, Zhang T, Jiang B, Mu W (2015) Recent novel applications of levansucrases. Appl Microbiol Biotechnol 99:6959–6969. doi: 10.1007/s00253- 015-6797-5

-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp. Biochem Eng J 79:187–193. doi: 10.1016/j.bej.2013.08.001

Mena-Arizmendi A, Alderete J, Águila S, Marty A, Miranda-Molina A, López-Mungía A, Castillo E (2011) Enzymatic fructosylation of aromatic and aliphatic alcohols by Bacillus subtilis levansucrase: Reactivity of acceptors. J Mol Catal B Enzym 70:41–48. doi: 10.1016/j.molcatb.2011.02.002

Muruganandan S, Srinivasan K, Gupta S, Gupta PK, Lal J (2005) Effect of mangiferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats. J Ethnopharmacol 97:497–500. doi: 10.1016/j.jep.2004.12.010

71 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Perez Oseguera MA, Guereca L, Lopez-Munguia A (1996) Properties of levansucrase from Bacillus circulans. Appl Microbiol Biotechnol 45:465–471. doi: 10.1007/s002530050714

Plaza M, Pozzo T, Liu J, Ara KZG, Turner C, Karlsson EN (2014) Substituent effects on in vitro antioxidizing properties, stability, and solubility in flavonoids. J Agric Food Chem 62:3321– 3333. doi: 10.1021/jf405570u

Rodriguez M, Gomez A, Gonzalez F, Barzana E, López-mungía A (1996) Selectivity of methyl- fructoside synthesis with -fructofuranosidase. Appl Biochem Biotechnol 59:167–175. doi: 10.1007/BF02787818

Rodríguez M, Gómez A, Gónzalez F, Barzana E, López-mungía A (1997) Stability of invertase in alcoholysis reactions with methanol. J Mol Catal B Enzym 2:299–306. doi: 10.1016/S1381- 1177(96)00063-X

Rouillard LF, Thiais AJ, Robin JR (1998) Cosmetic or pharmaceutical composition containing, as active ingredient, mangiferin or its derivatives, in pure or in plant extracts. U.S. Patent 5,824:320.

Sanchez GM, Re L, Giuliani A, Núñez-Sellés AJ, Davison GP, León-Fernandez OS (2000) Protective effects of Mangifera indica L. extract, mangiferin and selected antioxidants against TPA- induced biomolecules oxidation and peritoneal macrophage activation in mice. Pharmacol Res 42:565–573. doi: 10.1006/phrs.2000.0727

Selisko B, Ulbrich R, Schellenberger A, Müller U (1990) Invertase-Catalyzed Reactions in Alcoholic Solutions. Biotechnol Bioeng 35:1006–1010. doi:10.1002/bit.260351008

Straathof AJJ, Vrijenhoef JP, Sprangers EPAT, van Bekkum H, Kieboom APG (1988) Enzymatic Formation of -D-Fructofuranosides from Sucrose: Activity and Selectivity of Invertase in Mixtures of Water and Alcohol. J Carbohydr Chem 7:223–238. doi: 10.1080/07328308808058916 van Der Heijden AM, van Hoek P, Kaliterna J, van Dijken JP, van Rantwijk F, Pronk J (1999) Use of the yeast Hansenula polymorpha (Pichia augusta) to remove contaminating sugars from ethyl - D-fructofuranoside produced during sucrose ethanolysis catalysed by invertase. J Biosci Bioeng 87:82–86. doi: 10.1016/S1389-1723(99)80012-7 van Rantwijk F, Woudenberg-van Oosterom M, Sheldon RA (1999) Glycosidase-catalysed synthesis of alkyl glycosides. J Mol Catal B: Enzym 6:511–532. doi: 10.1016/S1381-1177(99)00042-9

Wang S, Liu G, Zhang W, Cai N, Cheng C, Ji Y, Sun L, Zhan J, Yuan S (2014) Efficient glycosylation of puerarin by an organic solvent-tolerant strain of Lysinibacillus fusiformis. Enzyme Micro Technol 57:42-47 doi: 10.1016/j.enzmictec.2014.01.009

Wu X, Chu J, Wu B, Zhang S, He B (2013a) An efficient novel glycosylation of flavonoid by - fructosidase resistant to hydrophilic organic solvents. Bioresour Technol 129:659–62. doi: 10.1016/j.biortech.2012.12.04

72 Chapter II: Functionaliation of natural compounds by enzymatic fructosylation

Wu X, Chu J, Xu T, He B (2013b) Isolation, identification and pharmacokinetic analysis of fructosyl puerarins from enzymatic glycosylation. J Chromatogr B Anal Technol Biomed Life Sci 935:70–74. doi: 10.1016/j.jchromb.2013.07.012

Wu X, Chu J, Liang J, He B (2013c) Efficient enzymatic synthesis of mangiferin glycosides in hydrophilic organic solvents RSC Advances 3:19027–19032. doi: 10.1039/C3RA42648C

Xiao J, Muzashvili TS, Georgiev MI (2014) Advances in the biotechnological glycosylation of valuable flavonoids. Biotechnol Adv 32:1145–1156. doi: 10.1016/j.biotechadv.2014.04.006

Yao LH, Jiang YM, Shi J, Tomás-Barberán FA, Datta N, Singanusong R, Chen SS (2004) Flavonoids in food and their health benefits. Plant Foods Hum Nutr 59:113–122.

Yu C, Xu H, Huang G, Chen T, Liu G, Chai N, Ji Y, Wang S, Dai Y, Yuan S (2010) Permeabilization of Microbacterium oxylans shifts the conversion of puerarin from puerarin-7-O-glucoside to puerarin-7-O-fructoside. Appl Microbiol Biotechnol 86:863–870. doi: 10.1007/s00253-009- 2341-9.

CHAPTER III: ENZYMATIC FRUCTOSYLATION OF FLAVONOIDS USING β-FRUCTOSIDASES FROM NON-SACCHAROMYCES YEASTS ISOLATED FROM FERMENTING MUST OF MEZCAL

Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

In order to find new β-fructosidases for the enzymatic fructosylation of flavonoids, two enzymatic screenings were carried out with non-saccharomyces yeasts isolated from the fermenting must of mezcal. This experimental work was carried out in collaboration with the group of Catalysis and Enzyme Molecular Engineering (CIMEs) at the Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés (LISBP) at INSA Toulouse, France.

First, we performed a screening of fructosyltransferase activity in the extracellular enzymatic extract of yeasts. Then, the positive strains were tested for the enzymatic fructosylation of a set of different class of flavonoids. The enzymatic extract of the yeast Rhodotorula mucilaginosa MB4 was able to fructosylate an isoflavone such as puerarin, thus it was selected for a deeper biochemical characterization.

The gene sequence of this β-fructosidase was searched in silico from the available genome of R. mucilaginosa. Then an optimization of the DNA gene sequence was performed to clone this gene in Pichia pastoris X-33 as a host, the synthetic gene was cloned and expressed. The produced enzyme was tested to investigate its potential for the fructosylation of flavonoids.

75 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

A. Introduction

Flavonoids are secondary metabolites that are widespread in plants. Their main functions are pigmentation and protection from abiotic and biotic stress of plants. Over 9,000 different structures have been identified in plants and classified into six subclasses (flavonols, flavones, isoflavones, flavanones, anthocyanidins and flavanol) on the basis of the structural variations observed in the rings of their common di-phenylpropane core structure (C6C3C6). Nowadays, flavonoids have received attention since they show a broad range of biological properties including their antioxidant, antitumor and anti-inflammatory activity (Quideau et al. 2011; Pandey et al. 2014). Epidemiological and clinical studies have revealed that flavonoids play an important role in the prevention and management of chronic diseases such as cancers, diabetes and certain cardiovascular diseases (Xiao et al. 2014; Hertog, 1993). However, most of the flavonoids have a poor solubility in water limiting their bioavailability. To overcome these limitations, flavonoid glycosylation has emerged as an alternative in order to modify their hydrophilic-lipophilic balance and access to a wider structural diversity (Xiao et al. 2014)

In the last two decades, chemical and enzymatic glycosylation methods have been proposed. Hence, chemical methods require protection and deprotection steps of the reactive hydroxyl groups, these chemical methods involve the use of toxic heavy metals as catalysts, hazardous purification steps and generate toxic waste (Agard, 2008; Desmet et al. 2012). Moreover, enzymatic glycosylation presents the advantage of using mild conditions, being regio- and stereo-selective and generating novel and more soluble structures (Hofer, 2016). Glycosylation is usually catalyzed by Leloir and non-Leloir glycosyltransferases (GTs, E.C. 2.4). Thus, the use of Leloir glycosyltransferases has been rapidly expanding but they present the disadvantage to use NDP-sugar substrate. These NDP-sugar substrates are not easily available and mostly are expensive. Alternatively, enzymatic glycosylation of flavonoids using cheap sugar donors such as sucrose has been explored for the synthesis of new gluco- and fructo- conjugates (Xu et al. 2016). However, the enzymatic fructosylation of flavonoids using sucrose as donor and the synthesis of new fructo-conjugates have been little explored so far (Herrera-González et al. 2017).

According to CAZy database, the enzymes able to perform transfructosylation reactions from sucrose are β-fructosidases and fructosyltransferases from microbial or plant origins.

These enzymes belong to the glycoside hydrolase families GH32 or GH68 (Cantarel et al. 2009; Lammens et al. 2009). Among GH32 family we find β-fructosidase or β-fructofuranosidase (EC

76 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

3.2.1.26), these enzymes catalyze the release of β-fructose from non-reducing termini of various β-D- fructofuranoside substrates (Alvaro-Benito et al. 2007; Lafraya et al. 2011). The main reaction catalyzed by β-fructosidases is sucrose hydrolysis but these enzymes can also transfer fructosyl units from sucrose to a suitable acceptor (mono, disaccharide or molecules different from sugars) depending on the transfructosylation versus hydrolysis ratio (T/H ratio). Transfructosylation may also result in the formation of different types of β-fructosidic linkages depending on the enzyme linkage specificity (Sangeetha et al. 2005; Lammens et al. 2009).

Recently, it has been reported that some β-fructosidases have the capacity to fructosylate different types of organic acceptors different from sugars to generate new fructo-conjugates. Wu et al. (2013a) using a β-fructosidase from Arthrobacter nicotianae XM6 CCTCC M2010164 were capable of achieving fructosylation of puerarin in the presence of 25% (v/v) of DMSO, with yields higher than 90% after 72 h of reaction (Wu et al. 2013a). This microbial β-fructosidase was capable to produce two new fructo-conjugates of puerarin, which have a new potential for cosmetic and pharmaceutical applications. However, in the literature there is few reports using β-fructosidases for the fructosylation of flavonoids (Herrera-González et al. 2017).

Therefore, in order to find new β-fructosidases for the fructosylation of flavonoids, extracellular enzymatic extracts of non-saccharomyces yeasts isolated from fermenting must of Mezcal were tested. From this screening, the extract of Rhodotorula mucilaginosa was retained and a β-fructosidase gene from Rhodotorula mucilaginosa was cloned and expressed for a deeper biochemical characterization.

77 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

B. Results and discussion

B.1. Screening of transfructosylation activity by β-fructosidases from non-saccharomyces yeasts isolated from fermenting must of Mezcal

A screening of transfructosylation activity in the enzymatic extract of non-saccharomyces yeasts isolated from fermenting must of Mezcal was performed. All of them belong to the CIATEJ collection.

Fructosyltransferase activity was evaluated in 121 enzymatic extracts from non-saccharomyces yeasts. The results are presented in table 15, only 62 enzymatic extracts from different species showed fructosyltransferase activity. Kluyveromyces marxianus (33), Torulaspora delbrueckii (22), Crytococcus albidus (2), Rhodotorula mucilaginosa (2), Candida parapsilosis (1), Candia apicola (2) and Zygosaccharomyces bisporus (2). The strains with fructosidase and fructosyltransferase activity in the enzymatic extract were similar to the strains reported by Arrizon et al. 2012. Therefore it may be inferred that these strains are able to produce fructooligosaccharides (FOS) such as 1-kestose and nystose and they have the capacity to elongate the fructosyl residue through different β-fructosyl linkages (Arrizon et al. 2012).

Afterwards, the positive strains were tested for fructosyltransferase activity in presence of 20 % of DMSO, only 15 enzymatic strains from 62 retain their fructosyltransferase activity in presence of this solvent, which were Rhodotorula mucilaginosa (MB4), Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Candida apicola (CC, MT3), Kluyveromyces marxianus (DV4, 1424, DH4, 1616, DA5, 2108, 717), Zygosaccharomyces bisporus (DG, MS3) and Candida parapsilosis (MF4).

In conclusion, 15 strains were able to retain their fructosyltransferase activity in presence of 20 % of organic solvent (DMSO). These strains were selected for the next screening, which consisted of the enzymatic fructosylation of different flavonoids.

78 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Table 15. Screening of fructosyltransferase activity in the enzymatic extract from non-saccharomyces yeast in absence of DMSO and presence of 20 % (v/v) of DMSO.

Number of positive strains with Number fructosyltransferase activity Species of Presence of 20 % strains Absence of DMSO (v/v) of DMSO Crytpcoccus albidus 3 2 1 Candida apicola 8 2 2 Candida boidini 2 - - Candida parapsilosis 1 1 1 Candida zemplenina 1 - - Deckera anomla 4 - - Hanseniaspora osmopila 3 - - Issachenkia orientalis 2 - - Kluyveromyces marxianus 36 33 7 Pichia anomala 1 - - Rodhotorula mucilaginosa 6 2 1 Schizosachcaromyces pombe 9 - - Torulaspora delbruekii 22 22 1 Zygosaccharomyces bisporus 21 2 2 Total strains 121 62 15

B.2. Screening of transfructosylation activity on different class of flavonoids

The objective of this screening is to select the best yeast enzymatic extract for the fructosylation of flavonoids in presence of 20 % (v/v) DMSO.

Table 16 presents a summary of the results of the enzymatic fructosylation of flavonoids using this 15 selected enzymatic extracts, only puerarin was fructosylated from all the set of different classes of flavonoids tested. The enzymatic extracts with positive results were: Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Kluyveromyces marxianus (DV4, DH4, DA5, 1616, DV3, 717), Rhodotorula muciliaginosa (MB4) and Candida apicola (CC, MT3). It is worth emphasizing that the strains producing of 1-kestose and their isomers are also positive in the screening of enzymatic fructosylation of puerarin (Arrizon et al. 2012), thus enzymatic promiscuity for the formation of β- fructosyl linkages is important for the fructosylation of flavonoids. The enzymatic extracts from Zygosaccharomyces bisporus (DG, MS3) and Candida parapsilosis (MF4) were not able to fructosylate flavonoids.

79 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Table 16. Screening of fructosylation of different flavonoids.

Number of Species Quercetin Catechin Luteolin Puerarin Hesperidin Strains Torulaspora 1 x x x yes x delbrueckii Kluyveromyces 7 x x x yes x marxianus 2 Candida apicola x x x yes x Rhodotorula 1 x x x yes x mucilaginosa 1 Candida parapsilosis x x x x x Zygosaccharomyces 2 x x x x x bisporus 1 Crytococcus albidus x x x yes x

Figure 11 shows the chromatogram for the screening of enzymatic fructosylation of puerarin by the enzymatic extract from yeasts at 5 mM puerarin, 409 mM sucrose in 100 mM acetate buffer pH 5 and 20 % (v/v) of DMSO during 24 h at 45 ºC and 600 rpm. On the HPLC chromatogram, the new peak (P1) appears just before puerarin’s peak with a retention time between 5.5 and 5.6 min. This new peak is not observed in the blank, neither in the reaction carried out with the enzymatic extract from Candida parapsilosis (MF4) and Zygosaccharomyces bisporus (DG, MS3). Thus, this new peak could correspond to a fructosylated flavonoid. The enzymatic extracts of the yeast species that have shown this new peak were Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Rhodotorula muciliaginosa (MB4), Kluyveromyces marxianus (DV4, DH4, DA5, 1616, DV3, 717), and Candida apicola (MT3, CC). In addition, the electrospray ionization mass spectrometry (ESI-MS) analysis in negative mode of each peak revealed that the product P1 has a value of [M-H]- [m/z] = 577.0 that corresponds to puerarin monofructoside (Figure 11). Thus, these results confirmed the fructosylation of puerarin. Different of monofructoside can be formed. Indeed, fructosylation can take place either on the glucosyl unit of puerarin or on one of the hydroxyl groups of the C or B cycle.

According to the literature, it is the first time that the fructosylation of puerarin by a β- fructosidase from non-saccharomyces yeasts is described. Two reports describe the fructosylation of puerarin with bacteria. The first one was published by Wu et al. 2013. These authors found a β- fructosidase from the bacterium Arthrobacter nicotianai XM6 isolated from contaminated soil with a yield of 91 % fructosyl puerarin in optimal conditions (Wu et al. 2013). The second report is by Wang et al. 2014 and shows that the Lysinibacillus fusiformis maltogenic amylase was able to fructosylate and maltosylate puerarin with a yield of 70 % (Wang et al. 2014).

80 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

In conclusion, the enzymatic extracts from Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Rhodotorula mucilaginosa (MB4), Kluyveromyces marxianus (DV4, DH4, DA5, 1616, DV3, 717), and Candida apicola (MT3, CC) contain an enzyme able to fructosylate puerarin. Therefore, it is necessary to purify the product and perform a structural characterization by NMR in order to confirm the position of fructosylation onto the molecule of puerarin.

81 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

P1 Blank

Absorbance (mAU) Absorbance

Time (min) (min)

((min) Absorbance (mAU)

Time (min)

) m/z= 415.1

P Relative abundance (% abundance Relative

) P1 m/z= 577.0 Relative abundance (% abundance Relative

Figure 11. a) LC Chromatograms from screening of enzymatic fructosylation of puerarin from enzymatic extract of yeast non-saccharomyces at 1 U mL-1, 5 mM puerarin, 409 mM of sucrose (100 mM acetate buffer pH 5 and 20 % (v/v) of DMSO) during 24 h at 45 ºC and 600 rpm. MS3 (---), 717(---), MT3 (---), DG (---), CC (---), 2108(---), DA5 (---), 1616(---), Blank (---), DH4 (---), 1424 (---), MB4 (---), MF4(---),DV4 (---), DW1(---) and DC4 (---). b) ESI-MS spectra in negative mode of new peak P1. (P) Puerarin; (P1), mono-fructosyl puerarin.

82 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

B.3. Gene search in silico from yeast genomes for new β-fructosidases

In this section, a gene sequence search for new β-fructosidase in available genomes was carried out. In the previous section (section B.2), six yeast species were selected based on their capacity to fructosylate puerarin. Thus, in order to find the gene of a new β-fructosidase, the available data (2017) of yeast genomes were mined.

Table 17 shows the yeast species that were previously selected. Wherein, Zygosaccharomyces bisporus is discarded because its genome has not been reported at NCBI, Cryctoccous albidus genome possesses no assembly, so in silico research has been ruled out.

Moreover, Torulaspora delbruekii and Candida apicola were discarded from this study because they are the subject of other PhD thesis in our research group. In addition, Kluyveromyces marxianus was discarded because this enzyme has already been reported. So, we have decided to study Rhodotorula mucilaginosa in order to perform the in silico search of the new β-fructosidase.

Table 17. In silico search β-fructosidase sequence parameters for selection of yeasts.

Sequence reported in Genome Fructosylation of Microorganism reported at Reference puerarin CAZy KEGG NCBI Torulaspora Hypothetical Gomez-Angulo et Yes No Yes delbruckeii sequence al., 2015 Vega-Alvarado et Candida apicola Yes No No Yes al. 2015 Kluveromyces Inokuma et al. Yes Inulinase Yes Yes marxianus 2015 Rhodoturula Deligios et al. Yes No No Yes mucilaginosa 2015 Zygosaccharomyces Yes No No No --- bisporus Cryptococcus Vajpeyi and albidus Yes No No Yes Chadran, 2016 (Naganishia albida)

83 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Figure 12 illustrates the dendrogram of the yeast species with β-fructosidase reported sequences in order to have a reference sequence for the in silico search in the genome. In Figure 12, the closer phylogenetic yeast with reported β-fructosidase sequences were Cryptococcus neoformans var. grubii H99 and Cryptococcus gatty WM276. The accession numbers of β-fructosidase sequences for Cryptococcus neoformans var. grubii H99 and Cryptococcus gatty WM276 are AFR94181.1 and ADV25067.1, respectively. Further, the nucleotide and amino acid sequence of the β-fructosidase were downloaded from the NCBI database. Then, the in silico search was performed using CLC Genomics Workbench 9.5, where a BLAST in the genome of Rhodotorula musilaginosa (GenBank: JWTJ00000000.1) was performed using β-fructosidases sequences from Cryptococcus neoformans var. grubii H99 and Cryptococcus gatty WM27.

Figure 12. Dendrogram built with the yeasts species with β-fructosidase reported and with Rhodotorula mucilaginosa with not reported β-fructosidase sequence.

84 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

The results of the BLAST in the equivalent genome from R. mucilaginosa indicated that only the β- fructosidase gene from Cryptococcus neoformans var. grubii H99 (accession number AFR94181.1) has been aligned with the R. mucilaginosa genome. Thus, it may be suggested that a β-fructosidase gene is present in the genome from R. mucilaginosa. Figure 13 shows an aligned section between 400- 1000 bp in the counting JWTJ0100029443 from the genome of R. mucilaginosa.

Figure 13. β-fructosidase sequence BLAST in genome from Rhodotorula mucilaginosa using a β-fructosidase from Cryptococcus neoformas var. grubii H99 (accession number AFR94181.1).

Afterwards the gene prediction in Sofberry was performed (http://www.softberry.com), using the counting JWTJ0100029443 and the nucleotide sequence from Cryptococcus neoformas var. grubii H99 (accession number AFR94181.1). Thus, table 18 shows the predicted gene of Rhodotorula mucilaginosa Furthermore, with the catalytic motif according to the GH32 family reported by coloured in blue (Lammens et al. 2009).

85 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Table 18. Predicted β-fructosidase from Rhodotorula mucilaginosa a) nucleotide and b) amino acid sequence. The catalytic domains are highlighted in blue and the signal peptide in red. a) ATGTTTCGCTCTCTCGTCCCCATTACCATCGCGGGTCTGGCATATCTCCTTCAGAGTCCAGCTCAATC GACAACGTCAAGTGCTGCCTCTGTCCCCACGGGCGTTCCCATCGAAGGAGACTACTCTGGACCTTAC CGCCCCAGAATTCATTTCTCACCCCCGAAAGGCTTTATGAACGACCCGAACGGTTGCCACCGCGAC CGCAACGGCACGTATCACCTCTACTACCAGTACAACCCGCTCGAGTACGTCGCCGGGAACCAGCAC TGGGGACACGCCACTTCGGACGACCTGTACCACTGGACGAACCAACCCATCGCCATCTTCCCGCCC AACTCGACCTCGCAGGTCTTCTCCGGTTCGGCAGTGCTCGACCCTAACAACACGTCGGGCTTCTTCC CGAACACGACCGACGGCGTCGTCGCCGTCTACACCCTCAACACGCCGACTCTCCAAGTCCAGGAGG TCGCGTACTCGACCGACGGCGGCTACAATTTCACGCCGTACGAGAACAACCCCGTCCTCTCTGTCGG CAGCAACCAGTTCCGTGACCCCAAGGTCTTCTGGTACGAAGACCACTGGGTCATGGCTGTCGCCGC CGCTAACGACTTTACCATCGAAATCTACACTTCGCCGAACCTCACGTCGTGGACTTTCGCCTCCAAC TTTACGCACCACGGTTTGCTCGGACTCGCGTACGAATGTCCGAACTTGGTCCAGGTGCCGTTCCAGG ACGACCCGTCCAAGTCGGCCTGGCTCATGTACATTTCGATCAACCCCGGCGCGCCACTCGGCGGCA GTGTCGGCCAGTACTTCCCGGGCGACTTTAACGGCACCCACTTTGTCGCGTACGACTCGGCGGCGA GGATCGCGGATTTTGCGAAGGACAACTATGCTTCGCAATGGTTCGCCGACACGGAGAACGGCGAGT CGATCTCGATCGCTTGGGCTTCCAACTGGCAGTACACTCAGCAAGTTCCTACATCAGCCCAAGCTTT CAGATCTGCGATGTCGTTGCCGCGCCGGAACTACCTTACGAACATCACCCGGCTCGGCTGGGATCTC GTCTCGCTTCCGTACGACCTCTCGCCGGTCGTCGGCCCGTCGCTCCTGTCGTCGTCCGAGGCCAACT CGACCGCCGACGTCGACTTTACCAACGTGACTTCGAACGCGGTCTGGTTCAGTCTGAACGTGACCCT CCCCGACGCCGCAATCCAGAACGCTTCGCTCATTTCGGCCGACGCATCGATCAACATCACCTTCCTC CCTTCGACCAAGTGCTCCTCCTCTTCGGGATCGGGGTCCGACTCACCAGCGGCGACCCTGACCTACT TCTACGCGGGTCTGACGAACGGAGCCCTCGCGCTCACTCGACCGGCGGCTTCCTCCTCGTGGGGAG CCGAGAACCCCTTCTTCACCGACAAGTTTTCGTACACGCTCGTCGACCCGCTCACGTCCCTCGTCGG CGTGTTTGATCGGTCGATGCTCGAGGTGTTTGTCAACGAGGGAGCACACTCGGCTACCATGTTGGTG TTCCCCGACTCGCCGGTCGGGAGTATGAAGGTCGCGACTGGGGGTTTGCCTGAGGGCACGCAGGTC AACCTGCAGGTCAACGGCCTCGAGTCGACTTGGCAGTCCTCGTGA b) MFRSLVPITIAGLAYLLQSPAQSTTSSAASVPTGVPIEGDYSGPYRPRIHFSPPKGFMNDPNGCHRDRNGT YHLYYQYNPLEYVAGNQHWGHATSDDLYHWTNQPIAIFPPNSTSQVFSGSAVLDPNNTSGFFPNTTDG VVAVYTLNTPTLQVQEVAYSTDGGYNFTPYENNPVLSVGSNQFRDPKVFWYEDHWVMAVAAANDFTI EIYTSPNLTSWTFASNFTHHGLLGLAYECPNLVQVPFQDDPSKSAWLMYISINPGAPLGGSVGQYFPGDF NGTHFVAYDSAARIADFAKDNYASQWFADTENGESISIAWASNWQYTQQVPTSAQAFRSAMSLPRRNY LTNITRLGWDLVSLPYDLSPVVGPSLLSSSEANSTADVDFTNVTSNAVWFSLNVTLPDAAIQNASLISAD ASINITFLPSTKCSSSSGSGSDSPAATLTYFYAGLTNGALALTRPAASSSWGAENPFFTDKFSYTLVDPLTS LVGVFDRSMLEVFVNEGAHSATMLVFPDSPVGSMKVATGGLPEGTQVNLQVNGLESTWQSS

86 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Additionally, a protein BLAST was performed and the homology with enzymes belonging to GH32 family was investigated to verify that the enzyme belongs to this family (Figure 14). As can be seen in the protein BLAST predicted that the β-fructosidase belongs to GH 32 family. Moreover the identity percentage for predicted β-fructosidase from Rhodotorula mucilaginosa indicates 94 % 60 % and 58 % of identity to a hypothetical protein from Rhodotorula sp., Cryptococcus gatty and Cryotococos neoformas, respectively. The protein BLAST further suggests that the predicted sequence from Rhodotorula mucilaginosa could be a functional β-fructosidase. First, it is important to remember the main characteristics of the GH32 family. According to the CAZy classification, GH32 and GH68 families belong to GH-J clan (Cantarel et al. 2009). Thus, the GH-J clan shares a β-propeller catalytic domain with three conserved acidic amino acids referred to as the “catalytic triad.” In the GH32 enzymes, these three residues (including the nucleophilic Asp, the acid-base Glu, and an Asp residue acting as a transition state stabilizer) are located within three conserved sequences, referred to as the WMNDPNG (β-fructosidase motif), EC, and RDP motifs, respectively (Lammens et al. 2009; Lafraya et al. 2011).

Table 19 shows some characteristics of the predicted protein such as, size of sequence in amino acids, isoelectric point (pI) and molecular weight. It is worth to say that pI and molecular weight were calculated by Compute pI/Mw tool Expansy. Furthermore, the invertase from S. cerevisiae was used as a control enzyme. As can be seen the size, pI, molecular weight are similar, according to the literature the sequence amino acid is in average between 500 to 570, pI 4.5-4.9 and the molecular weight is between 58.5 to 59.3 kDa (Nadeem et al. 2015). In addition, the predicted sequence from Rhodotorula mucilaginosa has three conserved motifs (WMNDPNG, EC, and RDP), which are characteristics from GH32 family (Lammens et al. 2009).

Table 19. Predicted β-fructosidases theoretical properties and catalytic motifs. Size Molecular Fructosidase RDP EC Enzyme pI (aa) weight (Da) motif Motif Motif Predicted β-fructosidase from 547 4.56 59263.53 FMNDPNG RDP EC Rhodotorula mucilaginosa Invertase from S. cerevisiae 512 4.58 58544.72 WMNDPNG RDP EC (control)

87 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Figure 14. BLAST protein performed at NCBI of predicted β-fructosidase from Rhodotorula mucilaginosa.

88 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Afterward, a structure homology-model was performed by SWISS-MODEL Workspace in order to propose a model of the structure of the predicted β-fructosidase. Table 20 shows the results obtained for the predicted sequence, it can be noticed that the homology structure-model for Rhodotorula mucilaginosa was similar to the one of S. cerevisiae (PDB 4eqv.1.A). Wherein, it was possible to observe in the model the β-propeller and β-sandwich domains characteristic of GH32 family (Lammens et al. 2009).

Table 20. Predicted β-fructosidase homology structure-model by SWISS-MODEL Workspace.

Enzyme Template Model

PDB 4eqv.1.A (Invertase from S. Predicted fructosidase from cerevisiae) Rhodotorula mucilaginosa Sequence identity 42 %

Table 21 shows a boxshade analysis for the catalytic domains between some reported and the predicted β-fructosidase. As it can be seen for the predicted β-fructosidase from Rhodotorula mucilaginosa in comparison to reported enzymes, the catalytic motifs are located in almost the same position, these results suggest that this sequence could lead to a functional enzyme.

In conclusion, it was possible to obtain a β-fructosidase sequence for Rhodotorula mucilaginoasa from in silico search. The bioinformatics analyses suggest that the sequence of the β-fructosidase could be a functional enzyme because it contains the main features characterizing the GH32 family including the conserved motifs, catalytic triad, structure and molecular weight.

89 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Table 21. Boxshade analysis of predicted β-fructosidase from Rhodotorula mucilaginosa (in red is highlighted the catalytic triad).

90 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

B.4. Cloning and expression of the new β-fructosidase from Rhodotorula mucilaginosa for enzymatic fructosylation of flavonoids

The results obtained from the cloning and expression of the new β-fructosidase from Rhodotorula mucilaginosa are presented and discussed in this section. Hence, it was possible to carry out transformation of E. coli DH5α with the synthetic gene of the β-fructosidase from R. mucilaginosa. Figure 15 shows propagation of plasmid in E. coli DH5α low-salt LB plates with ampicillin (1μg/μL); where it can be observed that pUC57-RhInv plates contains E. coli DH5α transformant colonies and negative control did not have colonies as it was expected.

Afterwards, a restriction analyses was performed in order to verify the presence of the gene RhInv in the construction pUC57-RhInv. Thus, figure 16 shows the electrophoresis gel and the presence of two bands could be visualized of approximately 3000 and 1600 bp, respectively. The first band corresponds to the empty vector pUC57 (3000 bp) and the second band of 1600 bp corresponds to the sequence of the β-fructosidase (RhInv) from R. mucilaginosa. Then, the band corresponding to (RhInv) gene was extracted from the gel for posterior ligation and expression in a vector (pGAPZB). Figure 17 shows electrophoresis of the purification gel of RhInv. In lane 2 and 3, we can observe the bands corresponding to the β-fructosidase from R. mucilaginosa with a size of 1600 bp approximately.

a) b)

Figure 15. Propagation of plasmid in E. coli DH5α Low-salt LB plates with ampicillin (1μg/μL) a)pUC57-RhInv and d) negative control.

91 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

1 2 3

10,000 bp

3000 2000 1500 1000 700

500

250

Figure 16. Electrophoresis gel of analysis of restriction of pUC57-RhInv, Lane 1 ladder 10 kbp, lane 2, 3 digest of pUC57-RhInv (β-fructosidase from R. mucilaginosa) using EcoRI and SalI.

1 2 3

10,000 bp

3000 2000 1500 1000 700 500

250

Figure 17. Electrophoresis gel for the purification of β-fructosidase gene Lane 1: ladder 10 kpb, Lane 2 and 3: Gene of β-fructosidase from R. mucilaginosa.

92 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Next, RhInv was introduced into the pGAPZB vector using a T4 ligase (figure 18). Electrophoresis gel shows that at initial time of this reaction, two bands were observed (Lanes 2 and 3 in Figure 18), which corresponded with empty pGAPZB vector (3,100 bp) and RhInv gene (1,600 bp). After 12 h of reaction with T4 ligase at 16 ºC, electrophoresis gel showed the disappearance of the band corresponding to RhInv and only one band can be observed per lane with an increase of the weight around 4,500 bp, showing that it was possible to link RhInv into the expression vector (pGAPZB- RhInv). The construct pGAPZB-RhInv was then propagated into competent cells of E. coli DH5α by electroporation. Figure 19 presents low-salt LB plates with zeocin (25 μg/μL) with positive colonies in pGAPZB-RhInv and pGAPZB, also in the negative control was not observed any colonies.

1 2 3 4 5

10,000 bp 4000 2000 1500

1000 700

500

250

Figure 18. Electrophoresis gel for RhInv ligation reaction into expression vector (pGAPZB). Lane 1: ladder 10 kbp, lane 2, 3, ligation at initial time of pGAPZB and RhInv, lane 4, 5 at final time of pGAPZB-RhInv.

a) b) c)

Figure 19. Propagation of the construct into E. coli DH5α in low-salt LB plates with zeocin (25 μg/μL) a) pGAPZB-RhInv, b) pGAPZB and c) negative control.

93 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

The construct pGAPZB-RhInv and the empty vector pGAPZB were linearized and they were introduced into competent Pichia pastoris X-33 cells by electroporation. Figure 20 shows zeocin resistant Pichia transformants colonies containing pGAPZB-RhInv, the empty vector pGAPZB and the negative control. Then, some colonies were subjected to the verification of the insert using PCR method, thus it is expected the weight of PCR product for the empty vector of 275 bp and for the construct (pGAPZB-Rh) a value of 1,928 bp (see Figure 21). As can be observed in line 3, a PCR product with an approximate size of 250 bp, which corresponds to the size of the amplification between the AOXI region and pGABZB of the empty vector; in line 2 a band with a size close to 2000 bp is observed (Figure 21), which suggests that the insert is found in the construction pGAPZB-Rh. Therefore, it can be suggested that zeocin resistant colonies have the pGAPZB-RhInv construct integrated into the genome.

a) b) c)

Figure 20. YPDS plates with 100 μg/mL of zeocin resistant Pichia pastoris X-33 transformants containing a) pGAPZB-RhInv, b) empty pGAPZB and c) negative control.

1 2 33 4

10,000 pb 8000 5000 3000 2000 1500 1000 500

Figure 21. Electrophoresis gel for PCR products. Lane 1: ladder 10 kbp, lane 2: pGAPZB-RhInv and RhInv, lane 3: pGAPZB and lane 4: negative control.

94 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Subsequently, the zeocin resistant colonies that resulted from the integration of plasmid pGAZB-RhInv and pGABZB were evaluated by phenotypic screening (Figure 22) using a colorimetric method. A change in the color of the medium from initial green (pH 6.5) to yellow (pH ≤ 6.0) indicates an acidification of the medium due to the formation of organic acids during the catabolism of glucose and fructose released in the sucrose utilization. A blue color (pH ≥ 7.5) suggest an increase of the pH as a consequence of the ammonia released by the catabolic oxidation of the nitrogen containing carbon sources yeast extract and peptone (Menéndez et al. 2013). Thus, as expected cells electroporated with the empty vector (pGAZB) and the negative control (Picha pastoris X-33) showed the non- saccharolytic phenotype revealed by blue color in the growth medium. Yellow color in the growth medium indicates colonies expressing active RhInv (Figure 22). Therefore, the colonies that contained active RhInv were selected in order to produce the enzyme by liquid fermentation.

a) b) c) d)

Figure 22. Phenotypic screening of zeocin resistant colonies transformed with a) negative control (P. pastoris X- 33), b) pGAPZB and c) pGAPZB-RhInv and d) pGAPZB-RhInv (lane 2 in blue is empty).

Table 22 shows the results obtained from the fructosidase activity in the supernatant from P. pastoris X-33, Pichia transformed with pGAPZB and pGAPZB-Rh. As expected only the supernatant from Pichia transformed with the construction pGAPZB-RhInv showed fructosidase activity with a value 28.0 ± 2.3 U mL-1. No fructosidase activities were detected from the supernatant from P. pastoris X-33 and Pichia transformed with the empty vector pGAPZB. As expected these results are in accordance to the previous results observed in the phenotypic screening.

95 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Table 22. Fructosidase activity in the supernatant from P. pastoris X-33, zeocin resistant Pichia transformed with pGAPZB and pGAPZB-Rh.

Fructosidase activity Strain -1) (U mL P. pastoris X-33 ND pGAPZB ND pGAPZB-RhInv 28.0 ± 2.3 ND: Non detected

In summary, it may be inferred from the results obtained from the cloning and expression of the β- fructosidase from Rhodotorula mucilaginosa (RhInv) that it was possible to express an active protein with fructosidase activity using P. pastoris as expression host.

96 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

B.5. Enzymatic fructosylation of flavonoids by a recombinant β-fructosidase from Rhodotorula mucilaginosa (RhInv)

The following section describes the results obtained from the enzymatic fructosylation of flavonoids using the recombinant β-fructosidase of Rhodotorula mucilaginosa in order to know its capacity to fructosylate different acceptors, such as puerarin, coniferyl alcohol and mangiferin.

Figure 23 shows the chromatograms obtained from the enzymatic fructosylation of puerarin, where the presence of a new peak (P1) can be observed with a retention time of 6.7 min, which does not appear at the beginning of the reaction. The mass spectrum of this peak reveals that the value of this peak is [M- H]- [m/z] = 577.2, which corresponds to the mass of fructosyl puerarin. Moreover, when coniferyl alcohol was tested as acceptor, the chromatogram revealed after 10 h of reaction a new peak (C1) just before the peak corresponding to coniferyl alcohol (C), which has a retention time of 8.3 min. The molar mass determined by mass spectrometry corresponds to a value of [M-H]- [m/z] = 341, which suggest the addition of a fructose moiety onto coniferyl alcohol (Figure 24). In addition, the enzymatic fructosylation of mangiferin was carried out. The results are shown in figure 25, and a small peak (M1) appears at the end of the reaction with a molar mass value of [M-H]- [m/z] = 583.2 and a retention time of 5.9 min. These results suggest the fructosylation of mangiferin. Therefore, it may be inferred that RhInv is able to fructosylate the tested acceptors. However, in the case of these three acceptors, only the synthesis of new mono fructosides was observed as result of the synthesis.

The substrate conversion for puerarin, coniferyl alcohol and mangiferin are summarized in table 23. As observed, the highest substrate conversion percentage was obtained with coniferyl alcohol (20 % ± 3), while mangiferin had the lowest value around 1.5 % ± 0.7 and puerarin obtained only 10 % ± 1.9. In the literature, Dudíková et al. (2007) reported that coniferyl alcohol could be fructosylated. They reported the fructosylation of coniferyl alcohol using Cryptococcus laurentii cell walls containing β- fructosidases; the reaction was carried out in the presence of 30% of acetone (v/v) used as co-solvent and 200 mM coniferyl alcohol, thus around 2% of conversion was achieved (Dudíková et al. 2007). However, the advantage of our method is the absence of a co-solvent. Moreover, the quantity of the mono fructosyl coniferyl alcohol was 2.5 mM using RhInv and 4 mM using Cryptococcus laurentii cell walls. In both cases the conversion is low; the ability to fructosylate this acceptor are similar using these β-fructosidases.

97 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

The synthesis of puerarin fructosides using β-fructosidases has been reported in the literature. Wu et al. (2013a) reported that mono- and di-fructosides were synthetized using a β-fructosidase from Arthrobacter nicotianae at 265.5 mM of puerarin after a reaction time of 72 h in the presence of 25% of DMSO as co-solvent with a substrate conversion of 91.0%. Indeed, this percentage of conversion is higher than the one obtained using RhInv (10 %). It suggests that β-fructosidase from Rhodotorula mucilaginosa (RhInv) is less efficient to carry out this reaction. In addition, the mono fructosyl puerarin was the only product; no traces of the di fructosides were detected as in the case of the work of Wu et al. (2013a).

In the case of mangiferin, percentage of conversion was much lower (1.5 %) than the value reported by Wu et al. (2013b). According to the literature, they achieved a percentage of conversion of 67.5% from 112 mM of acceptor using the β-fructosidase from Arthrobacter arilaitensis in DMSO (20% v/v), indicating that the β-fructosidase from Arthrobacter arilaitensis recognizes mangiferin more efficiently than β-fructosidase from Rhodotorula mucilaginosa (RhInv).

Table 23. Percentage of substrate conversion of different acceptors using RhInv at 1 U mL-1, 14 mM of acceptor, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm.

Compound Structure % Conversion OH

OH HO O OH Puerarin HO O 10 ± 1.9*

O OH O OH Coniferyl alcohol 20 ± 3* HO

HO O OH

O Mangiferin HO OH 1.5 ± 0.7* OH O HO OH OH

*The conversion rate was calculated from the amount of acceptor remaining. % conversion = 100*([acceptor] initial – [acceptor] final)/ [acceptor] initial.

98 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

(mAu) Absorbance Absorbance t= 10 h t=0 h

Time (min) b)

P m/z=415.2

)

Relative abundance (% abundance Relative

m/z=577.0

)

Relative abundance (% abundance Relative

Figure 23. a) LC Chromatograms from screening of enzymatic fructosylation of puerarin using RhInv at 1 U mL- 1, 14 mM puerarin, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI- MS spectra in negative mode of new peak. (P) Puerarin; P1, mono-fructosyl puerarin.

99 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Absorbance (mAu) Absorbance t=0 h

t=10 h

Time (min) b)

m/z=179.2

) Relative abundance (% abundance Relative

m/z=342.1

)

Relative abundance (% abundance Relative

Figure 24. a) LC Chromatograms from screening of enzymatic fructosylation of coniferyl alcohol using RhInv at 1 U mL-1, 14 mM coniferyl alcohol, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI-MS spectra in negative mode of new peak. (C) Coniferyl alcohol; (C1), mono-fructosyl coniferyl alcohol.

100 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

M M1

Absorbance (mAu) Absorbance

t=10 h t=0 h

Time (min) b)

M m/z=421.3

)

Relative abundance (% abundance Relative

)

m/z= 583.2

Relative abundance (% abundance Relative

Figure 25. a) LC Chromatograms from screening of enzymatic fructosylation of mangiferin using RhInv at 1 U mL-1, 14 mM mangiferin, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI-MS spectra in negative mode of new peak. (M) Mangiferin; (M1), mono-fructosyl mangiferin.

101 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

In summary, it may be inferred from the results of the reactions of the enzymatic fructosylation of flavonoids using the β-fructosidase from Rhodotorula mucilaginosa (RhInv) that it is possible to fructosylate acceptors other than sucrose but with low yields around 20%. The best yield was obtained in the case of coniferyl alcohol. Finally, it is necessary to biochemically characterize the enzyme and then perform the optimization of the enzymatic fructosylation reactions of flavonoids to further improve the conversion yields.

102 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

C. Experimental methods

C.1. Yeast propagation

Non-saccharomyces yeasts isolated from fermenting must of mezcal belong to the collection at CIATEJ were propagated in YPD medium (1% yeast extract, 2% peptone and 1 % dextrose (w/v) pH 4.5) over the night at 30 ºC and 250 rpm.

C.2. Production of β-fructosidases

Te induction medium for the production of β-fructosidases was prepared with 8 g L-1 of urea, 3 g L-1 of -1 K2HPO4 and 2 g L of MgSO4 the pH was adjusted at 4.5 and the media was sterilized at 121 ºC during 20 min. Moreover, the only source of carbohydrates was 20 g L-1 of Agave tequilana fructans (ATF) in pH 4.5; it was sterilized separately (105 ºC and 5 min). Then, 16-selected yeasts were inoculated in 100 mL of the induction medium with 2x106 cells mL-1 and they were added to the mineral medium sterilized before. The incubation time was 24 h at 30 ºC and 250 rpm. Afterward, the culture was centrifuged at 6000 rpm during 20 min at 4 ºC in order to obtain the supernatant (enzymatic extract).

C.3. Activity assay

C.3.1. Fructosidase activity

Fructosidase activity was evaluated by mixing 50 μL of sucrose 1% (w/v) in 100 mM acetate buffer pH 5 with 50 μL of each enzymatic extract during 15 min at 50 ºC, then in order to stop the reaction 100 μL of DNS was added and the mixture was heated at 95 ºC for 5 min, then it was placed in ice. The absorbance of the samples was measured at 540 nm. One unit (U) of enzymatic activity was defined as the amount of enzyme liberating 1 μmol of reducing sugars (Miller, 1959; Arrizon et al. 2012).

103 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

C.3.2. Fructosyltransferase activity

Fructosyltransferase activity was measured in the enzymatic extracts for selected yeast strains in order to confirm the capacity to perform the synthesis of fructooligosaccharides, which can be performed when the equilibrium of the reaction is displaced to a high concentration of sucrose and a low concentration of water. The reaction was carried out in 1 mL that contains 900 μL of 600 g L-1 of sucrose in acetate buffer pH 5 and 100μL of each enzymatic extract at 45 ºC, 100 rpm during 120 min. Then, the microtubes were heating at 95 ºC during 5 min, in order to stop the reactions. Then the samples were diluted 1:100 and analyzed by HPLC. It was used a DIONEX Ultimate 3000 equipped with a Shodex refractometer IR. The column was a Biorad Carbohydrate Analysis column (Animex HPX-87K, 300 x 7.8 mm, USA), the mobile phase was ultrapure water and the flow rate was 0.65 mL min-1; the temperature of the oven was 65 ºC. Glucose, fructose and sucrose were used as standards. Data acquisition and processing were performed with Chromeleon 6.8 version. For each reaction, the moles of sucrose, glucose and fructose were calculated. The fructosyltransferase activity was calculated considering the difference between moles of glucose and fructose (equivalent to the moles of fructose transferred, moles of sucrose transfructosylated). One unit of enzyme activity was defined as the amount of enzyme transferring 1 μmol of sucrose per minute (Arrizon et al. 2012).

C.4. Screening of enzymatic fructosylation of different flavonoids by enzymatic extracts from non-Saccharomyces yeasts

The screening of enzymatic fructosylation of different flavonoids was carried out with 5 different types of flavonoids such as quercertin, (+)-Catechin, Luteolin, Puerarin and Hesperidin. The final reaction volume of the mixture was 1 mL with a flavonoid concentration of 5 mM in 20 % of DMSO and 408 mM of sucrose (100 mM acetate buffer pH 5) at 45 ºC, during 24 h and 600 rpm. For all the enzymatic extract, the fructosidase enzymatic activity was 0.5 U.mL-1. Then, in order to stop the reaction, the microtubes were boiled at 95 ºC during 5 min.

C.5. Quantification of percentage of conversion of flavonoids by Liquid Chromatography-Mass Spectrometry (LC-MS)

Flavonoids and fructosylated products were analyzed by LC-MS using a Dionex Ultimate 3000 series chromatograph equipped with Dionex 340 UV/VIS detector coupled with a simple quadruple mass spectrometer (MSQ Plus, Thermo Scientific). The column was a Phenomex PFP C18 (Luna 5u, 100 A, 250 x 4.6 mm, USA) at 40 ºC. The mobile phase was composed of solvent A (Ultrapure water with

104 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal formic acid 0.05%) and solvent B (Acetonitrile with formic acid 0.05 %), the gradient started from 10 % B to 50 % over 20 min and from 50 % B to 95% B during 5 min, the flow rate was 1 mL min-1. The wavelengths in the UV detector were 254 and 350 nm. The mass spectrometer was in negative and positive mode with a voltage cone of 50 and 110 V and the range of mass was between 100 and 1000. Temperature of the electrospray was 450 ºC and the gas carrier was nitrogen.

C.6. Search of a new β-fructosidase gene sequence in silico from available genomes

In this section, the methodology for the search of a new of β-fructosidase gene sequence is described,it was carried out in silico from available genomes (2017). Figure 26 shows the general diagram of this procedure. The considerations for the search of a new sequence in silico were the following:

 Biochemical evidence of enzymatic fructosylation of puerarin in the enzymatic extracts  No reported fructosidase sequence at CAZy or KEGG database  Genome sequenced available in a database

Then, it was performed a dendrogram in PhiloT: a tree generator (http://phylot.biobyte.de) with the selected species yeast with fructosidase reported sequences in order to have a reference sequence for the in silico search in the genome. Thus, it was download the genome and the sequences of reported fructosidases from NCBI (https://www.ncbi.nlm.nih.gov). Afterwards, it was performed using BLAST tool in CLC Genomics Workbench 9.5 a BLAST in genome; it was carried out with β-fructosidase sequence close to the yeast according to the phylogenetic tree.

Thus, the alignment of the gene in the genome in a in a scaffold or counting were analyzed by Softberry (http://www.softberry.com) in order to gene prediction using the tool of FGENESH+ (Solovyev, 2007). Finally, in order to confirm if the predicted gene correspond to a β-fructosidase, it was carried out a bioinformatics analysis of the sequence.

105 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Yeast Download sequences of Dendrogram of yeast species Selection fructosidases reported from with fructosidases reported NCBI

Blast gene in genome in CLC Download genome from Genomics Workbench 9.5 NCBI

Gene Prediction by Softberry

Molecular model, pI, Search in predicted gen molecular mass GH32 Predicted gene Blast by SWISS-MPDEL Motif, catiltyc site and Boxshade NCBI, Pfam, Interpro signal peptide Analysis

Figure 26. General schema of in silico search of β-fructosidase gene in yeast genome.

106 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

C.7. Cloning and expression of β-fructosidase from Rhodotorula mucilaginosa in Pichia pastoris X-33

Cloning and expression of β-fructosidase from R. mucilaginosa was carried out using all the protocols reported in pGAPzα A, B, & C and pGAPZ A, B, & C Pichia expression vectors for constitutive expression and purification of recombinant proteins manual from Invitrogen.

β-Fructosidase gene from R. mucilaginosa (RhInv) was optimized in its codon usage for P. pastoris and synthetized into a puc57 plasmid (GenScript, USA) with flanking EcoRI and SalI. Then, E. coli DH5α was used as a cloning host for plasmid propagation and Pichia pastoris X-33 (wild-type) was used as the expression host. PUC57-RhInv plasmid was introduced into competent E. coli strain DH5α (Invitrogen) by electroporation (2100 V, 100 Ω, 25 μF). Then, E. coli DH5α transformants were grown at 37 ºC in low-salt LB (10 g L-1 peptone, 5 g L-1 yeast extract and 5 g L-1 NaCl) plates with ampicillin (1μg/μL) during an overnight. Ampicillin resistant transformants were inoculated in 2 mL of low-salt LB medium with ampicillin (1μg/μL) at 37ºC and 250 rpm for 14 h. Later, the DNA plasmid was isolated by miniprep (QUIAGEN plasmid miniprep Kit) for restriction analysis using EcoRI and SalI (Thermofisher). Afterwards, the purification of the gene was carried out by gel purification (QUIAGEN Gel purification kit).

Ligation of β-fructosidase gene to expression vector pGAPZB (Invitrogen) was carried out using T4 ligase (Sigma-Aldrich) at 16 ºC during 12 h of reaction. The E. coli DH5α was transformed with ligation reaction and inoculated in low-salt LB plates with Zeocin (25 μg/μL). Zeocin resistant transformant colonies were chosen for isolation of plasmid DNA by miniprep (QUIAGEN plasmid miniprep Kit) and for subsequent restriction analysis. Then, it was prepared 5-10 μg of plasmid DNA and BspHI was used to linearize the pGAPZB, pGAPZB-RhInv plasmids were introduced into competent P. pastoris X-33 cells by electroporation (1500 V, 200 Ω, 25 μF). Then, transformants cells were inoculated in YPDS plates (10 gL-1 yeast extract, 20 g L-1 peptone, 20 g L-1 dextrose and 1 M sorbitol) with Zeocin (100 μg/mL) and incubated at 30 ºC during 48 h. Zeocin resistant Pichia transformant colonies were subjected to the verification of the insertion using the PCR method. The primers were pGAP forward (5’-GTCCCTATTTCAATCAATTGAACAAC-3’) and 3'AOX1 (5’- GCAAATGGCATTCTGACATCC- 3’) as recommended in the invitrogen manual.

107 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Then, it was carried out a phenotypic screening was carried out in Zeocin resistant Pichia transformants expressing active RhInv. It was used a solid SPY medium (5 % (w/v) sucrose, 2 % (w/v) peptone, 1 % (w/v) yeast extract, and 0.025 % (w/v) bromothymol blue, 1.5 % (w/v) agar, pH 6.5) reported by Menéndez et al. (2013).

Thus, positive Zeocin resistant Pichia transformants expressing active RhInv were inoculated in the expression media (10 g L-1 yeast extract, 20 g L-1 peptone, 0.5 g L-1 glycerol and 50 g L-1 sucrose) at 30 º C, 200 rpm for 72 h (Hernández et al. 2018). Then, the culture was centrifuged at 6000 rpm and 4 ºC during 20 min. Finally, the fructosidase activity was measured (see section C.3.1) in the supernatant. The supernatant containing invertase activity was directly used in order to evaluate its capacity to fructosylate flavonoids.

C.8. Enzymatic fructosylation of flavonoids by a recombinant β-fructosidase (RhInv) from Rhodotorula mucilaginosa

The fructosylation of different flavonoids is carried out using a recombinant β-fructosidase (RhInv) from Rhodotorula mucilaginosa in order to know the capacity of this enzyme to fructosylate different acceptors. The reactions were performed using 877 mM of sucrose (300 g L-1), 14 mM of acceptor (puerarin, phlorizin, mangiferin and coniferyl alcohol) in 50 mM 100 mM acetate buffer pH 5.5, in presence of 1 U mL-1 of RhInv during 10 h at 40 ºC. The reactions mixtures were stopped by boiling at 95 ºC during 10 min. Then the samples were diluted with DMSO and analyzed by HPLC-MS (see section C.5). Percentage of conversion is calculated, taking into account the difference between initial and final concentration of acceptor.

% Conversion = ([Acceptortinitial]– [Acceptortfinal]/ [Acceptortinitial]) x100

108 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

D. Conclusions

In summary, this chapter achieved the second and third objective of this thesis. Wherein, it was possible to explore the potential of β-fructosidases from non-saccharomyces yeast isolated from the fermenting must of mezcal for the enzymatic fructosylation of flavonoids.

From 121 yeast strains, 62 positive enzymatic extracts were positive to fructosyltransferase activity. Only 15 enzymatic extracts from 62 retain their fructosyltransferase activity in presence of 20 % (v/v) DMSO. The species with fructosyltransferase activity in presence of DMSO were Rhodotorula mucilaginosa (MB4), Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Candida apicola (CC, MT3), Kluyveromyces marxianus (DV4, 1424, DH4, 1616, DA5, 2108, 717), Zygosaccharomyces bisporus (DG, MS3) and Candida parapsilosis (MF4).

The best enzymatic extract was from Rhodotorula mucilaginosa MB4, which is able to fructosylate an isoflavone such as puerarin and had a potential new β-fructosidase.

Then, it was possible to find a sequence of an active and not reported β-fructosidase from Rhodotorula mucilaginosa (RhInv) using an in silico gene search. The bioinformatics analysis confirmed that this sequence was a β-fructosidase or inveratase, which contained the three-conserved motif, size and structure related to GH32 family. Next, this sequence was cloned and expressed in Pichia pastoris X- 33 in order to explore its capacity for the enzymatic fructosylation of flavonoids.

Finally, the recombinant β-fructosidase from Rhodotorula mucilaginosa (RhInv) was tested for its capacity to fructosylate puerarin, coniferyl alcohol and mangiferin. The results showed that it is possible to fructosylate acceptors other than sucrose, but with low yields around 20%. The best yield is obtained in the case of coniferyl alcohol. Finally, it is necessary to biochemically characterize the enzyme and then perform the optimization of the enzymatic fructosylation reactions of flavonoids and thus improve the conversion yields.

109 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

E. References

Alvaro-Benito M, de Abreu M, Fernández-Arrojo L, Plou F, Jiménez-Barbero J, Ballesteros A (2007) Characterization of a beta-fructofuranosidase from Schwanniomyces occidentalis with transfructosylating activity yielding the prebiotic 6-kestose. J Biotechnol 132:75–81. doi: 10.1016/j.jbiotec.2007.07.939

Agard NJ (2008) Chemical approaches to glycobiology. ACS Symp Ser 990:251–271. doi: 10.1021/bk-2008-0990.ch012

Arrizon J, Morel S, Gschaedler A, Monsan P (2012) Fructanase and fructosyltransferase activity of non-Saccharomyces yeasts isolated from fermenting musts of Mezcal. Bioresour Technol 110:560–5. doi: 10.1016/j.biortech.2012.01.112

Cantarel BI, Coutinho PM, Rancurel C, Bernard T, Lombard V, Henrissat B (2009) The Carbohydrate- Active EnZymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Res 37:233–238. doi: 10.1093/nar/gkn663

Deligios M, Fraumene C, Abbondio M, Mannazzu I, Tanca, Addis MF, Uzzau S (2015) Draft genome sequence of Rhodotorula mucilaginosa, an emergent opportunistic pathogen. Genome Announc 3(2):e00201-15 doi: 10.1128/genomeA.00201-15

Desmet T, Soetaert W, Bojarová P, Křen V, Dijkhuizen L, Eastwick-Field V, Schiller A (2012) Enzymatic glycosylation of small molecules: Challenging substrates require tailored catalysts. Chem - A Eur J 18:10786–10801. doi: 10.1002/chem.201103069

Dudíková J, Mastihubová M, Mastihuba V, Kolarova N (2007) Exploration of transfructosylation activity in cell walls from Cryptococcus laurentii for production of functionalised β-d- fructofuranosides. J Mol Catal B Enzym 45:27–33. doi: 10.1016/j.molcatb.2006.11.003

Gomez-Angulo J, Vega-Alvarado L, Escalante-García Z, Grande R, Gschaedles-Mathis A, Amaya- Delgado L, Arrizon J, Sanchez-Flores A (2015) Genome Sequence of Torulaspora delbrueckii NRRL Y-50541, Isolated from Mezcal Fermentation. Genome Announc 3(4):e00438–15. doi: 10.1128/genomeA.00438-15

Hernández L, Menéndez C, Pérez ER, Martínez D, Dubiel A, Trujillo LE,, Ramírez R, Sbrino A, Mazola Y, Musacchio A, Pimentel E (2018) Fructooligosaccharides production by Schedonorus arundinaceus sucrose:sucrose 1-fructosyltransferase constitutively expressed to high levels in Pichia pastoris. J Biotechnol 266:59–71. doi: 10.1016/j.jbiotec.2017.12.008

Herrera-González A, Núñez-lópez G, Morel S, Amaya-delgado L, Sandoval G, Gschaedler A, Remaud-Simeion M, Arrizon J (2017) Functionalization of natural compounds by enzymatic fructosylation. Appl Microbiol Biotechnol 101:5223–5234. doi: 10.1007/s00253-017-8359-5

Hofer B (2016) Recent developments in the enzymatic O-glycosylation of flavonoids. Appl Biochem Biotechnol 100:4269–4281. doi: 10.1007/s00253-016-7465-0

110 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Inokuma K, Ishii J, Hara KY, Mochizuki M, Hasunuma T, Kondo A (2015) Complete genome sequence of Kluyveromyces marxianus NBRC1777, a nonconventional thermotolerant yeast. Genome Announc 3(2):e00389-15 doi: 10.1128/genomeA.00389-15

Menéndez C, Martínez D, Trujillo LE, Mazola Y, González E, Pérez ER, Hernández L (2013) Constitutive high-level expression of a codon-optimized β-fructosidase gene from the hyperthermophile Thermotoga maritima in Pichia pastoris. Appl Microbiol Biotechnol 97:1201– 1212. doi: 10.1007/s00253-012-4270-2

Miller GL (1959) Use of DinitrosaIicyIic Acid Reagent for Determination of Reducing Sugar. Anal Chem 31:426–428.

Nadeem H, Rashid MH, Siddique MH, Azeem F, Muzammil S, Javed MR, Ali A, Rasul I, Riaz M (2015) Microbial invertases: A review on kinetics, thermodynamics, physiochemical properties. Process Biochem 50:1202–1210. doi: 10.1016/j.procbio.2015.04.015

Lafraya Á, Sanz-Aparicio J, Polaina J, Marín-Navarro J (2011) Fructo-oligosaccharide synthesis by mutant versions of Saccharomyces cerevisiae invertase. Appl Environ Microbiol 77:6148–6157. doi: 10.1128/AEM.05032-11

Lammens W, Le Roy K, Schroeven L, Van Laere A, Rabijins A, Van den Ende W (2009) Structural insights into glycoside hydrolase family 32 and 68 enzymes: Functional implications. J Exp Bot 60:727–740. doi: 10.1093/jxb/ern333

Sangeetha PT, Ramesh MN, Prapulla SG (2004) Production of fructosyl transferase by Aspergillus oryzae CFR 202 in solid-state fermentation using agricultural by-products. Appl Microbiol Biotechnol 65:530–537. doi: 10.1007/s00253-004-1618-2

Solovyev VV (2007) Statistical approaches in Eukaryotic gene prediction. In Handbook of Statistical genetics Eds. Balding D., Cannings C., Bishop M.), Wiley-Interscience; 3d edition, 1616 p.

Vega-Alvarado L, Gómez-Angulo J, Escalante-García Z, Grande R, Gschaedler-Mathis A, Amaya- Delgado L, Sanchez-Flores A, Arrizon J (2015) High-Quality Draft Genome Sequence of Candida apicola NRRL Y-50540. Genome Announc 3(3):e00437–15. doi: 10.1128/genomeA.00437-15

Wang S, Liu G, Zhang W, Cai N, Cheng C, Ji Y, Yuan S (2014) Efficient glycosylation of puerarin by an organic solvent-tolerant strain of Lysinibacillus fusiformis. Enzyme Microb. Technol 86, 863-870 doi: 10.1016/j.enzmictec.2014.01.009

Wu X, Chu J, Wu B, Zhang S, He B (2013a) An efficient novel glycosylation of flavonoid by β- fructosidase resistant to hydrophilic organic solvents. Bioresour Technol 129:659–62. doi: 10.1016/j.biortech.2012.12.041

111 Chapter III: Enzymatic fructosylation of flavonoids using β-fructosidases from non-saccharomyces yeasts isolated from fermeting must Mezcal

Wu X, Chu J, Liang J, He B (2013b) Efficient enzymatic synthesis of mangiferin glycosides in hydrophilic organic solvents. RSC Adv 3:19027–19032. doi: 10.1039/c3ra42648c

Xiao J, Muzashvili TS, Georgiev MI (2014) Advances in the biotechnological glycosylation of valuable flavonoids. Biotechnol Adv 32:1145–1156. doi: 10.1016/j.biotechadv.2014.04.006

Xu L, Qi T, Xu L, Lu L, Xiao M (2016) Recent progress in the enzymatic glycosylation of phenolic compounds. J Carbohydr Chem 35:1–23. doi: 10.1080/07328303.2015.1137580

112

CHAPTER IV: ENZYMATIC FRUCTOSYLATION OF PHLORIZIN BY LEVANSUCRASE FROM Gluconacetobacter diazotrophicus

Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

In this chapter the enzymatic fructosylation of phlorizin by β-fructosidases from different sources and specificities such as GH32 and GH68 families were studied in order to accomplish the third aim of this thesis. This work was carried out in collaboration with the group of Catalysis and Enzyme Molecular Engineering (CIMEs) at LISPB (Laboratoire d’Ingénierie des Systèmes Biologiques et des Procédés) at INSA Toulouse, France.

The first part of this chapter describes the results obtained for the screening of the enzymatic fructosylation of phlorizin using three different enzymes. The enzymes employed were a levansucrase from Gluconacetobacter diazotrophicus (LsdA, 2.4.1.10), a sucrose:sucrose 1-fructosyltransferase from Schedonorus arundimaceus (Sa1-SSTrec, EC 2.4.1.99) and a β-fructofuranosidase from Rhodotorula mucilaginosa (RhInv, EC 3.2.1.26). Then, the best enzyme able to fructosylate the acceptor was selected in order to optimize the reaction conditions. The second part of the chapter presents the purification and the structure characterization of the new fructoside. Finally, the solubility and the antioxidant activity of the new fructo-conjugates were evaluated and compared to the properties of the aglycone. This chapter will be publish in an appropriate scientific journal.

114 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Enzymatic synthesis of novel phlorizin fructosides

Azucena Herrera-Gonzáleza,b, Gema Núñez-Lópeza,b Lorena Amaya-Delgadoa, Anne Gschaedlera, Georgina Sandovala, Magali Remaud-Simeonb, Sandrine Morel, Javier Arrizona, Lázaro Hernándezc* aCentro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C.- Unidad Zapopan. Camino Arenero 1227, El Bajio del Arenal, 4519, Zapopan, Jal. México b LISBP, Université de Toulouse, CNRS, INRA, INSA, Toulouse, France LISBP-INSA Toulouse 135 Avenue de Rangueil, 31077 Toulouse, France cCentro de Ingeniería Genetica y Biotecnología, Ave. 31 e/ 158 y 190 Cubanacán, Municipio Playa, 6162, La Habana, Cuba.

A. Summary

Phlorizin is a low soluble natural compound with several biological properties such as antioxidant capacity, antidiabetic and antipyretic properties. In order to increase its solubility, three different fructosyltransferases: the levansucrase from Gluconacetobacter diazotrophicus (LsdA), the sucrose:sucrose 1-fructosyltransferase from Schedonorus arundimaceus (Sa1-SSTrec) and the β- fructofuranosidase from Rhodotorula mucilaginosa (RhInv) were assayed for the fructosylation of phlorizin (25 mM) using sucrose (146 mM) as the fructosyl donor and the different enzymes at 1 U mL-1. LsdA was the only enzyme capable to fructosylate phlorizin reaching 40 % conversion of the acceptor substrate under this reaction condition. Under optimized concentrations of LsdA (5 U mL-1), sucrose (1.5 M) and phlorizin (25 mM) the conversion yield was increased up to 79.1 %. Mono-, di-, and trifructosides were synthesized in the ratio 11:3:1. The most abundant product was purified by chromatography and structurally characterized using 1D and 2D NMR analysis. The purified product with the molecular ion observed at m/z 597.2 [M-H]- was identified as mono-fructosyl-β-(26)- phlorizin. The mono-fructosylated product was 15.9 fold (30.5 g L-1 at 25°C) more soluble in water than the original substrate phlorizin (1.93 g L-1 at 25°C) and exhibited only a 1.4-fold reduction in the antioxidant capacity.

115 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

B. Introduction

Phlorizin (phloretin-2’-β-D-glucopyranoside) is a dihydrochalcone and an intermediate of the flavonoid biosynthetic pathway in plants. This compound is naturally found in the bark of the apple trees, peels of apples and strawberries (Ehrenkranz et al. 2005; Hilt et al. 2003). Phlorizin has interesting health-promoting properties, such as anti-diabetic, anticancer, antimicrobial and antioxidant (Rezk et al. 2002; Ehrenkranz et al. 2005; Xiao et al. 2017). However, phlorizin is poorly soluble in water (1 mg.mL-1 at 22 ºC), causing a decrease in the bioavailability with a loss of beneficial effects because of low assimilation (Pandey et al. 2013; Xu et al. 2016; Hofer, 2016). To overcome this limitation, the glycosylation the molecule could be an attractive solution

Currently, glycosylation of polyphenolic compounds can be performed by chemical or enzymatic synthesis. Chemical glycosylation of polyphenolic compounds requires several elaborated reactions of protection and deprotection of hydroxyl groups. This process is hazardous and generates toxic wastes (Desmet et al. 2012). The synthesis of glycosylated compounds using enzymes with high regio- and stereo-selectivity has emerged as a safer alternative (Xu et al. 2016; Desmet et al. 2012, Xu et al. 2016, Malbert et al. 2014, Thuan and Sohng 2013).Glycosyltransferases of Leloir and non-Leloir types have been successfully used for the glycosylation of dihydrochalcones. In the literature, two reports described the glycosylation of phloretin (Phlorizin aglycone). Pandey et al. (2013) reported the glycosylation of phloretin using an UDP-glycosyltransferase from Bacillus licheniformis, five different phloretin glucosides were identified phloretin 4’-O-glucoside, phloretin-2’-O-glucoside, phloretin 4’,4- O-diglucoside, phloretin 4,6’-O-diglucoside and phloretin 2’,4’,4-O-triglucoside. The in vitro enzymatic conversion of phloretin reached 95 % in the presence of 2 mM of phloretin and 16 mM of UDP-glucose after 12 h of incubation at 20 ºC (Pandey et al. 2013). In addition, Overwin et al. (2015) achieved the the glucosylation of phloretin by biotransformation using recombinant Escherichia coli cells containing amylosucrase gene from Neisseria polysaccharea ATCC 43768. The reaction enabled the synthesis of phloretin-4´-O-glucoside, α-D-diglucosyl-(14)-phloretin and α-D-triglucosyl-(14)- phloretin, with a coversion of 35 %, 32 % and 28 % respectively from 10 mM of phloretin in the culture medium. For biotechnological applications non-Leloir glycosyltransferases are of particular interest, as they do not require nucleotide-activated sugar donors, but use ubiquitous sucrose as an extremely cheap substrate. Moreover, to our knowledge, the enzymatic fructosylation of dihydrochalcones such as phlorizin has never been explored to date (Herrera-González et al. 2017).

116 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Enzymatic fructosylation can be performed by plant and microbial enzymes from GH 32 and GH 68 families. These enzymes are able to transfer fructosyl units from sucrose onto different acceptors, what results in the formation of different glycosides such as fructooligosaccharides and new-to-nature fructo-conjugates via the functionalization of alcoholic or phenolic compounds with a fructosyl unit (Lammens et al. 2009; Cantarel et al. 2009; Herrera-González et al. 2017).

In this study three different fructosyltransferases were tested for the fructosylation of phlorizin: the levansucrase from Gluconobacter diazathrophicus (LsdA), the recombinant fructosyltransferase from the plant Schedonorus arundimaceus (1SST, Sa1-SSTrec) and a yeast invertase from Rhodotorula mucilaginosa (RhInv), belonging to GH 68 and GH32 families. The levansucrase belongs to the GH 68 family, the main reaction of this enzyme is the synthesis of levan, which is a fructose polymer with β- (2→6) linkages (Lammens et al. 2009; Öner et al. 2016). Levansucrase catalyzes the transfer of the fructosyl moiety from sucrose onto different acceptors such as water (sucrose hydrolysis), glucose (exchange) and sucrose (initiator of polymerization) (Martínez-Fleites et al. 2005; Li et al. 2015; Raga- Carbajal et al. 2018). The enzyme is also able to transfer a fructosyl residue onto hydroxylated acceptors, such as aliphatic and aromatic alcohols or phenolic compounds (Herrera-González et al. 2017, Mena-Arizmendi et al. 2011). The plant enzyme sucrose:sucrose 1-fructosyltransferase (1-SST) belongs to the GH 32 family and catalyzes the transfer of the fructosyl residue of sucrose to the sucrose itself (used as acceptor) to obtain 1-kestose (GF2) β-D-fructofuranosyl-(2→1)-β-D-fructofuranosyl- (2→1)--D-glucopyranoside (Lammens et al. 2009; Hernández et al. 2018). Finally, the invertase from Rhodotorula mucilaginosa or β-fructofuranosidase (EC 3.2.1.26) belongs to the GH 32 family mainly catalyzed the sucrose hydrolysis of sucrose to D-glucose and D-fructose (Lincoln and More, 2017). The enzyme also catalyses transfructosylation when when high sucrose concentration are used (Plou et al. 2007). In the presence of 500 g L-1 of sucrose, a mixture of short fructooligosaccharides with β-(2→6) and β-(2→1) linkages was produced (Gutierrez-Alonso et al. 2009).

In the presence of phlorizin acceptor, the levansucrase from Gluconobacter diazathrophicus (LsdA) was the sole enzyme able to synthetize a new fructo-conjugate. The acceptor, sucrose and enzyme concentration were varied to improve the synthesis of this new fructoside. The fructo-conjugate was purified by preparative chromatography and its solubility and antioxidant activity were assessed.

117 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

C. Results and discussion

C.1. Enzyme selection for phlorizin fructoside production

Enzymatic fructosylation of phlorizin was attempted using three different enzymes a sucrose:sucrose 1- fructosyltransfersase (Sa1-SSTrec) from S. arundinacea, a levansucrase (LsdA) from Gluconacetobacter diazotrophicus SR4 and an invertase (RhInv) from Rhodotorula mucilaginosa at 25 mM phlorizin, 0.146 M sucrose in 50 mM phosphate buffer (pH 5.8) during 24 h, at 42 ºC and 1 U mL- 1 of enzyme. The reaction products were analyzed by LC/MS and the corresponding chromatograms are shown in Figure 27. With Sa1-SST, we did not observe any decrease phlorizin concentration after 24 h of reaction indicating that Sa1-SST does not fructosylate phlorizin. On the contrary, LsdA and RhInv produced new products. LC/MS chromatograms revealed the presence of products eluting before the phlorizin (S), which were not observed at the initial reaction time. In the case of LsdA, four new peaks named P1, P2, P3 and P4 were recorded at 12.4, 11.5, 11.1 min and 10.9 min retention times, respectively. The electrospray ionization mass spectrometry (ESI-MS) analysis in negative mode revealed that the products, P1 and P3, display exactly the same mass and as phlorizin monofructoside, [M-H]- [m/z] = 597.2, while P2 and P4 show a [M-H]- [m/z] of 759.4 and 921.2, which correspond to a di and a tri phlorizin fructoside, respectively (Figure 27). In the case of RhInv, only one fructoside (P1) is identified at a retention time of 12.4 min with a molar mass [M-H]- at [m/z] = 597.2, corresponding to phlorizin monofructoside. Table 24 shows the phlorizin conversion and the proportion of the fructosylated compounds. The highest phlorizin conversion (39 % ± 1.5) is reached with LsdA while RhInv converts only 6 % ± 2.3 of the flavonoid. In addition, LsdA 75.2 %, 17.9 % and 6.8 % of mono, di and tri fructoside, respectively, the mono fructosyl phlorizin being the principal product synthesized., LsdA presents the highest fructosylation efficiency and was thus was selected for reaction optimization.

118 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

S a)

P1 P1 P2 P4 P3

Absorbance (mAU) Absorbance P4 P3 P2

Time (min)

P1, P3 m/z= 597.2 b)

Relat ive P2 m/z= 759.2 abun danc e

(%) Relative abundance (%) abundance Relative

P4 m/z=921.2

Figure 27. a) LC Chromatograms from screening of enzymatic fructosylation of phlorizin at 25 mM, 0.146 M sucrose (50 mM phosphate buffer pH 5.8), 1 UmL-1, at 42 ºC during 24 h. Initial time t = 0 h (---), LsdA (---), 1- SST (---) and RhInv (---). b) ESI-MS spectra in negative mode of new peaks. (S) Phlorizin; P1, mono-fructoside; P2, di-fructoside; P3 mono-fructoside P4, tri-fructoside.

119 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Table 24. Conversion and percentage of fructosylated compounds for phlorizin in the enzyme selection.

Fructoside abundance (%)b Enzyme Substrate conversion (%)a Mono Di Tri LsdA 39 ± 1.5 75.2 17.9 6.8 Sa1-SST 0 - - - 6 ± 2.3 100 - - RhInv aThe % of conversion was calculated from the remaining acceptor. Phlorizin conversion=([phlorizin] initial – [phlorizin] final)/ [phlorizin] initial *100 b The % of fructosylated phlorizin was calculated using the formula (Pn peak area)/Σ(P1, P2…Pn peaks areas)*100, where 1≤n≤3

120 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

C.2. Effect of sucrose, phlorizin and enzyme concentration on phlorizin conversion

The effect of sucrose concentration on phlorizin conversion at constant phlorizin concentration is shown in figure 28. Phlorizin conversion increased with sucrose concentration. The best conversion reached 63.6 ± 3.6 % for 1 M sucrose and 25 mM of phlorizin after 24 h of reaction. In contrast, at 0.146M sucrose concentration, the conversion was only of 30 %. The high sucrose concentrations may prevent enzyme inhibition and could also improve the flavonoid solubilization, thus favoring its fructosylation to the detriment of natural acceptor glucosylation (ie glucose, sucrose, fructans and fructooligosaccharides). We further assessed the effect of phlorizin concentration (from 25 to 100 mM) onto phlorizin conversion at a fixed sucrose concentration of 1M (figure 29). The highest conversion (64 %) was observed at the lowest phlorizin concentration (25 mM), followed by 50 mM of this acceptor, and a strong decrease was observed at concentrations higher than 75 mM (figure 29). It may be suggested that phlorizin inhibits the enzyme.

Figure 28. Effect of sucrose concentration on phlorizin conversion. Reactions conditions: 1 U mL-1 of Levansucrase (LsdA), 25 mM phlorizin in 50 mM phosphate buffer pH 5.8, 42 ºC. Sucrose concentration () 0.146 M, (●) 0.5 M, () 1 M and (▼) 1.5 M.

121 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Figure 29. Effect of phlorizin concentration on phlorizin conversion. Reactions conditions: 1 U mL-1 of Levansucrase (LsdA), 1.5 M sucrose and 50 mM phosphate buffer pH 5.8, 42 ºC. Phlorizin concentration () 25 mM, (●) 50 mM, (▼) 75 mM and () 100 mM.

The effect of enzyme concentration on phlorizin conversion was studied in reactions containing 25 mM phlorizin, 1.5 M sucrose at 42 ºC (Figure 30). The acceptor conversion was monitored over time. As shown in figure 30, the acceptor conversion increased with increasing LsdA concentration. The highest value was obtained at 5 U mL-1 with 79 % ± 0.9 conversion after 8 h of reaction. Nevertheless, when the enzyme concentration was increased to 10 and 15 U mL-1 a maximum conversion of 80 % was achieved at 8 h and then decrease indicating a possible hydrolysis of the fructosides. According to these results, the best conditions for phlorizin fructosylation correspond to the use of 5 U mL-1 at 42 ºC with 25 mM phlorizin and 1.5 M sucrose.

122 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Figure 30. Effect of enzyme concentration on phlorizin conversion. Reactions conditions: 25 mM phlorizin, 1.5 M sucrose in 50 mM phosphate buffer pH 5.8, 42 ºC. () 1 U mL-1, (☐) 2.5 U mL-1, (●) 5 U mL-1, (▼) 10 U mL-1 and () 15 U mL-1.

123 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

C.3. Structural characterization of phlorizin mono-fructoside

Phlorizin fructosylation was performed in a larger volume and the mono-fructoside was subsequently purified for NMR characterization. The chemical structure of the compound, which corresponds to peak P1 was identified by mass spectrometry, 1H-NMR, 13C-NMR and 2D NMR. As previously seen, P1 shows a molar mass of 597.2 g mol-1 (m/z = 597.2 [M-H]-). Table 25 shows the chemical shifts from 13C and 1H NMR data of phlorizin and mono frucotosyl-phlorizin, wherein 13C spectrum from mono frucotosyl-phlorizin (P1) revealed twenty-five signals (two overlapped), ten signals were in the range 60-90 ppm and were assigned to the two monosaccharides (fructose and glucose). Compared with the 13C NMR spectrum of phlorizin, six additional signals are identified in the spectrum of fructosyl phlorizin. Notably, the signal of the carbon atom at 105.72 ppm is characteristic of the C2’’’ of the fructosyl moiety.

HMBC spectrum of P1 was then used to deduce the position of fructosylation (see Fig S4). The HMBC spectrum showed long-range correlations between C2’’’ of fructose (C 105.72 ppm) and H6’’ (H 3.81-4.07 ppm) of the glucosyl unit, demonstrating that the fructoside produced by LsdA corresponds to the β-D-mono-fructofuranosyl-(2’’’-6”)-phlorizin (Figure 31).

Figure 31. Enzymatic fructosylation of phlorizin with levansucrase from Glucconacetobacter diazotrophicus.

124 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Table 25. Chemical shifts from 13C and 1H data of phlorizin and -D-fructofuranosyl-(26)-phlorizin (Data were recorded in MeOD). Chemical shifts are given in ppm relative to the residual signal of the solvent at 3.34 ppm in 1H and 49.5 ppm in 13C. Coupling constants in Hz. Carbon atom Phlorizin β-D-fructofuranosyl-(2-->6)-phlorizin position C H (J, Hz) C H (J, Hz) 1 134.36 - 134.37 - 2,6 130.86 7.09 (d, 8,7 Hz, 2H) 130.89 7.10 (d, 8,7 Hz, 2H) 3,5 116.56 6.71 (d, 8,5 Hz, 2H) 116.59 6.71-6.73 (d, 8,9 Hz, 2H) 4 156.81 - 156.82 - 7 31.29 2.91 (t, 7,5 Hz, 2H) 31.26 2.91 (t, 7,8 Hz, 2H) 8 47.43 3.42 (m) 47.38 3.48 (m) 9 207.07 - 207.06 - 1’ 107.27 - 107.28 - 2’ 162.76 - 162.69 - 3’ 95.92 6.21 (d, 2,3 Hz, 1H) 96.08 6.25 (d, 2,3 Hz, 1H)

4’ 166.36 - 166.38 -

5’ 98.83 5.99 (d, 2,3 Hz, 1 H) 98.99 6.00 (d, 2,35 Hz, 1 H) 6’ 168.00 - 167.99 - 1’’ 102.52 5.07 (d, 7.2, 1H) 102.67 5.04 (d, 7,3 Hz, 1 H) 2’’ 75.17 3.50 75.22 3.51 3’’ 78.94 3.42-3.52 78.81 3.50 4’’ 71.55 3.42 71.59 3.49 5’’ 78.86 3.42-3.52 (m) 77.50 3.60 3.75 (dd, 5,4 Hz, 12,4 Hz, 1H) 3.81 (dd, 5,3 Hz, 11,6 Hz, 1H) 6’’ 62.89 62.70 3.94(dd, 2,2 Hz, 12,4 Hz, 1H) 4.07 (dd, 2,3 Hz, 11,6 Hz, 1 H) 3.58 (d, 12 Hz, 1 H) 1’’’ 62.70 3.67 (d, 12 Hz, 1H) 2’’’ 105.72 - 3’’’ 79.40 4.14 (d, 8 Hz, 1H) 4’’’ 76.96 4.07 (d, 7.3 Hz, 1H) 5’’’ 83.99 3.75 m 3.64 (d, 7 Hz, 1H) 6’’’ 64.41 3.71 (d, 12.3 Hz, 1H)

125 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

C.4. Water solubility and antioxidant activity of mono fructosyl phlorizin

The solubility of phlorizin and mono fructosyl phlorizin was determined (table 26). The results show that phlorizin monofructoside was 16 folds more soluble than phlorizin in water at room temperature (25° C). This is explained by a high number of hydroxyl groups due to the additional fructosyl group, resulting in a higher number of hydrogen bonds with water (Herrera-González et al. 2017). The effect of antioxidants on 2,2-diphenyl- 1-picrylhydrazyl (DPPH) radical scavenging is shown in table 27. Phlorizin showed a higher antioxidant activity (62%) in comparison with the β-D-fructofuranosyl-(26)-phlorizin (44%), indicating that the fructosyl unit linked the C6 of the glucosyl residue masks the radical scavenging capacity of the phenolic rings (Torres et al. 2011).

Table 26. Solubility of phlorizin and fructosyl phlorizin in water at 25 ºC

Compound Solubility (g L-1) Phlorizin 1.93 ± 0.03 Mono fructosyl phlorizin 30.57 ± 0.1

Table 27. Free radical scavenging of phlorizin and phlorizin mono-fructoside Compound Free radical scavenging activity (%)a

Phlorizin 62.13 ± 1.8 Phlorizin mono-fructoside 44.56 ± 8.4 Trolox (control) 87.04 ± 0.6 aFree radical scavenging activity of DPPH was considered as 100%

126 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

D. Experimental methods

D.1. Chemical materials

Phlorizin was supplied by Carbosynth Limited (Compton, UK). Sucrose, glucose, fructose, dimethyl sulfoxide (DMSO), formic acid, acetonitrile (ACN) and absolute ethanol were supplied by Sigma Aldrich Inc. (MO, USA). All substances were high purity grade (≥ 98%). Acetonitrile and ethanol were HPLC grade. C18-reversed phase silica gel was purchased by Sigma Aldrich Inc. (MO, USA) and

TLC Silica gel 60 RP-18 F254s was obtained from Merck KGaA, (Darmstadt, Germany).

D.2. Enzyme production

Levansucrase (E.C. 2.4.1.10) from Gluconacetobacter diazotrophicus (LsdA) was recovered from the culture supernatant of the native strain SRT4 as previously reported by Arrieta et al. (1996). Sucrose:sucrose 1-fructosyltransferase (E.C. 2.4.1.99) from Schedonorus arundimaceus (Sa1-SSTrec) was expressed in Pichia pastoris X-33 and purified from the culture supernatant by ion exchange chromatography as previously described by Hernandez et al. (2018). Invertase or β-D- fructofuranosidase (E.C. 3.2.1.26) from Rhodotorula mucilaginosa MB4 (RhInv) was cloned in pGAPZB vector Invitrogen and constitutively expressed in Pichia pastoris X-33. Fermentation was carried out using the following culture media 1 % (w/v) yeast extract, 2 % (w/v) peptone, 2 % (w/v) glycerol and 5 % (w/v) sucrose at 30 ºC and 200 rpm during 72 h (Hernandez et al. 2018). The culture was centrifuged (6000 rpm, 10 min) and the supernatant containing invertase activity was directly used in the reaction.

D.3. Enzyme activity assays

The β-fructosidase or fructosyltransferase activity of the studied enzymes was determined from initial rate of reducing sugar release (fructose and glucose) from a 1 % (w/v) sucrose solution in phosphate buffer 50 mM pH 5.8 for LsdA or 100 mM acetate buffer pH 5.5 for Sa1-SSTrec and RhInv, at 42 ºC. Reducing sugars were quantified by the dinitrosalicylic acid (DNS) method of Miller (1959). One unit (U) was defined as the amount of enzyme that caused the release of 1 μmol of glucose per min (μmol min-1) at 42 °C in 50 mM phosphate buffer (pH 5.8).

127 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

D.4. Enzyme selection for phlorizin fructoside production

The fructosylation of phlorizin was carried out using three different enzymes: a sucrose:sucrose 1- fructosyltransferase (Sa1-SSTrec) from Schedonorus arundimaceus, a levansucrase (LsdA) from Gluconacetobacter diazotrophicus and an invertase (RhInv) from Rhodotorula mucilaginosa. The reactions were performed using 0.146 M of sucrose, 25 mM of phlorizin in 50 mM phosphate buffer pH 5.8 for LsdA or 100 mM acetate buffer pH 5.5 for Sa1-SSTrec and RhInv in presence of 1 U mL-1 of enzyme during 24 h at 42 ºC. The reactions mixtures were stopped by boiling at 95 ºC during 10 min. Then the samples were diluted with DMSO and analyzed by HPLC-MS. Percentage of conversion of phlorizin was calculated, taking into account the difference between initial and final concentration of phlorizin.

% Conversion = ([phlorizintinitial]– [phlorizintfinal]/ phlorizintinitial) x100

D.5. Effect of sucrose, phlorizin and enzyme concentration on the percentage of conversion of phlorizin with LsdA

Fructosylation of phlorizin was optimized by varying sucrose concentration from 0.1 to 1.5 M (fructosyl donor) and phlorizin concentration from 25 to 100 mM (acceptor) in 50 mM of phosphate buffer pH 5.8 in a total reaction volume of 2 mL, in presence of 1 U mL-1 of LsdA at 800 rpm and 42 ºC. To follow the synthesis, 50 μL aliquots were taken at predefined time intervals and boiled at 95 ºC for 10 min in order to stop the reaction, then the samples were diluted with DMSO, filtered by 0.45μm nylon syringe filter and used for analysis by HPLC-MS. All the experiments were performed in triplicate. The effect of enzyme activity on the percentage of conversion was tested. Enzyme activity was varied from 1 to 15 U mL-1, using 25 mM of phlorizin and 1.5 M of sucrose in 50 mM of phosphate buffer pH 5.8 in a total reaction volume of 2 mL at 800 rpm and 42 ºC. Synthesis was followed as described before.

D.6. HPLC-MS analysis of fructosylation reaction mixture

HPLC-MS analysis were performed using an Ultimate 3000 series chromatograph equipped with a Dionex 340 UV/VIS detector and coupled with a simple quadruple mass spectrometer (MSQ Plus,

USA) column at 40 ºC, using a gradient of solvent A (water-formic acid 0.05% (v/v)) and solvent B

128 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

(acetonitrile-formic acid 0.05% (v/v)) the gradient started from 10 % B to 50 % B over 20 min and from 50 % B to 95% B during 5 min, the flow rate was 1 mL min-1. Phlorizin and phlorizin fructosides were quantified by UV detection at 254 nm. The mass spectrometer was in negative and positive mode with a voltage cone of 50, 80 and 110 V and the temperature of the electrospray ionization (ESI) ion source was 450ºC and the gas carrier was nitrogen. The mass spectrometer scanned from m/z 100 to 1,500. The data acquisition and processing were performed with Chromeleon™ 7.2 data systems.

D.7. Large-scale production of fructosyl phlorizin

Preparative synthesis of phlorizin-fructosides was carried out using 0.146 M (4g) of sucrose, 25 mM (1 g) of phlorizin in 80 mL of 50 mM phosphate buffer pH 5.8 at 42 ºC with 1 U mL-1 LsdA during 24 h with agitation (300 rpm). The course of reaction was monitored by HPLC-MS. When phlorizin conversion reached 40 %, reaction was stopped at 95°C for 10 min. Then, the reaction mixture was subjected to glass column chromatography (30*2 cm) packed with 100 g Purosorb PAD 910 Purolite®. The column was eluted with pure water to remove sugars. Phlorizin and phlorizin fructosides were eluted with a solution of ethanol/water (50:50, v/v). The fractions comprising phlorizin and phlorizin fructosides were concentrated on a rotary evaporator and lyophilized. Then, the sample was applied on a C18 silica gel column (30*2 cm) using as a mobile phase a mixture of water/acetonitrile (65:35). Purification process was monitored by thin layer chromatography (TLC). TLC was performed using

TLC Silica gel 60 RP-18 F254s from Merck KGaA, (Darmstadt, Germany) and water/acetonitrile (65:35) used as mobile phase. Detection was achieved by UV absorption (254 nm). Fractions containing products were pooled and concentrated in vacuum, and then the product was lyophilized (15 mg of mono-fructosyl phlorzin).

D.8. Structural analysis of phlorizin mono-fructoside

NMR was acquired on an Advance 500 MHz spectrometer (Bruker) operating at 500 MHz for 1H and 125 MHz for 13C. The data were processed using Topspin 3 Software. All measurements were performed at 298 K and chemical shifts were given in ppm relative to the residual signal of methanol.

129 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

D.9. Solubility determination of mono-fructosyl phlorizin

Phlorizin and mono-fructosyl phlorizin were solubilized until saturation in 300 μl of ultrapure water. Then, the solutions were incubated at 25° C during 12 h at 500 rpm followed by centrifugation at 13000 g during 10 min. The supernatant was diluted in DMSO and analyzed by HPLC to determine the solubility.

D.10. Antioxidant activity of mono-fructosyl phlorizin

The antioxidant activities of phlorizin and mono fructosyl phlorizin were estimated by DPPH assay as described by Lee et al. 2005. 150 µl of DPPH at 0.15 mM were mixed with 150 µl of phlorizin or phlorizin mono-fructoside at 6.69 mM, all of them in methanol (80 %). Then the reactions were incubated at 37° C for 30 min in the dark and the absorbance was measured at 517 nm. The assay was carried out in triplicate. The percentage of DPPH inhibition was calculated using the following equation.

% DPPH inhibition = ((1-(Asample / Acontrol)) x 100

Asample is the absorbance of phlorizin or phlorizin monofructoside, and Acontrol is the absorbance of DPPH solution. Trolox TM (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used as a positive control for the determination of the antioxidant activity.

130 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

D. Conclusions

Three enzymes with fructosyltransferase activity were tested in order to synthetize new fructosides of phlorizin. The best results were obtained with levansucrase from Gluconacetobacter diazotrophicus (LsdA), which is able to synthetize mono fructosyl phlorizin as a new substance from sucrose by enzymatic fructosylation in 79.1 % of conversion. The concentration of acceptor, donor and enzyme must be considered to improve the enzymatic fructosylation of phlorizin by LsdA, in particular sucrose concentration. This process has high biotechnological potential for the production of new compounds with nutraceutical and pharmaceutical applications.

131 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

E. Supplementary data

Figure S1. 1H-NMR spectrum of β-D-fructofuranosyl-(26)-phlorizin.

Figure S2. 13C spectrum of β-D-fructofuranosyl-(26)-phlorizin.

132 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Figure S3. HSQC spectrum of β-D-fructofuranosyl-(26)-phlorizin.

Figure S4. HMBC spectrum of β-D-fructofuranosyl-(26)-phlorizin.

C-2’’’-H-6’’ C-2’’’-H-6’’

133 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

F. References

Arrieta J, Hernández L, Coego A, Suárez V, Balmori E, Menéndez C, Petit-Glatron MF, Chambert R, Seleman-Housein, G (1996) Molecular characterization of the levansucrase gene from the endophytic sugarcane bacterium Acetobacter diazotrophicus SRT4. Microbiology 142:1077– 1085. doi: 10.1099/13500872-142-5-1077

Cantarel BI, Coutinho PM, Rancurel C, Lombard TBV, Henrissat B, (2009) The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics. Nucleic Acids Res 37:233– 238. doi:10.1093/nar/gkn663

Desmet T, Soetaert W, Bojarová P, Kařen V, Dijkhuizen L, Eastwick-Field V, Schiller A (2012) Enzymatic glycosylation of small molecules: Challenging substrates require tailored catalysts. Chem. - A Eur. J. 18:10786–10801. doi: 0.1002/chem.201103069

Ehrenkranz JRL, Lewis NG, Kahn CR, Roth J (2005) Phlorizin: A review. Diabetes Metab Res Rev 21:31–38. doi: 10.1002/dmrr.532

Gutiérrez-Alonso P, Fernández-Arrojo L, Plou FJ, Fernández-Lobato M (2009) Biochemical characterization of a β-fructofuranosidase from Rhodotorula dairenensis with transfructosylating activity. FEMS Yeast Res 9:768–773. doi: 10.1111/j.1567-1364.2009.00526.x

Herrera-González A, Núñez-López G, Morel S, Amaya-Delgado L, Sandoval G, Gschaedler A, Remaud-Simeon M, Arrizon J (2017) Functionalization of natural compounds by enzymatic fructosylation. Appl Microbiol Biotechnol 1–12. doi: 10.1007/s00253-017-8359-5

Hilt P, Schieber An, Yildirim C, Arnold G, Klaiber I, Conrad J, Beifuss U, Carle R (2003) Detection of Phloridzin in Strawberries (Fragaria x ananassa Duch.) by HPLC − PDA − MS / MS and NMR Spectroscopy. J Agric Food Chem 51:2896–2899. doi: 10.1021/jf021115k

Hofer B (2016) Recent developments in the enzymatic O-glycosylation of flavonoids. Appl Biochem Biotechnol 100:4269–4281. doi: 10.1007/s00253-016-7465-0

Lammens W, Le Roy K, Schroeven L, Van Laere A, Rabijns A, Van Den Ende W (2009) Structural insights into glycoside hydrolase family 32 and 68 enzymes: Functional implications. J. Exp. Bot. 60:727–740. doi: 10.1093/jxb/ern333

134 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Lee CH, Yang L, Xu JZ, Yeung SYV, Huang Y, Chen ZY (2005) Relative antioxidant activity of soybean isoflavones and their glycosides. Food Chem 90:735–741. doi: 10.1016/j.foodchem.2004.04.034

Malbert Y, Pizzut-Serin S, Massou S, Cambon E, Laguerre S, Monsan P, Lefoulon F, Morel S, Andre I, Remaud-Simeon M (2014) Extending the structural diversity of flavonoid glycosides with engineered glucansucrases. ChemCatChem 6:2282–2291. doi: 10.1002/cctc.201402144

Mena-Arizmendi A, Alderete J, Águila S, Marty A, Miranda-Mlina A, López-Munguía, Castillo E (2011) Enzymatic fructosylation of aromatic and aliphatic alcohols by Bacillus subtilis levansucrase: Reactivity of acceptors. J Mol Catal B Enzym 70:41–48. doi: 10.1016/j.molcatb.2011.02.002

Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. doi: 10.1021/ac60147a030

Öner ET, Hernández L, Combie J (2016) Review of Levan polysaccharide: From a century of past experiences to future prospects. Biotechnol Adv. doi: 10.1016/j.biotechadv.2016.05.002

Overwin H, Wray V, Hofer B (2015) Biotransformation of phloretin by amylosucrase yields three novel dihydrochalcone glucosides. J Biotechnol 211:103–106. doi: 10.1016/j.jbiotec.2015.07.002

Pandey RP, Li F, Kim E, Yamaguchi T, Park YI, Kim JS, Sohng JK (2013) Enzymatic Synthesis of Novel Phloretin Glucosides. Appl Environ Microbiol 79:3516–3521. doi: 10.1128/AEM.00409- 13

Rezk BM, Haenen GRMM, van der Vijgh WJF, Bast A (2002) The antioxidant activity of phloretin: The disclosure of a new antioxidant pharmacophore in flavonoids. Biochem Biophys Res Commun 295:9–13. doi: 10.1016/S0006-291X(02)00618-6

Thuan NH, Sohng JK (2013) Recent biotechnological progress in enzymatic synthesis of glycosides. J. Ind. Microbiol. Biotechnol. 40:1329–1356

Torres P, Poveda A, Jimenez-Barbero J, Para JL, Comelles F, Ballesteros AO, Plou F (2011) Enzymatic synthesis of α-glucosides of resveratrol with surfactant activity. Adv Synth Catal 353:1077–1086. doi: 10.1002/adsc.201000968

Xiao Z, Zhang Y, Chen X, et al (2017) Extraction, identification, and antioxidant and anticancer tests of seven dihydrochalcones from Malus “Red Splendor” fruit. Food Chem. 231:324–331. doi: 10.1016/j.foodchem.2017.03.111

135 Chapter IV: Enzymatic fructosylation of phlorizin by levansucrase from Gluconacetobacter diazotrophicus

Xu L, Qi T, Xu L, Lu L, Xiao M (2016) Recent progress in the enzymatic glycosylation of phenolic compounds. J Carbohydr Chem 35:1–23. doi: 10.1080/07328303.2015.1137580

136

CONCLUSIONS AND PERSPECTIVES

Conclusions and perspectives

Flavonoids and their glycosides are natural plant secondary metabolites exhibiting physico-chemical and biological properties that offer therapeutic and cosmetic applications. Glycosylation of flavonoids affects the solubility, stability, and the antioxidant activity of the aglycone. The chemical synthesis of flavonoid glycosides remains limited due to the complexity of glycochemistry. One way to facilitate the production of these compounds and open the route to a larger diversity of flavonoid glycosides is the development of enzymatic method.

Thus, the first achievement of this thesis consisted in writing a mini review on the enzymatic fructosylation of natural compounds. This bibliography study lists all the compounds which have been fructosylated, the reactions conditions, the enzymes and the physico-chemical properties of the new fructo-conjugates. Based on this knowledge, we found interesting to deepen the enzymatic fructosylation of flavonoids using enzymes belonging to GH32 and GH68 families. These enzymes present the advantage to use sucrose as donor of fructose, a cheap and abundant renewable substrate. By this way, different classes of phenolic compounds have been tested using -fructosidase and cell from bacteria. However, in the case of flavonoids only puerarin was efficiently fructosylated by β- fructosidases and cells from bacteria source as catalysts. The presence of co-solvents such as DMSO and ethanol in the reaction mixtures do not facilitate the fructosylation reaction.

The results presented in this PhD thesis open a new route to obtain new fructo-conjugates of flavonoids using enzymes from GH32 and GH68 families.

This PhD thesis had three main objectives:

 Screening of enzymes from non-saccharomyces yeasts isolated from Mezcal able to catalyze the transfructosylation of flavonoids

 In silico gene search, cloning and expression of new β-fructosidase from selected yeast genomes and assessment of their potential to catalyze flavonoid fructosylation.

 Comparison of the enzymatic fructosylation of phlorizin by different β-fructosidases from GH32 and GH68 families

138 Conclusions and perspectives

Regarding the first aim, sixty-two positive enzymatic extracts were shown to produce fructosyltransferase activity of 121 different yeast strains. Among them, only 15 enzymatic extracts retain their fructosyltransferase activity in the presence of 20 % (v/v) DMSO as co-solvent. The species showing fructosyltransferase activity in the presence of DMSO were Rhodotorula mucilaginosa (MB4), Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Candida apicola (CC, MT3), Kluyveromyces marxianus (DV4, 1424, DH4, 1616, DA5, 2108, 717), Zygosaccharomyces bisporus (DG, MS3) and Candida parapsilosis (MF4).

These 15 strains were then tested for the fructosylation of five different classes of flavonoids. The result showed that it was possible to synthetize a mono-fructosyl puerarin using the enzymatic extract of Torulaspora delbrueckii (DW1), Crytococcus albidus (DC4), Rhodotorula muciliaginosa (MB4), Kluyveromyces marxianus (DV4, DH4, DA5, 1616, DV3, 717), and Candida apicola (MT3, CC). Thus, it was the first time that the fructosylation of puerarin by a β-fructosidases from non- saccharomyces yeasts was described.

Then, genome mining enabled the identification of a sequence putatively coding for a β-fructosidase in the genome of Rhodotorula mucilaginosa (RhInv). Bioinformatics analysis revealed that the protein sequence shows 58% of identity with the invertase from Cryotococos neoformas and contains the three-conserved motif characteristic of the GH32 family confirming that the protein sequence likely encodes a functional β-fructosidase or invertase,. Subsequently, the gene was cloned, expressed in Pichia pastoris X-33 and its product was tested for its capacity to fructosylate puerarin, coniferyl alcohol and mangiferin. The results showed that it was possible to fructosylate acceptors other than sucrose in absence of co-solvents, but with low yields around 20%. The best yield was obtained in the case of coniferyl alcohol and only the presence of monofructosyl coniferyl alcohol was detected. This enzyme should be now biochemically characterized and the optimization of the flavonoid fructosylation could be investigated to improve the conversion yields and characterize by mass spectrometry and NMR the new fructosides.

Finally, the third objective of this thesis was accomplished. The capacity of a levansucrase from Gluconacetobacter diazotrophicus (LsdA, EC 2.4.1.10), a sucrose:sucrose 1-fructosyltransferase from Schedonorus arundimaceus (Sa1-SSTrec, EC 2.4.1.99) and a β-fructofuranosidase from Rhodotorula mucilaginosa (RhInv, EC 3.2.1.26) to fructosylate phlorizin was assessed. The best results were obtained with the levansucrase from Gluconacetobacter diazotrophicus (LsdA), which synthetizes β-D-fructofuranosyl–(26)-phlorizin as a new substance from phlorizin by enzymatic

139 Conclusions and perspectives

fructosylation with 79.1 % of conversion in the absence of co-solvents. Moreover, the concentration of acceptor, donor and enzyme were quite important to improve the enzymatic fructosylation of phlorizin by LsdA, in particular the use of high sucrose concentration probably enabled to better solubilize the flavonoid and may limit its inhibiting effect on enzyme activity. The mono- fructosylated product was 15.9 fold (30.5 g L-1 at 25°C) more soluble in water than the original substrate phlorizin (1.93 g.l-1 at 25°C), and exhibited a 1.4-fold reduction in the antioxidant capacity. As perspectives of this work, it will be important to isolate the other products observed in the reaction such as di-fructosyl phlorizin (P2), tri-fuctosyl phlorizin (P4) and the isomer mono fructosyl phlorizin (P3) in order to determine their structures. In addition, it will be interesting to evaluate the biological activities of this new fructoside in order to identify potential pharmaceutical applications.

In conclusion, the potential of the enzymes from GH32 and GH 68 families for the synthesis of novel fructosides that do not exist in nature was successfully explored. The pharmacological properties of these new compounds deserve to be determined in order to develop applications for the cosmetic and pharmaceutical industry. Moreover, it could be interesting to determine the three dimensional structure of the levansucrase from Gluconacetobacter diazotrophicus in order to initiate structure/function relationship studies, identify structural determinants involved in the enzyme specificity and initiate protein engineering to target and enhance the synthesis of new fructosides.

140

FIGURE AND TABLE CONTENTS

Figure and table contents

Figure and Table contents

Figure contents

Figure 1. General structure of flavonoids (Crozier et al. 2009)………………………………………16 Figure 2. Prominent glycosyl donors used in chemical synthesis (Desemet et al. 2012)……………..23 Figure 3. General mechanism for the synthesis of glycosidic bond by different enzymes (adapted from Xu et al. 2016)………………………………………………………………………………...... 25 Figure 4. General glycoside hydrolases a) inverting and b) retaining mechanisms. AH: a catalytic acid residue, B-: a catalytic base residue, Nuc: a nucleophile, and R: a carbohydrate derivative. HOR: an exogenous nucleophile, often a water molecule………………………………………………………..32 Figure 5. Synthetic and hydrolytic reactions catalyzed by Glycoside Hydrolases (Desmet et al. 2012)……………………………………………………………………………………………………33 Figure 6. Three-dimensional structure of invertase from Thermotoga maritime. The N-terminal β- propeller module, the five blades (numbered I–V), and the C-terminal β-sandwich module (dark red) were highlighted in colors (adapted from Alberto et al. 2004)………………………………...... 35 Figure 7. Three-dimensional structure of levansucrase from Gluconacetobacter diazotrophicus (adapted from Martínez-Fleites et al. 2005)………………………………………………………...... 37 Figure 8. Schematic drawing of the sugar-binding subsites from –n to +n nomenclature (adapted from Davies et al. 1997)……………………………………………………………………………………..38 Figure 9. Retaining mechanism form GH32 and GH68 families (adapted from Lammes et al. 2009)………………………………………………………………………………………………...... 39 Figure 10. Schematic diagram of hydrolysis and transfructosylation reactions catalyzed by enzymes from GH32 and GH68 families………………………………………………………………………...50 Figure 11. a) LC Chromatograms from screening of enzymatic fructosylation of puerarin from enzymatic extract of yeast non-saccharomyces at 1 U mL-1, 5 mM puerarin, 409 mM of sucrose (100 mM acetate buffer pH 5 and 20 % (v/v) of DMSO) during 24 h at 45 ºC and 600 rpm. MS3 (---), 717(- --), MT3 (---), DG (---), CC (---), 2108(---), DA5 (---), 1616(---), Blank (---), DH4 (---), 1424 (---), MB4 (---), MF4(---),DV4 (---), DW1(---) and DC4 (---). b) ESI-MS spectra in negative mode of new peak P1. (P) Puerarin; (P1), mono-fructosyl puerarin………………………………………………….82 Figure 12. Dendrogram built with the yeast species with β-fructosidase reported and with Rhodotorula mucilaginosa with not reported β-fructosidase sequence………………………………………………84 Figure 13. β-fructosidase sequence BLAST in genome from Rhodotorula mucilaginosa using a β- fructosidase from Cryptococcus neoformas var. grubii H99…………………………………………..85 Figure 14. BLAST protein performed at NCBI of predicted β-fructosidase from Rhodotorula mucilaginosa……………………………………………………………………………………………88 Figure 15. Propagation of plasmid in E. coli DH5α Low-salt LB plates with ampicillin (1μg/μL) a)pUC57-RhInv and d) negative control……………………………………………………………….91 Figure 16. Electrophoresis gel of analysis of restriction of pUC57-RhInv, Lane 1 ladder 10 kbp, lane 2, 3 digest of pUC57-RhInv (β-fructosidase from R. mucilaginosa) using EcoRI and SalI………………………...... 92 Figure 17. Electrophoresis gel for the purification of β-fructosidase gene Lane 1: ladder 10 kpb, Lane 2 and 3: Gene of β-fructosidase from R. mucilaginosa ……………………………………………….92 Figure 18. Electrophoresis gel for RhInv ligation reaction into expression vector (pGAPZB). Lane 1: ladder 10 kbp, lane 2, 3, ligation at initial time of pGAPZB and RhInv, lane 4, 5 at final time of pGAPZB-RhInv………………………………………………………………………………………...93 Figure 19. Propagation of the construct into E. coli DH5α in low-salt LB plates with zeocin (25 μg/μL) a) pGAPZB-RhInv, b) pGAPZB and c) negative control………………………………………………93

142 Figure and table contents

Figure 20. YPDS plates with 100 μg/mL of zeocin resistant Pichia pastoris X-33 transformants containing a) pGAPZB-RhInv, b) empty pGAPZB and c) negative control…………………………...94 Figure 21. Electrophoresis gel for PCR products. Lane 1: ladder 10 kbp, lane 2: pGAPZB-RhInv and RhInv, lane 3: pGAPZB and lane 4: negative control………………………………………………….94 Figure 22. Phenotypic screening of zeocin resistant colonies transformed with a) negative control (P. pastoris X-33), b) pGAPZB and c) pGAPZB-RhInv and d) pGAPZB-RhInv (lane 2 in blue is empty)…………………………………………………………………………………………………..95 Figure 23. a) LC Chromatograms from screening of enzymatic fructosylation of puerarin using RhInv at 1 U mL-1, 14 mM puerarin, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI-MS spectra in negative mode of new peak. (P) Puerarin; P1, mono-fructosyl puerarin…………………………………………………………………………………………………99 Figure 24. a) LC Chromatograms from screening of enzymatic fructosylation of coniferyl alcohol using RhInv at 1 U mL-1, 14 mM coniferyl alcohol, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI-MS spectra in negative mode of new peak. (C) Coniferyl alcohol; (C1), mono-fructosyl coniferyl alcohol……………………………………………………..100 Figure 25. a) LC Chromatograms from screening of enzymatic fructosylation of mangiferin using RhInv at 1 U mL-1, 14 mM mangiferin, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm. b) ESI-MS spectra in negative mode of new peak. (M) Mangiferin; (M1), mono- fructosyl mangiferin……………………………………………………………………………….….101 Figure 26. General schema of in silico search of β-fructosidase gene in yeast genome……………. 106 Figure 27. a) LC Chromatograms from screening of enzymatic fructosylation of phlorizin at 25 mM, 0.146 M sucrose (50 mM phosphate buffer pH 5.8), 1 UmL-1, at 42 ºC during 24 h. Initial time t = 0 h (---), LsdA (---), 1-SST (---) and RhInv (---). b) ESI-MS spectra in negative mode of new peaks...... 119 Figure 28. Effect of sucrose concentration on phlorizin conversion. Reactions conditions: 1 U mL-1 of Levansucrase (LsdA), 25 mM phlorizin in 50 mM phosphate buffer pH 5.8, 42 ºC. Sucrose concentration () 0.146 M, (●) 0.5 M, () 1 M and (▼) 1.5 M………………………………….....121 Figure 29. Effect of phlorizin concentration on phlorizin conversion. Reactions conditions: 1 U mL-1 of Levansucrase (LsdA), 1.5 M sucrose and 50 mM phosphate buffer pH 5.8, 42 ºC. Phlorizin concentration () 25 mM, (●) 50 mM, (▼) 75 mM and () 100 mM…………………………..….122 Figure 30. Effect of enzyme concentration on phlorizin conversion. Reactions conditions: 25 mM phlorizin, 1.5 M sucrose in 50 mM phosphate buffer pH 5.8, 42 ºC. () 1 U mL-1, (☐) 2.5 U mL-1, (●) 5 U mL-1, (▼) 10 U mL-1 and () 15 U mL-1……………………………………………………….123 Figure 31. Enzymatic fructosylation of phlorizin with levansucrase from Glucconacetobacter diazotrophicus………………………………………………………………………………………....124 Figure S1. 1H-NMR spectrum of β-D-fructofuranosyl-(26)-phlorizin…………………………....132 Figure S2. 13C spectrum of β-D-fructofuranosyl-(2-->6)-phlorizin…………………………..………132 Figure S3. HSQC spectrum of β-D-fructofuranosyl-(26)-phlorizin……………………………….133 Figure S4. HMBC spectrum of β-D-fructofuranosyl-(26)-phlorizin……………………………....133

143 Figure and table contents

Table contents

Table 1. Flavonoids classification: structure and examples…………………………………………..18 Table 2. Enzymatic glycosylation of flavonoids by Leloir glycosyltransferases……………………...27 Table 3. Enzymatic glycosylation of flavonoids by non-Leloir glycosyltransferases………………....28 Table 4. Enzymatic glycosylation of flavonoids by glycoside hydrolases (GHs)………………….….29 Table 5. The established Glycoside Hydrolases clans of related families in the CAZy database (adapted from http://www.cazy.org/)…………………………………………………………………..31 Table 6. Enzymes members and some features of GH32 family (adapted form CAZy, http:// www.cazy.org/)…………………………………………………………………………………..….….34 Table 7. Enzymes members and some features of GH68 family (adapted form CAZy, http:// www.cazy.org/)………………………………………………………………………………………....36 Table 8. Fructosylation of alkyl alcohols from sucrose by microbial enzymes…………….………….55 Table 9. Fructosylation of aromatic alcohols from sucrose by microbial enzymes…………………...58 Table 10. Fructosylation of alkaloids from sucrose by microbial enzymes………………….………..60 Table 11. Fructosylation of flavonoids from sucrose by microbial enzymes………………….………64 Table 12. Solubility of glucosylated and fructosylated compounds……………………..…………….66 Table 13. Antioxidant activity of fructoside and glucoside compounds determined by DPPH (2, 2- diphenyl-1-picrylhydrazyl) radical scavenging activity IC50 …………………………………..………66 Table 14. Pharmacokinetics properties of glucosyl and fructosyl compounds…………..…………….68 Table 15. Screening of fructosyltransferase activity in the enzymatic extract from non-saccharomyces yeast in absence of DMSO and presence of 20 % (v/v) of DMSO……………………..……………...79 Table 16. Screening of fructosylation of different flavonoids…………………………………...…….80 Table 17. In silico search β-fructosidase sequence parameters for selection of yeast…………...…….83 Table 18. Predicted β-fructosidase from Rhodotorula mucilaginosa a) nucleotide and b) amino acid sequence. The catalytic domains are highlighted in blue and the signal peptide in red…………….….86 Table 19. Predicted β-fructosidases theoretical properties and catalytic motifs……………………….87 Table 20. Predicted β-fructosidase homology structure-model by SWISS-MODEL Workspace……..89 Table 21. Boxshade analysis of predicted β-fructosidase from Rhodotorula mucilaginosa (in red is highlighted the catalytic triad)……………………………………………………………………….…90 Table 22. Fructosidase activity in the supernatant from P. pastoris X-33, zeocin resistant Pichia transformed with pGAPZB and pGAPZB-Rh…………………………………………………….……96 Table 23. Percentage of substrate conversion of different acceptors using RhInv at 1 U mL-1, 14 mM of acceptor, 877mM of sucrose (100 mM acetate buffer pH 5.5) during 10 h at 40 ºC and 600 rpm……………………………………………………………………………………………….……..98 Table 24. Conversion and percentage of fructosylated compounds for phlorizin in the enzyme selection……………………………………………………………………………………………….120 Table 25. Chemical shifts from 13C and 1H data of phlorizin and -D-fructofuranosyl-(26)-phlorizin (Data were recorded in MeOD). Chemical shifts are given in ppm relative to the residual signal of the solvent at 3.34 ppm in 1H and 49.5 ppm in 13C. Coupling constants in Hz………………………………………………………………………………………………...……125 Table 26. Solubility of phlorizin and fructosyl phlorizin in water at 25 ºC……………………..……126 Table 27. Free radical scavenging of phlorizin and phlorizin mono-fructoside………………...……126

144

ABBREVIATIONS

Abbreviations

Asp: Aspartic acid AOXI: Alcohol Oxidase promotor ATF: Agave tequilana Fructans AUC(0-t): time concentration curve AUC(0-∞): area under the time concentration curve BLAST: Basic Local Alignement Search Tool bp: Bases pair CAZy: Carbohydrate-active enzymes Cmax: Peak serum concentration DMSO: Dimethyl sulfoxide DNS: Dinitrosalicylic acid DPPH: 2, 2-diphenyl-1-picrylhydrazyl E. C.: Enzyme Commission number ESI-MS: Electrospray Ionization Mass Spectrometry FOS: Fructooligosaccharides GH: Glycoside hydrolase Glu: Glutamic acid GT: Glycosyltransferases HPLC: High Performance Liquid Chromatography kDa: kilo Dalton KEGG: Kyoto Encyclopedia of Genes and Genomes LB: Lysogeny Broth LPH: Lactase Phloridzin Hydrolase Mw: Molecular weight NCBI: National Center for Biotechnology Information ND: Non-Detectable NMR: Nuclear Magnetic Resonance RhInv: Invertase from Rhodotorula mucilaginosa PCR: Polymerase Chain Reaction PDB: Protein Data Bank pI: Isoelectric point t1/2 : Half-time elimination TLC: Thin Layer Chromatography U: Unit of enzyme activity correspond to μmol of fructose liberate per minute UDP-glucose: Uridine Diphosphate Glucose UV: Ultra Violet v/v: volume per volume w/v: weight per volume

146