Purification and Characterization of levansucrase from newly isolated strain of Zymomonas mobilis
SIDRA SHAHEEN
Dr. A. Q. Khan Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi, Karachi-75270, Pakistan 2017
THIS THESIS IS SUBMITTED IN THE
FULFILLMENT OF REQUIREMENT
FOR THE DEGREE OF DOCTOR OF
PHILOSOPHY
IN
BIOTECHNOLOGY
THIS THESIS IS DEDICATED TO
MY BELOVED PARENTS
Mr. Muhammad Asghar and Mrs. Maqsoodan Bibi
MY LOVELY DAUGHTER
Sarah Owais
AND MY DEAREST HUSBAND
Mr. Muhammad Owais TABLE OF CONTENTS
SECTION DESCRIPTION EPAGE
ACKNOWLEDGEMENT i-ii
ABBREVIATIONS iii-iv
ABSTRACT v-ix
ABSTRACT (ENGLISH) v-vi ABSTRACT (URDU) vi-ix
Chapter 1 INTRODUCTION 1-25
1.1 Zymomonas mobilis 1-2 1.2 Microbial Extracellular Polysaccharide (EPS) 2
1.2.1 General Characteristics of EPS 2-3
1.2.2 Chemistry of EPS 3
1.2.3 Applications of EPS 3-4 1.2.4 Classification of EPS 5
1.2.4.1 Bacterial Heteropolysaccharides 5 1.2.4.2 Bacterial Homopolysaccharides 5 1.2.4.2.1 Glucans 6-7 1.2.4.2.2 Fructans 7-8 1.3 Background of Levan 9 1.3.1 Microbial Levan: The Chemical Structure 9-10 1.3.2 Properties of Levan Polysaccharide 10 1.3.2.1 Physical and Biochemical Characteristics 10 1.3.2.2 Biological Characteristics 10-11 1.3.3 Applications of Levan 11 1.3.3.1 Medical and Pharmaceutical 11-12 1.3.3.2 Prebiotics 12 1.3.3.3 Personal Hygiene 12 1.3.3.4 Levan in Marine Aquaculture 13 1.3.3.5 Levan as a Packaging Films 13
1.3.3.6 Levan in Food Industry 13-14 1.3.3.7 Additional Applications 14 1.4 Industrial Production of Levan 14 1.4.1 Fructosyltransferases 15-16 1.4.1.1 Levansucrase 16-17 1.4.1.2 Reaction Mechanism of Levansucrase 17-18 1.4.1.3 Transfructosylation Reaction 18-19 1.4.1.4 Specificity of Levansucrase for Donor and Acceptor 19 Residues 1.5 Purification of Levansucrase 19-20 1.6 Structural and Functional Analysis of Levansucrase 20-21 1.6.1 Signal Peptide and N-terminal Variable Region of 22 Levansucrase 1.6.2 Catalytic Domain of Levansucrase 22 1.6.2.1 Catalytic Residues of Levansucrase 22 1.6.2.2 Catalytic Site of Levansucrase 23 1.6.2.3 Topology of Levansucrase Active Site 23-24 1.6.2.4 Calcium Binding Site of Levansucrase 24-25 1.6.2.5 C-terminal Domain 25
Chapter 2 RESEARCH DESIGN 26-27
2.1 27 Schematic Representation of Production, Characterization and Purification of Levansucrase
Chapter 3 MATERIALS AND METHODS 28-51
3.1 General Section 28 3.2 Experimental Session 28 3.2.1 Sample Collection, Screening and Isolation of 28 Levansucrase Producing Strains 3.2.1.1 Sample Collection 28 3.2.1.2 Isolation and Screening of Levan Producing strain 29 3.2.1.3 Culture Identification 29-34 3.2.1.4 Preservation of Bacterial Isolate 34 3.2.2 Production of Levansucrase 34 3.2.2.1 Selection of Growth Medium for Levansucrase 34 Production 3.2.2.2 Research Setup to Formulate Modified Medium for 35-38 Maximum Production of Levansucrase from Z. mobilis KIBGE-IB14 3.2.3 Partial Purification of Crude Levansucrase 38 3.2.3.1 Batch Precipitation 38 3.2.3.2 Gradient precipitation 39 3.2.4 Desalting of Precipitated Levansucrase 39 3.2.5 Ultrafiltration of Precipitated Levansucrase 39 3.2.6 Size Exclusion Chromatography 39-40 3.2.7 Characterization of levansucrase 40 3.2.7.1 Reaction Time 40 3.2.7.2 Temperature Profile 41 3.2.7.3 pH Profile 41 3.2.7.4 Selection and Ionic Strength of Buffer 41 3.2.7.5 Enzyme Kinetics 41 3.2.7.6 Effect of Metal Ions 42 3.2.7.7 Effect of Solvents 42 3.2.7.8 Effect of Surfactants 42 3.2.7.9 Thermal Stability 43 3.2.7.10 Storage Stability 43 3.2.8 Molecular Weight Estimation of Levansucrase 43 3.2.8.1 Sample Preparation 44 3.2.8.2 Preparation of Gel 44 3.2.8.3 Visualization of Protein Bands 44-45 3.2.8.4 Glycoprotein Staining of purified Levansucrase 45-46 3.2.9 Relative Amino Acid Composition Analysis 46-49 3.3 Analytical Section 49 3.3.1 Assay for Levansucrase 49-50 3.3.2 Estimation of Total Protein 51 3.3.2.1 Total Protein Assay for Levansucrase 51 Chapter 4 RESULTS AND DISCUSSION 52-99
4.1 Isolation, Screening and Identification of 53-56 Levansucrase Producing Strain 4.2 Selection of an Optimum Medium 56-58 4.3 Optimization of Cultivation Conditions for Production 59 of Levansucrase 4.3.1 Selection of Physical Parameters for Levansucrase 59-60 Production 4.3.1.1 Effect of Incubation time 60-61 4.3.1.2 Effect of Temperature 61-62 4.3.1.3 Effect of pH 62-63 4.3.2 Influence of Carbon Source on Levansucrase 64 Production 4.3.2.1 Effect of Sucrose 64-65 4.3.3 Optimization of Nitrogen Sources for Levansucrase 65 Production 4.3.3.1 Effect of Yeast extract 65-66 4.3.3.2 Effect of Peptone 66-67 4.3.4 Influence of Micronutrients for Levansucrase 67 Production
4.3.4.1 Effect of Dipotassium Hydrogen Phosphate (K2HPO4) 68
4.3.4.2 Effect of Calcium Chloride (CaCl2.2H2O) 69 4.4 Purification of Levansucrase 70-71 4.4.1 Partial Purification of Levansucrase 72-75 4.4.2 Desalting of Partially Purified Levansucrase 76 4.4.2 Gel Permeation Chromatography 76-79 4.5 Biochemical Characterization of Purified 80 Levansucrase 4.5.1 Enzyme-Substrate Reaction Time Profile 80-81 4.5.2 Temperature Activity Profile 81-82 4.5.3 pH Profile and Selection of Buffer 82-84 4.5.4 Selection of Ionic Strength of Buffer 84-85 4.5.5 Kinetic Analysis 85-86 4.5.6. Stability Studies 86 4.5.6.1 Thermal Stability 87-88 4.5.6.2 Storage Stability 88-89 4.5.7 Effect of Metal Ions 89-91 4.5.8 Effect of Organic Solvents 92-93 4.5.9 Effect of Surfactants 93-94 4.6 Approximate Molecular Weight and Zymography of 94-96 Purified Levansucrase 4.7 Amino acid Analysis of Levansucrase 97-99
Chapter 5 CONCLUSIONS 100-101
Chapter 6 REFERENCES 102-119
APPENDICES 120-136
APPENDIX-I (Research Instruments) 120 APPENDIX-II (Reagents Index) 121-122 APPENDIX-III (Gram’s staining) 123-124 APPENDIX-IV (Spore Staining) 125 APPENDIX-V (Buffer Preparation) 126-127 APPENDIX-VI (Preparation of Reagents for SDS- 128-130 PAGE)
APPENDIX-VII (Preparation of Reagents for Enzyme 131
Assay) APPENDIX-VIII (Reagents Preparation for Total 132-133
Protein Assay) APPENDIX-IX (List of Publications) 134-136 LIST OF FIGURES
FIGURES TITLE PAGE NO.
1 Schematic figure of the repeating moieties of dextran, 7 Mutan, Alternan, Glucan and Reuteran.
2 Figure shows the structure of (A) Levan and (B) Inulin 8 comprises of fructan backbone with their branching points.
3 Schematic representation of hydrolysis of sucrose into 17 glucose and fructose by levansucrase.
4 Schematic depiction illustrates the acceptor reaction 18 catalyzed by levansucrase.
5 Schematic illustration of fructansucrase protein from 21 lactic acid bacteria. This figure depicts four different regions (i) N-terminal signal sequence (ii) N-terminal variable region (iii) Catalytic core (iv) C-terminal region sometimes attached with LPXTG cell wall).
6 Topology of glycoside hydrolase. A, B and C indicated 24 as Open cleft, Pocket and Tunnel respectively.
7 Growth of Z. mobilis KIBGE-IB14 on YPD agar plate. 53
8 Phylogenetic tree of Zymomonas isolates representing the 56 16S rDNA sequence similarity against the data available in GenBank database.
9 Levansucrase production from Z. mobilis KIBGE-IB14 at 61 various time intervals.
10 Production of levansucrase from Z. mobilis KIBGE-IB14 62 at different temperatures.
11 Levansucrase production from Z. mobilis KIBGE-IB14 at 63 various pH values.
12 Production of levansucrase from Z. mobilis KIBGE-IB14 65 at various substrate concentrations.
13 Levansucrase production from Z. mobilis KIBGE-IB14 at 66 different yeast extract concentrations.
14 Levansucrase production from Z. mobilis KIBGE-IB14 at 67 different peptone concentrations.
15 Production of levansucrase from Z. mobilis KIBGE-IB14 68
at various di-potassium hydrogen phosphate (K2HPO4) concentrations.
16 Production of levansucrase from Z. mobilis KIBGE-IB14 69
at various calcium chloride (CaCl2.2H2O) concentrations.
17 Purification scheme of levansucrase from Z. mobilis 71 KIBGE-IB14.
18 Gel permeation chromatography of levansucrase from Z. 79 mobilis KIBGE-IB14.
19 The effect of reaction time on catalytic activity of 81 levansucrase from Z. mobilis KIBGE-IB14.
20 The effect of temperature on levansucrase activity from 82 Z. mobilis KIBGE-IB14.
21 The influence of pH dependence on catalytic activity of 84 levansucrase from Z. mobilis KIBGE-IB14.
22 The effect of ionic strength of sodium phosphate buffer 85 (pH-6.0) on catalytic activity of levansucrase from Z. mobilis KIBGE-IB14.
23 Lineweaver-Burk plot and Michaelis-Menten constant of 86 levansucrase from Z. mobilis KIBGE-IB14.
24 Thermal stability profile of levansucrase from Z. mobilis 88 KIBGE-IB14.
25 Storage stability profile of levansucrase from Z. mobilis 89 KIBGE-IB14.
26 Electrophoretic pattern and zymogram of purified sample 96 of levansucrase from Z. mobilis KIBGE-IB14.
27 Amino acid profile of levansucrase produced by Z. 99 mobilis KIBGE-IB14. LIST OF TABLES
TABLES TITLE PAGE NO.
1 Microbial EPS and their applications. 4
2 Composition of YPD agar medium. 29
3 Modified experimental scheme to attain maximum 36-37 Levansucrase yield from Z. mobilis KIBGE-IB14.
4 Composition of modified medium designed for 38 levansucrase production by Z. mobilis KIBGE-IB14.
5 Composition of electrophoresis gel for SDS-PAGE. 44
6 Amino acids standard solution used. 47
7 Morphological and biochemical analysis of Z. mobilis 54-55 KIBGE-IB14.
8 Taxonomic classification of Z. mobilis KIBGE-IB14. 55
9 Levansucrase production on various reported and 58 newly formulated media.
10 Partial purification of levansucrase from from Z. 74 mobilis KIBGE-IB14 with different precipitating agents of various concentrations.
11 Partial purification of levansucrase from Z. mobilis 75 KIBGE-IB14withgradientprecipitationof ammonium sulphate.
12 Complete purification profile of levansucrase 78 produced by Z. mobilis KIBGE-IB14.
13 Effect of different metal ions on the catalytic activity 91 of levansucrase from Z. mobilis KIBGE-IB14.
14 Influence of different organic solvents on levansucrase 93 activity obtained from Z. mobilis KIBGE-IB14.
15 Impact of different surfactants on catalytic activity of 94 levansucrase from Z. mobilis KIBGE-IB14.
16 Amino acid composition of levansucrase produced by 98 Z. mobilis KIBGE-IB14. ACKNOWLEDGMENT
In the name of Allah, the Most Gracious, the Most Merciful.
First and Foremost praise is to ALLAH ALMIGHTY, the greatest of all, on whom ultimately we depend for sustenance and guidance. I would like to thank ALMIGHTY ALLAH for giving me opportunity, determination and strength to do my research. His continuous grace and mercy is with me throughout my life and ever more during the tenure of my research.
Also, I cannot forget the ideal man of the world and most respectable personality for whom Allah created the whole universe, Prophet Hazrat Muhammad Mustafa (Peace Be Upon Him), ideal personality and the ultimate source of knowledge for whole mankind.
This thesis is the culmination of my journey of Ph.D. which was just like climbing a high peak step by step accompanied with encouragement, hardship, trust and frustration. When I found myself at top experiencing the feeling of fulfillment, I realized though only my name appears on the cover of this dissertation, a great many people including my family members, well-wishers, my friends and colleagues have contributed to accomplish this huge task.
First of all, I would like to thank and express my deep and sincere gratitude to my supervisor Dr. Nadir Naveed Siddiqui, Scientific Officer, KIBGE, University of Karachi for his continuous support, guidance and encouragement. I appreciate all his contributions of time, support and ideas.
No research is possible without infrastructure and requisite materials and resource. I would like to extend my sincere regards to Dr. Abid Azhar (Director General, KIBGE) for all institutional support regarding required facilities to conduct this work.
I do not have words to acknowledge Dr. Shah Ali Ul Qader (Professor, University of Karachi) for all his back support especially in bench work and paper publication. He is a great mentor, his guidance and encouragement always motivated me not only in my research work but in every step and problems of my life.
I would also like to pay my humble regards to Dr. Afsheen Aman (Assistant Professor, KIBGE) for her boundless help and encouragement especially during the purification process of my research work which would be impossible without her help and guidance.
i
My highest and special thanks go to my friends and colleagues Dr. Asma Ansari, Dr. Sidra Pervez, Ms. Faiza Shahid and Ms. Urooj Javed for their strong support during the work of the thesis and their great patience to me. I cannot express my deepest feeling and high appreciation through this acknowledgement since they deserve much more.
In my daily work I have been blessed with a friendly and cheerful group of fellow students. I am thankful to my colleagues, Dr. Zainab Bibi, Dr. Asad Karim, Dr. Asif
Nawaz, Ms. Tayyaba Abdullah, Ms. Bushra Zaidi, Ms. Hafsa Sattar, Ms. Safia Shakir, Ms. Fehmina Khan, Ms. Fariha Ibrahim, Ms. Zahida Shabbir and Mr. Muhsin Ullah Khan for their support, discussions and suggestions. I am extremely thankful to Dr. Rizma Khan who helped me in preparing the thesis write-up with her full strength and sincerity. I would not forget to acknowledge my senior colleague Ms. Samina Iqbal for providing bacterial strain used in my research work.
I also appreciate the support of technical faculty (KIBGE), Mr. Shahab Ahmad, Mr. Zaheer Ahmed, Mr. Shahid and Ms. Nafeesa Bano for all the things that facilitated smooth work of my research. Furthermore, I also acknowledge the support of Mr. Shuja Ahmad, for his help in urdu translation of abstract.
Years ago my parents used to say, "We wish our daughter (Sidra) to be a doctor". Although they meant some other doctor, but I am sure they would be the most happiest and proud persons for my title of Ph.D. My sincere and profound gratitude are due to my beloved parents Mr. Muhammad Asghar and Mrs. Maqsoodan Bibi. Their prayers and moral support will always boost my progress. I salute you both for the selfless love, care, pain and sacrifice you did to shape my life. A warm thanks to my siblings Muhammad Waseem Asghar and Muhammad Faheem Asghar as well as my bhabhi Mrs. Samina Waseem who support me in taking care of my daughter and kept encouraging me towards success.
I would like to express my deep appreciation to my parents and in laws, Mr. Liaquat Ali, Mrs. Nayyar Liaquat, Ms. Jaweria Liaquat and specially Ms. Maria Liaquat for their valuable support, motivation and care of my daughter.
Specially and most sincerely, I would like to pay my warmest tribute to a very special person, my beloved husband, Mr. Muhammad Owais for his continued and unfailing love, support and understanding during my pursuit of Ph.D. degree that made the completion of thesis possible. You were always around at times I thought that it is impossible to continue. I consider myself the luckiest in the world to have such a supportive and caring husband. Last but not least, I deeply appreciate my daughter, my little angel, Sarah Owais for abiding my ignorance and the patience she showed during my thesis writing.
Sidra Shaheen
ii ABREVIATION
(NH4)2SO4 Ammonium Sulphate g Gravitational Force
APS Ammonium per sulphate
CFF Cell Free Filtrate
C-terminal Carboxyl Terminus
EC# Enzyme Commission Number
EDTA Ethylenediaminetetraacetic Acid
GPC Gel Permeation Chromatography kDa Kilo Daltons
KIBGE Dr. A. Q. Khan Institute of Biotechnology and Genetic Engineering
U mg-1 Units per milli grams mM milliMoles
N-terminal Catalytic Domain Terminal
OD Optical Density
PAGE Poly Acryl Amide Gel Electrophoresis rDNA Ribosomal Deoxyribo Nucleic Acid rpm Revolutions per minute
SDS Sodium Dodecyl sulphate
TEMED N,N,N′,N′-Tetramethylethylenediamine
UV Ultra-violet
-1 m L milli Liter PCSIR Pakistan Council of Scientific and Industrial Research
GOD-PAP Glucose Oxidase Method
RA Relative Activity
iii GH Glycoside hydrolase
FTF Fructosyltransferase
SDB Sample Diluting Buffer g L-1 gram per Liter Mbp Megabase pair
EPS Extracellular Polysaccharide
-1 mgL milli gram per Liter nm Nanometer
FFases Fructosylfuranosides
Lsc Levansucrase
FOS Fructo-oligosaccharide
Asp Aspartic acid
Asn Asparagine
Glu Glutamic acid
DEAE Diethylaminoethyl cellulose
M L-1 Moles per Liter
SE Standard Error dNTP Deoxynucleotide triphosphate
BLAST Basic Local Alignment Search Tool
iv Abstract
In all stages of biochemical and metabolism reactions, the enzymes play major role as biocatalysts. In industrial utilization, microbial enzymes are predominant as compared with other natural and synthetic enzymes. Isolation, production, characterization as well as applications of several microbial enzymes have continuously progressed in bioindustry.
The current study covers the hyper production and purification of levansucrase from
Zymomonas mobilis KIBGE-IB14. This bacterial isolate could be a plausible candidate for different biotechnological and industrial processes. Preliminary step presented the screening of bacterial isolate. Production of maximum levansucrase resulted in the selection of Z. mobilis KIBGE-IB14 among different microbial species. The manipulation of different fermentation parameters enhanced the yield of levansucrase and higher titers of enzyme was achieved by using sucrose (15%) as a substrate with pH-6.5 at 30°C for 24 hours. In case of chemical parameters, yeast extract (1.0%), peptone (0.2%), K2HPO4
(1.5%) and CaCl2.2H2O (0.01%) were found to be the most suitable macro and micronutrients.
Different precipitating agents including ammonium sulphate, ethanol and PEG 4000 were used for the partial purification of levansucrase. Ammonium sulphate was selected as a suitable precipitating agent among all precipitants. Gradient precipitation by ammonium sulphate (50%) resulted in 1.6 times increase in purification of levansucrase. After desalting, the final purifcation of levansucrase was achieved using Sepharose CL-6B chromatographic system which purified the enzyme upto 12.13 fold with 1.77% yield.
SDS-PAGE electrophoresis and zymography revealed that the apparent molecular weight of purified levansucrase from Z. mobilis KIBGE-IB14 was approximately 130.0 kDa.
The catalytic performance of levansucrase during kinetic analysis was observed in
100.0 mM sodium phosphate buffer (pH-6.0) after 5.0 minutes of incubation with a Vmax
v -1 -1 and Km values of 21381 U mg and 0.02308 moles L , respectively. Levansucrase quite stable at 25°C and 35 °C as the enzyme retained its activity approximately 83% and 92% for 120 minutes, respectively. Storage stability of levansucrase suggested that the enzyme was stable at -80°C up to 180 days with the residual activity of 94%. Some of the metal + + + +2 +2 +2 +2 +2 ions including K , Na , Cs , Ba , Ca , Cu , Mg and Mn accelerated the activity
+2 +2 of levansucrase at 1.0 mM concentration whereas, other metal ions including Co , Hg ,
+3 +3 Fe and Al showed an inhibitory effect on enzyme activity. In the case of organic solvents, isopropanol (1.0 mM) acted as an activator while, at 10.0 mM concentration of
DMSO, formaldehyde and chloroform were found to be inhibitors. Non-ionic surfactants
(Triton X-100, Tween-20 and Tween-80) did not have any significant effect on levansucrase activity whereas, anionic detergent (SDS) exhibited a slight inhibition.
Amino acid analysis of levansucrase from KIBGE-IB14 revealed that this enzyme is a combination of non-polar (Proline, Alanine and Valine), polar (Glycine and Tryptophan) and basic amino acids (Lysine and Arginine).
To summarize the whole research study, it makes evident that an improved kinetic analysis and successful purification system for levansucrase has been developed. The present investigation provides amino acid composition of levansucrase which could be used as a preliminary data for its molecular characterization. Thus, it is concluded that the titer and performance of levansucrase biosynthesized by Z. mobilis KIBGE-IB14 has been enhanced and it could be used for a wider range of applications in many industrial bioprocesses.
vi Abstract (urdU)
vii
viii
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Chapter 1 Introduction
1.1 Zymomonas mobilis
A bacterium, Zymomonas mobilis is considered as one of the unique organism in the microbial domain due to its diverse functions including ethanol production, sugarcane fermentation, exopolysaccharide formation and considered as a spoilage organism
(Erdal et al., 2017; Yang et al., 2016b; Swings and De Ley, 1977). These are Gram’s negative rod shaped bacteria which was originally isolated from different sources such as African palm wine and sugarcane. In general, its length is about 2-6 μm while diameter is of 1-1.4 μm (Baratti and Bu'Lock, 1986).
On the basis of taxonomical and molecular evaluation of bacterium, genus
Zymomonas has only one species that is Zymomonas mobilis with three subspecies namely mobilis, pomaceae and francensis (Coton et al., 2005; Swings and De Ley,
1977). The genome size of Z. mobilis is ~2.056416 to 2.200722 Mbp which encodes about 1,998 protein coding genes (Zhao et al., 2016; Seo et al., 2005).
Z. mobilis is facultative anaerobe which utilizes sucrose as a carbon source. The unique biochemical property of Z. mobilis shows that it metabolizes glucose and fructose by Entner–Deudoroff (E–D) pathway which is the general characteristic of aerobic microorganisms (Sawides et al., 2001). Therefore, the biochemistry of this microbe not only exhibits an extreme exclusivity but its growth pattern, energy production, culture and cultivation techniques are also very distinct. All these extraordinary properties attract Z. mobilis to the scientific, industrial and biotechnological spheres with greater extent. It is considered as an ideal microorganism for the development of bacteriological procedures due to its stimulation towards physical and chemical environmental conditions, coupling and uncoupling of energy mechanisms for product generation as well as its restriction for
1 product formation (Doelle et al., 1993).
In addition to these properties, Z. mobilis also exhibits slimy granular layer around the microbial cell in the presence of elevated levels of CO2 or ethanol concentrations. This extracellular coating provides potential to Z. mobilis to produce biopolymer i.e. levan. Levan is a fructose polymer having -(2→6) bond which is produced by the action of levansucrase in the presence of sucrose as a substrate
(Gunasekaran and Raj, 1999). Microbial levan has been used in various industrial zones for different applications. Therefore, a great interest has been developed in the improvement of levan production by microorganisms using various new techniques.
Further information related to the polymer as well as its properties, applications and production is discussed below.
1.2 Microbial Extracellular Polysaccharide (EPS)
The natural polysaccharide has huge potential in different sectors that triggered its interest in various fields due to several advantages over synthetic and chemical polymers. This polymeric substance possesses unique properties that make it superior over other chemically synthesized polymers such as biodegradable, biocompatible, non-toxic and eco-friendly. Biologically, polysaccharides can be produced by both prokaryotic and eukaryotic microbial sources which mainly include bacteria, fungi, yeasts and algae respectively. Microbial polysaccharide is widely classified into structural, intracellular and extracellular polysaccharides referred to as EPS.
1.2.1 General Characteristics of EPS
Exopolysaccharides (EPS) are polymers of high molecular weight which are
synthesized extracellularly by various microorganisms. All bacterial polysaccharides
were named as EPS by Sutherland (1972) that can be differentiated from each other in terms of molecular mass, solubility, viscosity and type of linkage. The
primary role of 2 exopolysaccharides is the protection of microorganisms as they are responsible for the morphological organization of the cellular matrix (Flemming and Wingender, 2001).
The bacterial exopolysaccharides are sectioned into two major forms that are capsular or slime EPS depending on their association with the cell wall. The capsular polysaccharides are attached to the microbial cell whereas, the slime EPS remains free from cellular material (Gehr and Henry, 1983).
1.2.2 Chemistry of EPS
Monosaccharides (aldehyde or ketones) are the building blocks of carbohydrates which are linked to each other by glycosidic bonds resulted in the formation of oligosaccharides (sucrose and panose) or polysaccharides (dextran, levan and starch).
In the field of glycol technology, polysaccharides are become extremely significant due to its structural divergence and functional adaptability. Polysaccharides are diverse to each other on the basis of either type of sugar or structural connectivity of molecule (De Vuyst et al., 2001).
1.2.3 Applications of EPS
Numerous exopolysaccharides have been documented but some of them indicated the medical and industrial significance with potent commercial value. The limited application of these polysaccharides has been mainly due to their high production cost compared to the commercial value. However, some approaches to combat this problem are use of low-cost substrates, optimization of fermentation conditions and development of high yielding strains (Rehm, 2010). On the other hand, unique properties of bacterial exopolysaccharides as compared to the traditional polysaccharides (plants) reduce the production cost by producing high quality products that aids valuable applications (Costerton et al., 1999). The foremost applications of some of these natural polymers are enlisted in Table-1.
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Table-1: Microbial EPS and their applications (Moscovici, 2015; van Kranenburg et al., 1999).
Biopolymer Components Microbial Source Major Applications
Levan Fructose Bacillus, Streptococcus, In sweet confectionary (viscosifier and stabilizer) Pseudomonas, Zymomonas In confectionary products (moisturizer and viscosifier) Dextran Glucose Leuconostoc mesenteroides In medicine (blood plasma extender, cholesterol lowering agent and micro-carrier) In food industry (viscosifier, stabilizer, emulsifier) Glucose, Mannose In crude-oil recovery Xanthan and Glucuronic Xanthomonas In paints, pesticides and detergents, pharmaceuticals and acid cosmetics Guluronic acid In immobilization matrix Pseudomonas aeruginosa and Alginate and Mannuronic In coating of roots of seedlings and plants to prevent Azotobacter vinelandii acid desiccation In human medicine (heal burns or surgical wounds) Cellulose Glucose Acetobacter spp. In nutrition (natural non-digestible fibers) In audio-visual equipment (acoustic membranes)
Rhizobium meliloti and As a gelling agent Curdlan Glucose Agrobacterium Radiobacter In immobilization matrix Glucuronic acid and Streptococcus In ophthalmic surgery (replacer of eye fluid) Hyaluronic acid N-acetyl equii and Streptococcus In wound healing glucosamine zooepidemicus In cosmetic industry (lotions, moisturizing agent)
4 1.2.4 Classification of EPS
On the basis of structure and monomeric composition of bacterial EPS, it is divided into two classes: heteropolysaccharides and homopolysaccharides.
1.2.4.1 Bacterial Heteropolysaccharides
These polysaccharides are made up of various sugar residues such as fructose, glucose, galactose, rhamnose and some charged groups such as phosphate, acetate or glycerolphospate. Glycosyltransferases synthesize these types of heteropolysaccharides at
-1 the cytoplasmic membrane of prokaryotes in amounts below 100 mgL (Welman and
Maddox, 2003; Bounaix et al., 2009). In general, heteropolysaccharides are acidic
(anionic) in nature because of the presence of different monomeric sugars and non- carbohydrate components. There are many enzymes which are responsible for the synthesis of heteropolysaccharides. These sugar residues are mainly produced by lactic acid bacteria in which Lactobacillus sp. (Oleksy and Klewicka, 2016), Lactococcus strains (Cerning, 1990) and Streptococcus sp. (Stingele et al., 1996) are predominant.
1.2.4.2 Bacterial Homopolysaccharides
Homopolysaccharides are composed of same kind of repetitive units of sugar monomers containing majority of pentose and hexose moieties. These sugar components mainly formed two kinds of polymers either glucan or fructan by polymerizing glucose or fructose residues respectively (Monsan et al., 2001). Both polymers differ from each other on the basis of type or degree of branching. The single type of sucrase enzyme is necessary for the formation of bacterial homopolysaccharides. They are biosynthesized by the action of donor (sucrose) and acceptor (polymer chain, sucrose, raffinose, or water) molecules in the presence of sucrose as a substrate on the outside of the cell (van Hijum, 2004).
5 1.2.4.2.1 Glucans
Glucans are polymers of glucose entities known as an important constituent of bacterial and fungal cell walls. They are catalyzed by glucansucrases that cleave sucrose into two subunits (glucose and fructose) and synthesize glucan polymer by adding glucosyl unit to a growing glucan chain. There is a range of bacterial glucans because of the considerable variation in the type of branching, degree of association, linkages, size and conformation of glucan chains that make them different from each other in solubility and various other physical characteristics (Monchois et al., 1999).
Different kinds of glucans are produced by lactic acid bacteria containing α- glucopyranosyl moieties with various types of linkages (Figure-1). These include:
(a) Dextran: A backbone of α-(1→6) linkage with different α-(1→2), α-(1→3) and
α-(1→4) branching points formed by L. mesenteroides, and Lactobacillus hildegardii
(Funane et al., 2001; Maina et al., 2008).
(b) Mutan: A linear unbranched polyglucose with α-(1→3) linkage produced by
Streptococci sp.
(c) Alternan: D-glucopyranosyl residues linked by alternating α-(1→6) and α-
(1→3) bonds with α-(1→3) branch linkages in L. mesenteroides.
(d) Reuteran: It is composed of α-(1→4) glucosidic bonds with α-(1→6) branch linkage synthesized by Lactobacillus reuteri (van Leeuwen et al., 2009).
(e) Glucan: It is a linear polysaccharide main branch bonded by α-(1→2) with α-
(1→6) linkage formed by L. mesenteroides strains (Van Geel-Schutten et al., 1999).
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Figure-1: Schematic figure of the repeating moieties of Dextran, Mutan, Alternan, Glucan and Reuteran (Korakli and Vogel, 2006).
1.2.4.2.2 Fructans
Bacterial fructans are the chain of fructose molecules (polymer) which are produced by fructosyltransferases (FTFs). They are belonging to the second class of sucrase type of enzymes that breaks sucrose and forms fructan polymer by coupling a fructosyl unit to a growing chain of fructan residues, sucrose or water (He et al.,
2015).
FTF enzymes can be produced by both natural sources of enzymes that are plants and microorganisms. Plants require two types of FTF enzymes for the synthesis of fructan. The first enzyme is responsible for the cleavage of sucrose as well as addition of fructosyl unit to another sucrose molecule which makes the primer for the second FTF enzyme that connects the fructosyl unit of sucrose to the primer moiety
7
(Vijn and Smeekens, 1999). In bacteria, fructan is synthesized in a similar manner but it requires single FTF enzyme to cleave substrate and primer synthesis which further elongates the chain of fructose residues that synthesize the polymer (Velázquez‐ Hernández et al., 2009)
Fructans may contain different types of linkages i.e. -(2→1), -(2→6) with
-(2→1→6) branching points of various combinations. Those fructans whose backbone is composed of -(2→6) fructosyl units are known as levan (Figure-2A).
However, the chain of fructans having -(2→1) linked fructosyl units are known as inulin (Figure-2B). Mostly, low molecular weight (ranging from 4 to 10 kDa) fructans of inulin are predominantly produced by plants, whereas, bacteria synthesize high
4 molecular weight (ranging from 10 to 10 kDa) fructans of both levan as well as inulin (Vijn and Smeekens, 1999).
(A)
T
(B)
Figure-2: Figure shows the structure of (A) Levan and (B) Inulin comprises of fructan backbone with their branching points (Cuskin, 2013).
8 1.3 Background of Levan
Levan is a non-structural polymer of fructose residues linked via -(2→6) carbons present in numerous microorganisms and a small number of plant species. This is a nano sized molecule of spherical form that provides it a better stability and significantly low intrinsic viscosity rather than of linear molecules (Arvidson et al., 2006).
The story of levan was initiated by a traditional Japanese food named as
‘natto’ which was known for its health benefits. Levan was presumed as one of the most attractive component of natto for the researchers which were engaged with health promoted natural products. Lippmann is considered as the first who coined the term “lävulan” for the gum (levan) retrieved from molasses in the sugar beet industry
(Lippmann, 1881). Later on, in 1902, an Australian bacteriologist Greig-Smith introduced the term “levan” when he discovered a levorotatory compound having similar properties as dextran.
1.3.1 Microbial Levan: The Chemical Structure
Levan is one of the natural fructose polymer which is mostly associated with -(2→6) linkages having D-glucosyl residue at the end of the chain (Cuskin, 2013; Han, 1990)
(Figure-2A). Hence, it is also termed as (2→6)--D fructans. Despite of its linear structure, most of the bacterial levans are branched through -(2→1) bonds. These are short branch chains usually made up of only one fructose unit. Moreover, levan is the derivative of sucrose as it represents a series of homologous oligosaccharides and polysaccharides.
Generally, levan is produced by various organisms which consist of homologous morphology. The difference between these organisms lies in the variety of monomeric
units and repeating units at branch points. In levan, the carbohydrate is 9 present in the furanose form which is the quality of few natural polymers. The significance of this property is that it helps in the final molecular conformation of polymer (Gupta et al., 2011).
1.3.2 Properties of Levan Polysaccharide
1.3.2.1 Physical and Biochemical Characteristics
Levan is an unusual crystalline polysaccharide of white in colour with strong adhesive properties. The distinctive nature of this biopolymer lies in its morphological characteristics such as compact spherical structure, about 75-200 nm in diameter which do not swell in water. Generally, the properties of levan are similar to another polymer named as dextran. Levan belongs to the group of levorotatory polysaccharide that exhibits amorphous nature with varying solubility in solutions. It is considered to be readily soluble in hot water but insoluble in absolute alcohol. This property of high solubility is due to the branching criteria of levan that is -(2→6) linkage instead of
-(2→1) bond (Srikanth et al., 2015). Moreover, levan is non-reducing polymer which is easily hydrolyzed by acid but restricted to enzymatic hydrolysis by the action of amylase and invertase. The biochemical characteristic of levan reveals that it does not support any colour by iodine but hydrogen chloride makes a purple indication that differentiates it from other fructose free polysaccharides. The viscosity and molecular weight of this biopolymer (levan) distinctly increases in aqueous solutions of various salts but it reduces when the temperature rises (Kang and Cottrell, 1979).
1.3.2.2 Biological Characteristics
The stimulation and inhibition of tumor as well as permeability of cell for a cytotoxic agent directed much attraction of researchers to the biological properties of biopolymer. These features may be triggered by suppression of normal inflammatory
10 7 response. The levan with molecular weight of 10 encourages infection but when polymer degrades its effect becomes disappeared. According to the study, the virulence of bacteria by intraperitoneal injection increases when levan is administered intravenously to mice. This effect may be due to the permeability and inhibition of release of blood components into the peritoneal area (Leibovici and Stark, 1985).
1.3.3 Applications of Levan
There is a great attraction towards levan due to its exclusive properties that makes it different and unique from other polysaccharides. However, the use of levan based items in commercial market has been limited but its increasing demand in the market directed many companies to produce them in large quantities. Following are the few applications of levan.
1.3.3.1 Medical and Pharmaceutical
One of the most interesting medical application of levan is its use as a levan based thin films for the healing treatment of burned or damaged tissues. Sturzoiu et al. (2011) reported the role of levan in the healing of burned or damaged tissues by activating metalloproteinase. Moreover, the polysaccharide levan has also performed a dominating role in laser based technologies. In 2011, Sima et al. prepared films with a gradient of levan and oxidized levan for the targeting of drugs, micro-sensors and tissue regeneration by using matrix-assisted pulsed laser evaporation (MAPLE). It has been suggested that levan derivatives like phosphated, sulphated and acetylated levan can be used as an anti-
AIDS agent. In a drug delivery system, levan functions as a coating substance in its preparation (Gupta et al., 2011). In addition, levan has the ability to restrict the adherence of leucocytes to the blood vessels during an inflammatory response thus helps in the reduction of inflammation (Sedgwick et al., 1984).
11
Another unique feature of levan is the suppression of weight as it has been proved when it was added in the diets of laboratory rats. As a result of this, a significant loss in white fat development, serum triglycerides, free fatty acids as well as cholesterol was also observed (Kang et al., 2004). Moreover, different studies showed the impact of levan on the reduction of cholesterol level which demonstrates its beneficial effect on health (Belghith et al., 2012).
1.3.3.2 Prebiotics
Prebiotics is the substance that nourishes the growth of healthy and beneficial bacteria in the colon and restricts the harmful microorganisms. The role of levan or levan type fructooligosaccharides on intestinal flora and probiotic microbes have been investigated by many researchers. Among them, one of the study showed the reshaping of microbiota in human fecal samples through microbiome and metabolomic analysis (Adamberg et al., 2015). Furthermore, other studies have been reported that the levan showed beneficial effects on the intestinal flora of farmed animals (Li and Kim, 2013; Zhao et al., 2013).
1.3.3.3 Personal Hygiene
The outstanding properties of levan makes it an effective moisturizer having several properties such as it is anti-inflammatory, counteracts irritation, reduces the darker pigmented patches on the skin and increases the skin firmness by smoothing and eliminating the wrinkles on the skin (Kim et al., 2004; Kim et al., 2005).
The major application of levan in hygiene and cosmetology of hairs is as a hair holding and fixative products. In order to increase the hair holding properties, levan solution is added to the existing hair maintenance products such as fixative gels, mousses, muds, hair spray resins and anti-frizz solutions. Moreover, a levan based
12 surfactant with cationic substituents is developed by Rhodia Inc. for the utilization in the formation of a conditioning shampoo (Gunn et al., 2009).
1.3.3.4 Levan in Marine Aquaculture
The prevention of fish diseases and environmental stress in aquaculture requires the use of antibiotics, drugs and chemicals. Unfortunately, these substances exhibited limited effectiveness with environmental stress by bioaccumulation. Therefore, researchers are keen to substitute prophylactic antibiotics with harmless alternatives such as immunostimulants in order to stimulate non-specific immunity of aquatic fish species (Siwicki et al., 1994). Several immunostimulants are being used which are based on carbohydrate polymers and their derivatives. According to different studies, microbial levan also possess immune stimulating activities in aquaculture
(Rairakhwada et al., 2007).
1.3.3.5 Levan as a Packaging Films
The safe, healthy and environmental friendly packaging requires bio-based packaging materials for food industry (Vijayendra and Shamala, 2014). Packaging films composed of only levan are extremely hard for practical use. This brittleness can be overwhelmed by the addition of clay and other plasticizers. An excellent oxygen barrier is another exclusive property of levan that makes it tempting to be used as a packaging material (Chen et al., 2014).
1.3.3.6 Levan in Food Industry
In food industry, levan has been used in the replacement of fat (Roberts and Garegg,
1998). A patent application explains the enhancement of flavor, texture and mouthfeel of dairy items due to the fat like properties of fructans (Booten et al., 1998). The benefits of levan to be used as a fat substitute lies in its distinctive features such as
13 high molecular weight and volatile nature. Therefore, levan is too large as it cannot be perceived by taste makers and its volatility is too low which cannot emit noticeable aromas. Levan can also be used as a carrier for micronutrients in the form of nanoparticles with the coating of cobalt, selenium and iron. (Bondarenko et al., 2016).
It has been also demonstrated that the shelf life of bread can be increased by using levan in the form of microgel (Jakob et al., 2012)
1.3.3.7 Additional Applications
Despite of levan applications in a wide range of sectors such as food, feed, cosmetics and pharmaceuticals, some more uses of this natural polymer have been documented by other industries especially chemical and biotechnological industries. Levan has multifarious applications in industries such as in domestic use as a surfactant, in separation of proteins as an aqueous two-phase system and in gel filtration assembly as a molecular sieve (Srikanth et al., 2015). Unfortunately, the industrial usage of levan is limited due to several challenges which mainly include weak polymer stability in a chemical solution and its purification scheme which is highly sophisticated (Gupta et al., 2011).
1.4 Industrial Production of Levan
Levan highlights numerous applications in various industrial and technological firms.
With respect to current and future prospects of levan, it is very crucial to find out its optimization of production as well as alternative sources in order to boost several sectors (Dos Santos et al., 2013). The industrial and biological importance of this natural polysaccharide has been the subject of an inclusive research to improve enzymatic activities. Hence, the role of its catalyzing enzyme (levansucrase) which belongs to the group of fructosyltransferases is necessary to understand.
14 1.4.1 Fructosyltransferases
In nature, fructooligosaccharides are produced by the catalytic action of enzymes which possess activity of transfructosylation. These enzymes are termed as 1F- fructosyltransferases [FTases, E.C. 2.4.1.9, E.C. 2.4.1. 99, and E.C. 2.4.1.100], or 3- fructofuranosidases [FFases, E.C. 3.2.1.26] (Yun, 1996). Fructosyltransferases are mainly present in various higher plants and are also produced by few microbes such as
Aspergillus sp. and Aureobasidium sp. The enzymatic and physicochemical parameters of fructosyltransferases such as specificity of substrate, molecular mass or stereoselectivity are different from each other on the basis of source or even strain
(Mutanda et al., 2014).
Plant fructosyltransferases investigated that they can synthesized oligosaccharides that are structurally different from each other. The fructooligosaccharides formed by these enzymes are comparatively small. There is a limited production of plant fructosyltransferases due to seasonal environmental conditions (Antošová and Polakovič, 2001). Microbial fructosyltransferases are polymerases which are involved in the formation of microbial fructans such as levan, inulin and fructooligosaccharides. In structural point of view, microbial fructosyltransferase proteins are grouped in seven clusters which are phylogenetically related to each other. Moreover, these proteins also share the catalytic domain of glycoside hydrolases 68 family (Velázquez-Hernández et al., 2009).
In general, the reaction catalyzed by fructosyltransferase involves in the transfer of a fructosyl group to sucrose or raffinose to a variety of acceptor molecules.
Some of acceptor molecules include water (sucrose hydrolysis), sucrose or raffinose
(production of tri or tetrasaccharide respectively), different monosaccharides and disaccharides (Yang et al., 2016a).
15
In bacteria, fructosyltransferases include two enzymes that are levansucrase and inulosucrase which catalyzes the formation of levan and inulin polymers respectively (van Hijum, 2004).
1.4.1.1 Levansucrase
An extracellular enzyme, levansucrase (Lsc) or sucrose: 2, 6-β-D-fructan: 6-D-FTF
[E.C.2.4.2.10] belongs to the superfamily of β-fructosidase. This enzyme consists of five bladed β-propeller fold which has the ability to catalyze the hydrolysis of sucrose into glucose and fructosyl moieties with the release of glucose and polymerization of fructosyl residues to form levan polymer. This saccharolytic enzyme not only plays an important role in the formation of levan polymer but also contributes to the primary metabolic pathways for the bacterial utilization of sucrose in the form of an energy source (Han, 1990).
From the production point of view, it can be isolated by both Gram’s positive and Gram’s negative bacteria which mainly includes Z. mobilis, Leuconostoc mesenteroides, Bacillus subtilis and Pseudomonas syringae (Song et al., 1999;
Morales-Arrieta et al., 2006; Seibel et al., 2006b and Hettwer et al., 1998). There is an intriguing fact that both Gram’s negative and Gram’s positive bacteria varying from each other on the basis of biochemical and genetic parameters like enzymatic characteristics, peptide categorization, protein localization and transcriptional activation or inhibition. According to the study, the levansucrase protein named as
LevU of Z. mobilis was associated with membrane. It was also elucidated from mutational analysis of levansucrase that the sucrolytic activity of this enzyme could be uncoupled from its activity of polymerization (Song et al., 1999).
16
Levansucrase belongs to the glycoside hydrolase family 68 (GH 68) enzymes that cleave the glycosidic bonds between the sugar residues in carbohydrates
(Koshland and Stein, 1954). Two sugar binding subsites, subsite +1 and subsite -1 constitutes the active site of GH68 family whose nomenclature is given by Davies et al. 1997. GH 68 enzymes break the glycosidic bond between subsite -1 and subsite +1 by double displacement mechanism (Sinnott, 1990).
1.4.1.2 Reaction Mechanism of Levansucrase
Levansucrase, a fructosyltransferase enzyme that catalyzes four types of reactions including exchange, hydrolysis, transfructosylation and polymerization depends on the kind of acceptor molecule. The exchange reaction occurs when the fructose component of sucrose transfers to a free glucose (Hettwer et al., 1995) but when the enzyme transmits the fructosyl residue of sucrose to water molecule then hydrolysis reaction takes place (Yun, 1996). Both sucrose as well as sucrose analogues can be used by levansucrase as they are kinetically favorable. The energy is released by the breakdown of sucrose which is enough for the transferring of fructosyl unit of sucrose to another sucrose or acceptor molecule. As a result of which, a high energy bond is formed which is known as glucopyranosyl-fructofuranosyl linkage (Figure-3)
(Chambert et al., 1974; Seibel et al., 2006a)
Figure-3: Schematic representation of hydrolysis of sucrose into glucose and fructose by levansucrase (Strube, 2011).
17
The levan polymer as well as levan-type or inulin-type FOSs are synthesized in the third type of reaction named as transfructosylation (Euzenat et al., 1998;
Trujillo et al., 2001). Levansucrase has the ability to produce a large number of hetero-FOSs when other acceptors are present such as monosaccharides (Baciu et al.,
2005), disaccharides (Park et al., 2003), sucrose analogues (Seibel et al., 2006b), oligosaccharides (Meng and Fütterer, 2008), glycerol (Gonzalez-Munoz et al., 1999) or methanol (Kim et al., 2001) (Figure-4).
Figure-4: Schematic depiction illustrates the acceptor reaction catalyzed by levansucrase (Strube, 2011).
1.4.1.3 Transfructosylation Reaction
The amino acid residues located at the subsite +1 are diverse between Gram’s positive and Gram’s negative bacteria as described by the 3-D structures and the sequence alignment of glycoside hydrolase family 68. However, these differences do not describe the selectivity of reactions (hydrolysis, transfructosylation and polymerization) and even not the formation of end products (Hernandez et al., 1995).
The reaction occurs either in the proportionate or disproportionate manner. The high molecular mass levan is synthesized by the levansucrase of B. subtilis (Chambert et al., 1974), B. megaterium (Homann et al., 2007) and Lactobacillus reuteri (Ozimek et al., 2006a; 2006b) through the proportionate method. In this process, substrate
18 remains attached to the enzyme in polymerization stage while the chain becomes elongated in a continuous fashion. On the other hand, levansucrase of Z. mobilis synthesize the short-chain fructooligosaccharides such as ketose and nytose
(Hernandez et al., 1995). This type of reaction must be processed in a disproportionate manner such that the polymerized chain released from the active site of enzyme after each transfructosylation step (Kralj et al., 2008).
1.4.1.4 Specificity of Levansucrase for Donor and Acceptor Residues
It has been reported that the subsite +1 and subsite -1 of the active site of levansucrase has different specificities for acceptor molecules. The subsite +1 is highly specific for glucose (from sucrose or raffinose), fructose (sucrose residue), glycopyranosides (from galactose, xylose, mannose or fucose) and disaccharides (from maltose, lactose) whereas, subsite -1 shows its affinity towards fructose moieties only (Hernandez et al., 1995; Song et al., 1999; Seibel et al., 2005). Therefore, this specific feature provides the opportunity to levansucrase to use a number of substrates as fructosyl donors like sucrose, analogues of sucrose, raffinose or even stachyose (Park et al., 2003; Seibel et al., 2006b; Meng and
Fütterer, 2008). There are various qualities of sucrose that makes it most suitable as a substrate which mainly includes vast accessibility, high purity, cheap, and high energy glycosidic bond (Monchois et al., 1999). This bond energy of sucrose is significantly very high as it has been suggested that the Gibbs energy value essential for the hydrolysis of sucrose is higher than other saccharides which is about ΔGoA = -26.5 kJ/mol (Goldberg and Tewari, 1989; Tewari and Goldberg, 1991).
1.5 Purification of Levansucrase
There are various salts, solvents and polymers available for the partial purification of
levansucrase which mainly incude ammonium sulphate (van Hijum et al., 2001),
manganese chloride (Vigants et al., 2001) protamine sulphate (Euzanat et al., 1998),
19
polyethylene glycol (Oseguera et al., 1996) and acetone (Ammar et al., 2002).
Nevertheless, it is also very essential that levansucrase must be purified by means of
chromatography techniques such as gel filtration or anionic exchange
chromatography. In size exclusion chromatography, the columns that have been used
for the purification of levansucrase are sephacryl S-300 (Cote et al., 1989; Vigants et
al., 2003; Goldman et al., 2008), sephadex (Tanaka et al., 1979) and (TMAE)-
Fraktogel (Hettwer et al., 1995). The anionic columns which are commonly used in
the purification technique of levansucrase are hydroxyapatite (Gonzy-Treboul et al.,
1975) or DEAE-cellulose (Esawy et al., 2008) columns. SDS-PAGE is a technique
which employs for the assessment of levansucrase purity and its molecular weight.
The enzyme exists in a monomer form with the molecular weight of around 55 kDa
(Sangiliyandi et al., 1998; Abdel-Fattah et al., 2005; Tanaka et al., 1978). Whereas,
there are some microbial sources which revealed that the levansucrase present in
dimer form (Goldman et al., 2008) with the molecular weight of about 94 kDa
(Yanase et al., 1992; Ohtsuka et al, 1992). Some studies speculated the increase
molecular weight of levansucrase which might be due to the association of product
(levan) to the enzyme (Crittenden and Doelle, 1994; Hettwer et al., 1995; Oseguera et
al., 1996).
1.6 Structural and Functional Analysis of Levansucrase
According to the amino acid sequencing of fructansucrases, its overall structure is
categorized into four different regions (Figure-5) which includes:
(a) Signal peptide (b) N-terminal region having variable length (c) Conserved stretch
of catalytic core region composed of 500 amino acids shared by all family members of
GH68 (carbohydrate-active enzyme website: http://afmb.cnrs-mrs.fr/CAZY) (d) C- terminal variable region, sometimes attached with a cell wall binding domain (LPXTG). 20
Figure-5: Schematic illustration of fructansucrase protein from lactic acid bacteria. This figure depicts four different regions (i) N- terminal signal sequence (ii) N-terminal variable region (iii) Catalytic core (iv) C-terminal region attached with LPXTG cell wall (Côté and Robyt, 1982).
21 1.6.1 Signal Peptide and N-terminal variable Region of Levansucrase
There is an N-terminal signal sequence which allows these enzymes to secrete as bacterial fructansucrases belong to the extracellular enzymes (Figure-5). Upon secretion of fructansucrases by Gram’s positive bacteria, the signal peptide containing precursor is broken down (Bezzate et al., 2000). The size and length of N-terminal domain diverges between fructansucrases. The function of this domain has not been assigned.
1.6.2 Catalytic Domain of Levansucrase
1.6.2.1 Catalytic Residues of Levansucrase
The structural description of SacB levansucrase as well as its inactive mutant by B. subtilis has been described by high resolution crystallography. These structures revealed that levansucrase possesses an unusual five-fold propeller topology composed of a negatively charged and deep central pocket (Meng and Fütterer, 2003).
The residues of central pocket consist of highly conserved sequence elements which include mainly glutamic acid and aspartic acid residues that are Asp86 (Figure-5A),
Asp247 (Figure-5E), and Glu342 (Figure-5G). In 2005, the three dimensional structure of levansucrase of Gluconacetobacter diazotrophicus was documented
(Martínez-Fleites et al., 2005). The levansucrase structure of G. diazotrophicus and B. subtilis represents the same five bladed propeller fold. Levansucrases of both organisms are structurally related to each other as their three dimensional positions of catalytic residues are superimposable.
22 1.6.2.2 Catalytic Site of Levansucrase
The three-dimensional structures of B. subtilis SacB (Meng and Futterer, 2003) and
G. diazotrophicus LsdA (Martínez-Fleites et al., 2005) revealed that levansucrase constitutes two subsites that are +1 and -1 for the binding of substrate (sucrose) in the active site (Davies et al., 1995). In short, the sucrose cleaves between +1 and -1 subsites forming a covalent intermediate (fructose) at subsite -1. After that, this sucrose coupled to the acceptor molecule (fructan). In GH68 levansucrase family, the subsite +1 is different between Gram’s positive and Gram’s negative bacteria (van
Hijum et al., 2002).
1.6.2.3 Topology of Levansucrase Active Site
The specificity and mode of action of glycoside hydrolases depends mainly upon the topology of the active site of enzyme. Glycoside hydrolases revealed three main active site topologies which include open cleft, pocket topology and tunnel topology.
In open cleft topology, internal regions of polysaccharides are present which makes enzyme as an endo-enzyme. Pocket topology usually provides the space for monosaccharide. However, in tunnel topology, the chain of biopolymer proceeds through the substrate binding tunnel which is the exciting feature of processive exo- enzymes (Figure-6). Therefore, a list of oligosaccharides is formed by its endo-mode of action, monosaccharides are released by exo-acting enzymes while exo-acting processive enzymes usually involves in the production of monosaccharides (Davies and Henrissat, 1995).
23
Figure-6: Topology of glycoside hydrolase. A, B and C indicated as Open cleft, Pocket and Tunnel respectively. (Active sites are represented in red color). (Davies and Henrissat, 1995).
1.6.2.4 Calcium Binding Site of Levansucrase
There is an evidence that a bound metal ion, probably calcium exists in the levansucrase by the analysis of the three dimensional structure of B. subtilis (Meng and Fütterer, 2003).
+2 It has been discovered that the acidic amino acid i.e. Asp339 coordinates the Ca ions in the enzyme structure. In order to observe the role of Asp339 residue, mutational analysis of corresponding amino acid residues in the levansucrase (Asp500Asn) and inulosucrase
(Asp520Asn) of L. reuteri has been carried out (Ozimek et al., 2005). The results of both mutants indicated that the optimal temperature decreases as well as there is a significant
+2 reduction in the affinity for Ca binding. Moreover, levansucrase is dependent on calcium ions for the activity of enzyme (Jacques, 1984). It has been revealed by the sequence alignment of GH68 family members that most of the enzymes in Gram’s positive bacteria has conserved residues for the binding of calcium ions but are absent in the enzymes of Gram’s negative microbes (Ozimek et al., 2005). The three dimensional structure of Gram’s negative bacteria (G. diazotrophicus) found that it contains a disulphide bridge which
24 has a similar role as the calcium binding site plays in Gram’s positive bacteria of fructansucrases (Martínez-Fleites et al., 2005).
1.6.2.5 C-terminal Domain
It was claimed that the product size or specificity of enzyme can be affected by the C- terminal region of fructansucrases. This has been recognized by an observation that a larger fructan polymer (levan) with an increase number of branches is produced by the levansucrase of B. subtilis (Chambert et al., 1992). A distinct function of C-terminal domain of fructansucrases is that it helps enzymes in the adhesion to the cell wall of producer microbe. Both fructansucrases i.e. levansucrase (Van Hijum et al., 2004) and inulosucrase (Van Hijum et al., 2002) of L. reuteri 121 consists of a common C- terminal cell wall-anchoring element named as LPXTG (Navarre and Schneewind,
1994) (Figure-5). The bacterial surface exhibits protein that performs a variety of functions in microbial cell like infection process and adhesion of tissue cells (Ton-
That et al., 1997; Sillanpää et al., 2000). It has also been shown that homopolysaccharides produced by the sucrase enzymes anchored to the cell surface might also play a role in the adherence of microbes to the surface including teeth and intestinal lining (Rozen et al., 2001)
25
CHAPTER 2 Research Design
The ultimate goal of current investigation is to obtain enhanced production of
levansucrase biosynthesized from Z. mobilis KIBGE-IB14, its purification and kinetic
evaluation which provides improved enzyme with pivotal biochemical and molecular
information desirable for multifarious applications of levansucrase in different
industrial segments.
In consideration of above target, the current research was planned with the following
objectives:
Isolation and screening of bacterial isolates for the hyper production of
levansucrase. Optimization of different physical and chemical fermentation conditions for
maximum production of levansucrase using one variable at a time approach. Partial purification and desalting of levansucrase required for characterization
process. Purification and characterization of levansucrase to determine its kinetic
behavior for industrial utilization. Amino acid analysis of levansucrase which provides quantitative parameter in
the characterization of enzyme. The whole research strategy is demonstrated in flow chart scheme:
26
2.1 Schematic representation of production, characterization and purification of levansucrase
27
Chapter 3 Materials and Methods
3.1 General Section
The required instruments used in this research study is mentioned in Appendix-I. The analytical grade chemicals and reagents which were purchased from Sigma, Merck,
Oxide and BDH is detailed in Appendix-II. Initially, all glasswares were washed and scoured by cleansing agent and bristle brush respectively. Subsequently, they were washed several times with tape water and rinsed with double deionized water at the end of washing procedure. Thereafter, these washed glassware were oven-dried before its usage for practical procedures. In the whole experimental work, double deionized and MilliQ water was used to formulate all chemicals and buffer solutions.
3.2 Experimental Session
The bacterial strain used in this study was obtained from microbial culture bank of
KIBGE, University of Karachi, Pakistan by follow the procedure described below:
3.2.1 Sample Collection, Screening and Isolation of Levansucrase Producing Strains
Several bacterial strains were isolated from different samples. All isolated strains were screened for levansucrase production on Yeast Peptone Dextrose (YPD) agar medium.
Among these natural isolates, the strain that showed the maximum levan production was selected. After selection, the strain was further identified by morphological, biochemical (Holt et al., 1994) and molecular analysis.
3.2.1.1 Sample Collection
The bacterial strains were isolated from molasses and soil samples of different sugar refineries and vegetative fields located in Karachi, Pakistan, respectively.
28 3.2.1.2 Isolation and Screening of Levan Producing Strain
For the isolation of bacterial culture, 1.0 g molasses and soil were weighed and -1 -1 incorporated in 9.0 ml of normal saline (8.5 g L NaCl) preparing a tube of 10
-1 -6 dilution. Serial dilutions of 10 to 10 were prepared by shifting 1.0 ml solution
-1 -6 from 10 dilution tube to other consecutive tubes up to 10 dilution in a step wise manner. The culture sample (0.1 ml) was transferred to YPD agar plate from the last -5 -6 two dilution tubes (10 and 10 ) which were then spread on agar plate by the help of sterilized glass rod (Table-2). After 24 hours of incubation, morphologically different colonies were observed on plate. All of these contrasting colonies were further incubated for 24 hours to screen out the maximum levan producing strain.
Table 2: Composition of YPD agar medium
Medium Components* %
Yeast extract 0.3
Peptone 0.1
Dextrose 2.0
Agar 2.0
*pH of medium was adjusted to 6.0±0.2 before sterilization.
3.2.1.3 Culture Identification
After purification of levan producing strain by consecutive streaking on YPD agar medium, the strain was identified according to “Bergey’s Manual of Determinative
Bacteriology” (Holt et al., 1994) on the basis of morphological and biochemical analysis. For further confirmation of the strain 16S rDNA sequence analysis was performed.
29 Morphological Analysis
In morphological analysis, bacterial colony, cell morphology and spores arrangement were observed for the identification of bacterial isolate. Colony Morphology
The culture was grown on YPD agar plate at 30°C for 24 hours to examine its morphological characteristics. The different morphological properties of culture colonies which mainly include color, surface, shape, margin and elevation were analyzed. Cell Morphology
The Gram’s staining method was used for the investigation of type, shape and arrangement of bacterial cell culture (Gephart et al., 1981). In this method, a drop of
-1 normal saline (8.5 g L NaCl) was mixed with bacterial culture on the center of glass slide which then spread by the help of wire loop allowing it to dry in air and heat fixed. The culture smear was stained by using Gram’s staining solutions (Appendix-
III). After staining, the microscopic examination of culture was achieved by observing the slide in microscope. Arrangement of Spores
The presence and location of spores were analyzed by spore staining technique following modified method of Schaeffer and Fulton (1933) (Appendix-IV). Biochemical Analysis
In biochemical characterization, the bacterial isolate was identified by performing different biochemical tests like catalase, oxidase, nitrate reduction, MP-VP and citrate utilization test. The sugar fermentation reaction and gas production was observed by
30 testing selective sugars such as glucose, xylose, sucrose, maltose, fructose, galactose and lactose. The growth of bacterial culture was also monitored by inoculating the -1 culture on sabouraud dextrose agar plate as well as NaCl (70.0 g L ) containing medium (Holt et al., 1994). The late logarithmic phase of the bacterial isolate was selected for performing all of these biochemical tests. 16S rDNA Sequence Analysis
The bacterial isolate was confirmed on molecular basis by performing 16S rDNA sequence analysis. The DNA was isolated by using genomic DNA extraction kit method. The presence of isolated DNA was confirmed by conducting agarose gel electrophoresis and quantify by nanodrop photometer (P-Class IMPLEN, Germany).
The polymerase chain reaction (PCR) was performed for the amplification of DNA gene with universal primers. The sequencing of amplified product was carried out after DNA amplification. Thereafter, the sequence of isolate was compared with other prokaryotic 16S rDNA sequences using similarity rank analysis service which was provided by National Center for Biotechnology Information (NCBI) (http://www. ncbi.nlm.nih.gov/BLAST/). Extraction of DNA
The isolation of bacterial genomic DNA was carried out by using DNA extraction kit
® method with minor modifications (Promega Corpora , USA). The manufacturer’s protocol was used for the preparation of reagents. In order to harvest the overnight bacterial culture, cells were centrifuged at 13,000 ×g for 2.0 minutes. Nuclei lysis solution (600 µl) was added in the genomic DNA and pipetted the sample gently until the cells were resuspended. The sample was incubated at 80°C for 5.0 minutes to lyse the cells and then cooled to room temperature. In this cell lysate, RNase (3 µl)
31 solution was added and mixed well by inverting the tube for 2-5 times. The prepared sample was incubated at 37°C for 15 to 60 minutes allowing it to cool until the room temperature was achieved. Protein precipitation solution (200 µl) was added to the
RNase treated cell lysate and vortex vigorously at high speed for 20 seconds to mix the solution with cell lysate. This sample was incubated on ice for 5.0 minutes and then centrifuged at 13,000 × g for 3.0 minutes. The supernatant having DNA was transferred to a microcentrifuge tube (1.5 ml) containing room temperature isopropanol (600 µl). The sample was mixed gently by inversion until a DNA of thread like strand was visible. The tube was again centrifuged at 13,000 × g for 2.0 minutes. The supernatant was discarded and tube was drained on clean absorbent paper. The room temperature ethanol (70%, 600 µl) was added in the tube and inverted it several times in order to wash the DNA pellet. Again the sample was centrifuged at 13,000 × g for 2.0 minutes. When ethanol was aspirated, the tube was drained on clean absorbent paper to air dry the pellet for 10 to15 minutes. Finally,
DNA rehydration solution (100 µl) was added in the tube to rehydrate the DNA and incubated the solution overnight at 4°C. After rehydration, the DNA was stored at -
20°C. Amplification of Bacterial 16S rDNA
16S rDNA gene was amplified by using bacterial universal primer set with the sequence as follows:
32
Forward Primer: 5'-GAGTTTGATCCTGGCTCAG-3'
Reverse Primer: 5'-AGAAAGGAGGTATCCAGCC-3'
The PCR reaction master mix contained DNA composed of DNA template (50.0 ng), dNTPs mix (200 M), Taq DNA polymerase (2.5 U), primers (0.2 μM) and PCR reaction buffer (10X). The thermal cycling was conducted in BioRad T100™ for
DNA amplification by following steps:
3 Step Protocol Cycle Step Cycles Temperature Time
Initial Denaturation 95°C 5.0 minutes 1
Denaturation 95°C 1.0 minute
Annealing 55°C 1.0 minute 35
Extension 72°C 1.0 minute
Final extension 72°C 10.0 minute 1
Hold
Agarose Gel Electrophoresis
The amplified PCR product was visualized in 1.0% agarose gel which contain 0.5 µg -1 -1 ml final concentration of ethidium bromide (10.0 mg ml ). The electrophoresis
-1 technique was conducted at 50 volts cm using 1.0 Kb DNA ladder (Thermo
Scientific). After electrophoresis, the bands were visualized under UV light in a gel documentation system (Gel Doc 2000, Universal Hood, BioRad Laboratories Inc.,
Italy).
33 Purification and Sequencing of PCR Product
The PCR product was purified through product purifying kit (Wizard SV Gel and
PCR Cleanup System, Promega) by following manufacturer’s guidelines. DNA
Sequencer Genetic Analyzer (ABI-3130; Applied BioSystem) was used for sequencing of purified DNA. Construction of Phylogenetic Tree
Clustal-W program of Mega 6.0 software was used for the sequence alignment of selected strain and construction of phylogenetic tree was carried out by likelihood method with 1000 bootstrap values (Tamura et al., 2013). The sequences used for phylogenetic tree were retrieved from NCBI Genbank database.
3.2.1.4 Preservation of Bacterial Isolate
YPD medium with glycerol (15%) was used to store the purified bacterial isolate at -
20°C. The isolate was also preserved in preservative vials (MW&E Viabank culture bead storage system, Lakewood Biochemical Co. Inc., USA). The bacterial culture was maintained and routinely sub-cultured on YPD agar slants at 4°C.
3.2.2 Production of Levansucrase
3.2.2.1 Selection of Growth Medium for Levansucrase Production
Levansucrase production was carried out by submerged fermentation technique in stepwise manner. Initially, levansucrase production from Z. mobilis KIBGE-IB14 was investigated in various previously reported media formulations. The composition of different investigated media is presented in Table-9. The media were autoclaved at
115°C for 20.0 minutes under 15 lb pressure. In order to investigate maximum enzyme production, a new modified medium was designed by standardization of medium-4 components (Table-4).
34 3.2.2.2 Research Setup to Formulate Modified Medium for Maximum
Production of Levansucrase from Z. mobilis KIBGE-IB14
The maximum production of levansucrase was optimized by varying several physical and chemical conditions which are briefly mentioned in Table-3. All medium components were altered in a step by step manner to achieve optimization. The batch fermentation with one-variable-at-a-time approach was used to select the inducer constituents mainly include carbon and nitrogen sources as well as mineral elements.
35 Table-3: Modified experimental scheme to attain maximum levansucrase yield from Z. mobilis KIBGE-IB14.
Variable Parameters Constant Parameters Range of Variable Physical Factors Parameters
Growth Temperature Step 1 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 20°C-60°C chloride, 0.01; Di-potassium hydrogen phosphate, 1.5 pH-6.5; Incubated for 24 hours. Fermentation Time Step 2 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 6.0-96.0 hours chloride, 0.01; Di-potassium hydrogen phosphate, 1.5 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours. Growth medium pH Step 3 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 5.0-9.0 chloride, 0.01; Di-potassium hydrogen phosphate, 1.5 Growth temperature, 30°C; Incubated for 24 hours.
Macronutrients
Sucrose Step 4 (%): Peptone, 0.2; Yeast extract, 1.0; Calcium chloride, 0.01; Di- 5.0-25% potassium hydrogen phosphate, 1.5 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours.
Nitrogen Source Step 5 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 0.1-0.5% Peptone chloride, 0.01; Di-potassium hydrogen phosphate, 1.5 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours. Yeast extract Step 6 (%): Sucrose, 15.0; Peptone, 0.2; Calcium chloride, 0.01; Di- 0.5-2.0% potassium hydrogen phosphate, 1.5 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours.
36
Micronutrients
(NH4)2SO4 Step 7 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 0.5-2.5% chloride, 0.01; Di-potassium hydrogen phosphate, 1.5 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours.
K2HPO4 Step 8 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 0.5-2.5%
chloride, 0.01 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours.
KH2PO4 Step 9 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Calcium 0.5-2.5% chloride, 0.01; Di-potassium hydrogen phosphate, 1.5 pH-6.5; Growth temperature, 30°C; Incubated for 24 hours.
CaCl2.2H2O Step 10 (%): Sucrose, 15.0; Peptone, 0.2; Yeast extract, 1.0; Di- 0.005-1.5%
potassium hydrogen phosphate, 1.5
pH-6.5; Growth temperature, 30°C; Incubated for 24 hours.
37 The formulated modified medium after the optimization of different physical and chemical parameters is presented in Table-4.
Table-4: Composition of modified medium designed for levansucrase production by Z. mobilis KIBGE-IB14.
Medium Components* %
Sucrose 15
Yeast extract 1.0
Peptone 0.2
K2HPO 4 1.5
CaCl2.2H2O 0.01
*About 6.5±0.2 pH of medium required prior to sterilization
3.2.3 Partial Purification of Crude Levansucrase
The partial purification of levansucrase was carried out by using three different techniques included salt, solvent and polymer precipitation methods. In order to acquire such approach, different precipitating agents were used like ammonium sulfate, ethanol and polyethylene glycol (PEG).
3.2.3.1 Batch Precipitation
This process requires the gradual incorporation of precipitant in cell free fluid with constant stirring at 4°C allowing it to dissolve completely. After the final dissolution of precipitant, this mixture was refrigerated overnight at the same temperature to acquire complete equilibration. Precipitated enzyme was separated from the mixture by centrifugation technique with required conditions of 40248 × g at 4°C for 15.0 minutes. Thereafter, partially precipitated enzyme was re-dissolved in 100.0 mM sodium phosphate buffer of pH-6.0.
38 3.2.3.2 Gradient precipitation
In gradient precipitation, the 10% increment batches of precipitant were repeatedly
added each time to the cell free filtrate until 60% saturation was reached. After each
precipitation step, precipitants were removed to analyze its enzyme activity and total
protein by standard assay procedure.
3.2.4 Desalting of Precipitated Levansucrase
PD-10 pre-packed column (GE healthcare, UK) containing Sephadex G-25 was used
for desalting of partially purified levansucrase. Column equilibration was achieved by
washing it extensively with 100.0 mM sodium phosphate buffer at pH-6.0. After five
times washing of column, 2.5 ml sample was loaded and centrifuged at 4°C, 716 × g
for 2.0 minutes. Enzyme activity and total protein content was determined from eluent
by standard assay method. This eluent was stored at -20°C for further purification.
3.2.5 Ultrafiltration of Precipitated Levansucrase
® Ultrafiltration of levansucrase was done by using Centricon centrifugal filter device
(Ultracel YM-10, Millipore Corporation, USA) of 10.0 kDa. Each time loaded 2.0 ml
sample volume and centrifuged at 4°C, 2,862 × g until desired volume of
concentrated sample was achieved.
3.2.6 Size Exclusion Chromatography
The gel permeation chromatography (Econopump EP-1, Bio-Rad, USA) encompasses the packing of gel i.e. Sepharose CL-6B (GE Healthcare Biosciences AB, Sweden) in a column (1.5x55 cm) of glass. The traces of preservatives from the gel was removed by washing it extensively with deionized water.
39
Equilibration of column was done with 100.0 mM sodium phosphate buffer (pH- 6.0) prior to load the sample. After ultrafiltration, concentrated levansucrase sample (1.0 ml) was obtained. -1 This sample was then loaded on column having flow rate of 0.5 ml min . At the same time, about 0.5 ml of each fraction was collected by the automated fraction collector (2110 Fraction collector, Bio-Rad, USA) associated with the chromatography. Each obtained fraction was evaluated on the basis of its enzyme activity and total protein according to the standard assay protocols. Those fractions that retained their enzyme activity were first pooled and then concentrated by lyophilization.
3.2.7 Characterization of Levansucrase
Enzyme characterization based on kinetic behavior is crucial for determining its
potential commercial applications. The appropriate conditions for catalytic activity of
levansucrase produced by Z. mobilis KIBGE-IB14 was identified by the investigation
of different parameters. The presented data is a mean value of three observations as
the experimental work was conducted in triplicate manner.
3.2.7.1 Reaction Time
An enzyme substrate assay was performed to determine the rate of reaction for
various time intervals ranging from 5.0 to 60.0 minutes while keeping other
conditions constant that is temperature: 35°C; pH-6.0 and substrate concentration:
250.0 mM.
40 3.2.7.2 Temperature Profile
To determine the effect of temperature on activity of levansucrase, an enzyme assay was performed at a range of temperature from 25°C to 65°C while substrate (250.0 mM) and buffer conditions (potassium phosphate buffer, 100.0 mM, and pH-6.0) were kept constant.
3.2.7.3 pH Profile
A range of pH values from 5.0 to 9.0 was investigated by using different buffers to analyze the influence of pH on the activity of levansucrase. These buffers include citrate, sodium phosphate, acetate and glycine-sodium hydroxide buffer with the pH ranging from 5.0-6.0, 6.0-8.0, 7.0-8.0 and 8.0-9.0 respectively (Appendix-V). All investigated buffers share the same ionic strength (100.0 mM) with other conditions such as temperature (35°C) and substrate concentration (250.0 mM) were kept constant.
3.2.7.4 Selection and Ionic Strength of Buffer
The influence of ionic concentration of the selected buffer on levansucrase activity was observed by altering the molar strength of sodium phosphate buffer (pH-6.0) ranging from 25.0 mM to 200.0 mM.
3.2.7.5 Enzyme Kinetics
The maximum rate of reaction (Vmax) for sucrose hydrolysis and binding affinity of sucrose to levansucrase (Km) was estimated by altering the substrate concentration from 0.025 M to 0.4 M. To achieve this goal, substrate (sucrose) was prepared in
100.0 mM sodium phosphate buffer (pH-6.0). Lineweaver-Burk Plot was applied in the calculation of Vmax and Km (Lineweaver and Burk, 1934).
41 3.2.7.6 Effect of Metal Ions
+ + + 2+ 2+ 2+ 2+ 2+ Various monovalent (K , Na and Cs ), divalent (Ba , Co , Mn , Zn , Cu ,
2+ 2+ 2+ 2+ 3+ 3+ Ca , Ni , Hg and Mg ) and trivalent (Al and Fe ) ions were used to investigate their impact on the activity of levansucrase. For this purpose, enzyme was pre-incubated with 1.0, 5.0 and 10.0 mM solutions at 37°C. After 60.0 minutes of enzyme incubation, these samples were retrieved to conduct their enzymatic activity at 35°C. All of the observed metal ions required in this process were in the form of chloride salts. The percent relative activity of levansucrase in the presence of different metal ions was calculated by comparing it with control (enzyme untreated with any metal ion). The activity of enzyme was accomplished by standard assay conditions.
3.2.7.7 Effect of Solvents
The influence of organic solvents on levansucrase activity was determined by incubating levansucrase with various organic solvents such as ethanol, methanol, isopropanol, formaldehyde, chloroform and DMSO. The concentrations of solvents used were 1.0, 5.0 and 10.0 mM for 60.0 minutes at 37°C. Enzyme without exposed to any solvent was considered as control and taken as 100%.
3.2.7.8 Effect of Surfactants
Levansucrase was incubated by several surfactants including Sodium dodecyle sulfate
(SDS), Triton X-100, Tween-80 and Tween-20 with 1.0, 5.0 and 10.0 mM concentrations at 37°C for 60.0 minutes to evaluate their effect exerted on activity of enzyme. Levansucrase free from any surfactant treatment was taken as control and its value was considered as 100%.
42 3.2.7.9 Thermal Stability
The pre-incubation of enzyme at various temperature ranges between 25°C to 55°C was studied to examine the temperature stability profile of levansucrase. For the investigation of thermal stability of enzyme, samples were drawn after 30.0, 60.0,
90.0 and 120.0 minutes interval. After that, percent residual activity of obtained samples were determined. The control was considered as enzyme without any treatment and its value taken as 100%.
3.2.7.10 Storage Stability
The storage stability of levansucrase requires the preparation of enzyme aliquots of
0.5 ml for storing them at various temperatures i.e. 4°C, 37°C, -80°C up to 06 months.
The enzyme activity of each aliquot was observed after every consecutive day by following the standard assay procedures.
3.2.8 Molecular Weight Estimation of Levansucrase
Sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE) was performed using mini-protein II Dual Slab Cell system (BioRad). PageRuler Broad
Range Unstained Protein Ladder (5kDa-250kDa) (Thermo Scientific) was used to reveal the molecular weight of levansucrase. The molecular weight of enzyme was indicated by the comparison of partially purified levansucrase with protein marker of known molecular weight. During this experimental research, the technique of SDS-
PAGE was conducted as the method described by Laemmli (1970) with certain modifications using acrylamide of 4% (w/v) and 12% (w/v) for stacking and separating gels respectively (Appendix-VI).
43 3.2.8.1 Sample Preparation
The sample was prepared by mixing partially purified levansucrase in equal volume
(1:1) with the sample diluting buffer. Modified Laemmli (1970) method was applied to conduct SDS-PAGE technique at constant current of 80.0 mV.
3.2.8.2 Preparation of Gel
The gel composition formulated for SDS-PAGE electrophoresis is covered in Table-5.
Table-5: Composition of electrophoresis gel for SDS-PAGE.
Gel Components Stacking Gel (4%) Resolving Gel (12%)
Solution A 1.66 ml 8.0 ml
Solution B ------5.0 ml
Solution C 1.26 ml -----
Solution E 100 µl 300 µl
Double deionized water 6.8 ml 6.7 ml
SDS (10%) 100 µl 200 µl
TEMED 10 µl 10 µl
3.2.8.3 Visualization of Protein Bands
After performing SDS-PAGE electrophoresis, half portion of the gel was stained by incubating the gel in Coomassie Brilliant Blue R-250 staining solution for overnight with constant stirring. The gel was destained by placing it in the destaining solution until the background becomes clear and protein bands have visualized. Gel
Documentation system (Gel Doc 2000, Universal Hood, BioRad Laboratories Inc.,
44
Italy) with Quantity One Quantitative Software calculated the approximate molecular
mass of levansucrase.
3.2.8.4 Glycoprotein Staining of purified Levansucrase
Purified levansucrase fraction was detected on SDS-PAGE using Periodic acid-
Schiff’s staining method (Siddiqui et al., 2013).
Procedure
After completion of electrophoresis, In-situ levansucrase activity band was detected by staining another half portion of the gel through periodic acid- Schiff staining method with few adjustments. Gel was washed thrice with 100.0 mM phosphate buffer (pH 6.0) containing 0.5% Triton X-100 and 0.005% CaCl2 for 20 minutes and then incubated it in the buffer of same composition with 15% sucrose at 37°C for 18.0 hours. Afterwards, 70% ethanol was used to wash the gel three times with time interval of 15.0 minutes. Gel was incubated in 0.7% periodic acid (freshly prepared) and 5.0% acetic acid solution for 60.0 minutes at 37°C. After incubation, the gel was again washed with 0.2% sodium metabisulphite for 5.0 minutes. To remove the yellow background, gel was washed thrice with double deionized water until the background becomes clear. Subsequently, the gel was stained by placing it in the Schiff’s reagent (Sigma, USA) for 15 minutes in
dark.
45
Gel was washed three times with a solution of 0.6% sodium metabisulphite and 3% acetic acid and then placed on shaking until activity bands (magenta colour) were visualized against cleared background. A PageRuler Broad Range Unstained Protein Ladder (Thermo Scientific) was considered as molecular weight standard to estimate the approximate molecular weight of levansucrase. The computational analysis of molecular weight was revealed by Quantity One Quantitative Software bundled with Gel Documentation system (Gel Doc 2000, Universal Hood, BioRad Laboratories Inc., Italy).
3.2.9 Relative Amino Acid Composition Analysis
Sample was sent to PCSIR laboratories, Karachi, Pakistan for the assessment of
relative amino acid content of purified levansucrase by o-phthalaldehyde (OPA)
derivatization method which is described below: Columns
Analytical column Shim-pack Amino-Na
Ammonia trap column: ISC-30Na Analytical column: Shim-pack Amino-
Li Ammonia trap column: ISC-30Li
Amino Acid Standard Solution
The calibration and detection of fluorescence was carried out by standard solution of
amino acid (Sigma-Aldrich) which contains +4.0% concentration of amino acids
present in 0.1N HCl. The prepared standard solution was stored at 4°C. A list of
amino acids which were used as standards are presented below in Table-6.
46 Table 6: Amino acids standard solution used.
Molecular Concentration Amino Acids -1 Weight (nM ml ) L-Alanine 89.09 25.0
Ammonium Chloride 53.49 25.0
L-Arginine 174.2 25.0
L-Aspartic Acid 133.1 25.0
L-Cystine 240.3 12.5
L-Glutamic Acid 147.1 25.0
Glycine 75.07 25.0
L-Histidine 155.2 25.0
L-Isoleucine 131.2 25.0
L-Leucine 131.2 25.0
L-Lycine 146.2 25.0
L-Methionine 149.2 25.0
L-Phenyalalnine 165.2 25.0
L-Proline 115.1 25.0
L-Serine 105.1 25.0
L-Theronine 119.1 25.0
L-Tyrosine 181.2 25.0
L-Valine 117.2 25.0
47 Geofman Parameters
Oven temperature: 60°C SV = 1: (M.P – A) -1 T-Flow: 0.5 ml minute λ - Em: 450 nm λ - Ex: 350 nm SENS. = 3 (low): MAGNI. = 4 Preparation of Mobile Phase
A-Mobile phase
A-Mobile phase was composed of sodium citrate (0.2 N) with the pH adjustment of
about 3.2 by using perchloric acid having 7.0% ethanol. B- Mobile phase
B-Mobile phase consist of sodium citrate (0.6 N) and boric acid (0.2 M). With the
help of 4.0 N NaOH, final pH of the solution was adjusted to pH-10.0. C- Mobile phase
C- Mobile phase was made up of NaOH (0.2 M).
Preparation of Reaction Solution
Buffer Solution
In buffer solution, 122.1 g of sodium carbonate was dissolved in double deionized
water of 2.5 L. After that, about 70.7 g of boric acid was added and shaken
vigorously.
48 Reaction Solution A
In reaction solution A, sodium hypochlorite (0.2 ml, 7.0-10.0%) was added in to buffer solution (500.0 ml) and was shaken vigorously. Reaction Solution B
First, a solution was prepared by dissolving o-phthalaldehyde (0.4 g) in ethanol (7.0 ml). This prepared solution was added to reaction solution A (450.0 ml) and then finally, N-Acethylcysteine (500.0 mg) was added into it and was shaken vigorously. Acid Hydrolysis
In the ratio of 1:1, purified levansucrase was mixed with HCl (12.0 N) to make a final concentration of 6.0 N HCl. Sample tube that has been vacuumed and sealed was kept at 110°C for 24 hours. The sample was transferred in a round bottom flask after incubation. In rotary evaporator, HCl was evaporated. After several times washing with double deionized water, the volume of the sample was made up to 0.60 ml. Pre- column derivatization was achieved by OPA. The protein sample was stored at 4°C for further analysis. Filtration
Before loading on the amino acid analyzer (LC 10A/C-R7A, Shimadzu, USA), acid treated and washed sample (0.02 ml) was filtered through 0.45 μm filter membrane.
3.3 Analytical Section
3.3.1 Assay for Levansucrase
The reagents prepared for assay of levansucrase is mentioned in Appendix-VII.
49 Procedure
Levansucrase units were determined by incubating 0.1 ml of enzyme with 0.5 ml sucrose (250.0 mM) prepared in 100.0 mM sodium phosphate buffer of pH-6.0. The reaction tube was incubated at 35°C for 5.0 minutes. The reaction was stopped after incubation by adding sodium hydroxide (0.1 ml) in solution. After the digestion of sucrose in the reaction mixture, the glucose was liberated and estimated using glucose oxidase (GOD-PAP) method (Trinder, 1969a;
1969b). The absorbance was measured at 546 nm. In the assay of enzyme detection, levansucrase was detected by glucose oxidase peroxidase (GOD-PAP) kit according to the producer’s guidelines.
Unit Definition
“One unit of levansucrase is expressed as the amount of the enzyme that releases
1.0 µM of reducing sugar as glucose per minute under standard assay conditions”
Unit Calculation
50 3.3.2 Estimation of Total Protein
The total protein of liberated enzyme was determined by Lowry’s et al. (1951)
biochemical assay using Bovine serum albumin (BSA) as standard.
3.3.2.1 Total Protein Assay for Levansucrase
The reagents for total protein assay is enlisted in Appendix-VIII.
Procedure
Test tubes were characterised as S1, S2, S3, S4, S5, S6, S7, S8, S9 and S10 holding 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 ml of standard solution (working solution) respectively. Separate tube that contained 0.025 ml sample was marked as test (T). All standard tubes and test (T) were made up to final volume of 1.0 ml by using double deionized water. Another tube was marked as blank containing 1.0 ml double deionized water. In all tubes, 5.0 ml Reagent-C (freshly prepared) was incorporated, vigorously mixed and then incubated at room temperature for 15.0 minutes. Afterwards, Add Reagent -E of 0.5 ml in all test tubes and re-incubate them for 15.0 minutes in dark. Optical density was monitored at 650 nm against blank reagent. Standard curve was plot against the absorbance values of all standard tubes.
-1 The formula of total protein (mg ml ) was expressed as given below:
51
CHAPTER 4 Results and Discussion
A significant progress has been made in discovering and developing new bacterial polysaccharides producing enzymes possessing extremely functional properties.
Levan is a natural polymer of fructose linked by β-(2→6) glycosidic bond which is produced by transfructosylation reaction in the presence of levansucrase (Korneli et al., 2013). Levansucrase is one of the industrially promising enzyme that offers a variety of industrial applications as viscosifier, stabilizer, emulsifier, surface finishing agent, encapsulating agent, sweetener and gelling or water binding agent in the field of cosmetics, foods and pharmaceuticals (Rairakhwada et al., 2010; Jang et al., 2001;
Han, 1990). There is a wide range of microorganisms that can produce levansucrase which include Bacillus sp., Streptococcus sp., Zymomonas sp. and Aspergillus sp.
(Ghaly et al., 2007). Although, levan has potential applications but the amount of levan produced is not equal to the other biopolymers which is mainly due to the inefficiency of producer organism. However, for large scale production of levan, Z. mobilis is considered as a potential candidate among many levan producing organisms
(Reiss and Hartmeier, 1990).
In industries, most enzymatic applications do not require homogeneous preparation of enzyme. Instead, they need enzyme with certain degree of purity depending upon its final application in industry. Purification of enzymes allows the determination of primary amino acid sequence as well as three dimensional structure of enzyme. This enables the better understanding of detailed study of functions and kinetic mechanisms for the action of enzyme (Ghosh et al., 1996; Taipa et al., 1992).
Therefore, the current study is designed to focus on the purification and characterization of levansucrase produced from indigenously isolated Z. mobilis.
52
4.1 Isolation, Screening and Identification of Levansucrase Producing Strain
Levansucrase producing strains were isolated from different sources and grown on
YPD medium as described in methodology section. After 24 hours of incubation at
30°C, twenty one (21) different morphological contrasting colonies were appeared on
YPD agar plates. Among these colonies, only five (05) colonies showed slimy mucoid appearance of levan polysaccharide production. These colonies were further incubated for 24 hours at 30°C to observe maximum levan production. Based on this screening procedure, a colony which showed maximum levan production was selected for pure culture study (Figure-7). The data obtained by morphological and biochemical analysis (Table-7) revealed that the selected colony was of phylum
Proteobacteria (Table-8). Further identification of the strain was confirmed on the basis of 16S rDNA sequences analysis (Figure-8). This strain assigned NCBI
GenBank accession # HM102366 and was designated as Z. mobilis KIBGE-IB14. The standardization of culture conditions for levansucrase production was carried out by using this selected strain.
Figure-7: Growth of Z. mobilis KIBGE-IB14 on YPD agar plate.
53 Table-7: Morphological and biochemical analysis of Z. mobilis KIBGE-IB14.
Parameters Zymomonas mobilis KIBGE-IB14
a Colony Morphology
Configuration Straight Rods Size Short Margin Entire Elevation Slightly convex Surface Mucoid
Colony color White to cream Opacity Translucent
Microscopic Characteristics
Gram’s staining Negative Cell shape Short rods single, pairs and in triplets Motility Motile Spore No Spore former
Growth Factors
Oxygen requirement Facultative anaerobe Optimum pH 6.5 Optimum temperature 30°C
b Sugar Fermentation Reactions
Glucose +ve Fructose +ve Galactose -ve
Xylose -ve Maltose -ve Lactose -ve Sucrose +ve
Biochemical Properties
Catalase +ve Citrate test -ve
54 Indole test -ve Methyl red -ve Vogues proskauer test (acetoin) -ve Acid production (from ethanol) +ve Gas production +ve Nitrate reduction into nitrite -ve Growth in NaCl (0.5%) +ve c Starch hydrolysis -ve Growth on Sabourand dextrose agar
-ve (SDA)
a Bacterial isolate was grown on YPD agar plate and colony morphology was observed.
b -1 Sugars were incorporated in concentration of 10.0 g L in c -1 media. 5.0 g L concentration was incorporated into medium.
Table-8: Taxonomic classification of Z. mobilis KIBGE-IB14.
Taxonomic Classification
Kingdom Bacteria
Phylum Proteobacteria
Class Alphaproteobacteria
Order Sphingomonadales
Family Sphingomonadaceae
Genus Zymomonas
Species Z. mobilis
Bionomial name Zymomonas mobilis
55
Figure-8: Phylogenetic tree of Zymomonas isolate representing the 16S rDNA sequence similarity against the data available in Genbank database. Z. mobilis KIBGE-IB14 is represented by bold accession number.
4.2 Selection of an Optimum Medium
The enzyme activity as well as growth of strain is highly affected by fermentation conditions such as medium formulation, pH, temperature and others. The strain requires a rich medium including macronutrients and micronutrients for fastidious growth that promotes production of enzyme. Therefore, various reported media were used for the production of levansucrase from Z. mobilis KIBGE-IB14 (Table-9).
Different nutrients having various concentrations were present in all these media as an essential component for bacterial growth as well as enzyme production. All investigated media indicated distinct growth pattern and enzyme production due to the
56 specific nutritional requirements of Z. mobilis KIBGE-IB14. It was found that
-1 maximum enzyme yield was achieved in medium-5 (3900 U mg ) followed by
-1 -1 -1 medium-4 (3033 U mg ) > medium-3 (2655 U mg ) > medium-2 (2890 U mg ) >
-1 medium-1 (1908 U mg ). The minimum enzyme activity was observed in medium-1 as compared to other media might be due to the low amount of carbon source and lack of nutrients such as peptone, K2HPO4, (NH4)2SO4, MgSO4.7H2O and CaCl2.2H2O in medium. In medium-2 and medium-3, less enzyme yield was observed as compared to medium-4 and medium-5 might be due to the alkaline and acidic pH of media respectively as optimum pH for the growth of Z. mobilis KIBGE-IB14 is about 6.5
(Table-7). Medium-5 is a modified enriched medium which was designed by altering the nutrients composition supplemented in medium-4 through one variable constant at a time approach. Medium-5 contains all the essential nutrients and conditions suitable for both the growth of Z. mobilis as well as production of levansucrase.
57
Table-9: Levansucrase production on various reported and newly formulated media. * -1 Media Medium components Medium composition (%) pH Enzyme Activity (U mg )
Sucrose 5.0 a Yeast extract 1.0 6.0 1908 Medium-1 KH2PO4 0.2 Sucrose 2.0 Yeast extract 0.1 b Medium-2 KH2PO4 0.1 7.2 2890 MgSO4.7H2O 0.05 (NH4)2SO4 0.1
Sucrose 15 Yeast extract 0.7 c KH2PO4 0.25 4.9 2655 Medium-3 MgSO4.7H2O 0.1 (NH4)2SO4 0.16
Sucrose 15 Yeast extract 0.2
d Casein hydrolysate 0.2 Medium-4 6.5 3033 KH2PO4 0.2
MgSO4.7H2O 0.2 (NH4)2SO4 0.2 Sucrose 15 Yeast extract 1 e Medium-5 Peptone 0.2 3900 6.5 K2HPO 4 1.5 CaCl2.2H2O 0.01
a b c *All media were incubated at 30°C. Standard deviation (n=3). Ananthalakshmy and Gunasekaran. 1999. Schreder et al., 1934. Bekers et al., d e 2002. Kirk and Doelle 1992. Modified medium designed in current study.
58
4.3 Optimization of Cultivation Conditions for Production of Levansucrase
The successful application and commercialization of any biological product needs fermentation parameters to be optimized. It is necessary to provide appropriate biochemical and biophysical environment for the propagation of any microorganism. The variation in these different phsico-chemical parameters directly influence the microbial growth which ultimately affect the production of enzyme. These above-mentioned parameters not only affect the cost of production process but also utilization of synthesized product. It has been emphasized by different researchers that various growth parameters are significant for the production of enzyme (Sivaramakrishnan et al., 2006;
Sarikaya and Gürgün, 2000). Different physical factors which mainly includes fermentation time, incubation temperature and pH of growth medium should be optimized in order to enhance the enzyme production. In addition, other medium components such as carbon sources, nitrogen sources, trace elements as well as growth stimulators with their appropriate concentrations are also involved in the hyper-production of enzyme. The batch, continuous or fed batch operational methods are available to alter these conditions in either stepwise or randomized manner. Hence, the present study was aimed to optimize medium composition for increased yield of levansucrase from isolated strain of Z. mobilis
KIBGE-IB14.
4.3.1 Selection of Physical Parameters for Levansucrase Production
The optimization of biophysical environment plays vital role in the production of enzymes (Gupta et al., 2003; Bose and Das, 1996). These mainly includes the standardization of physical growth parameters such as cultivation time, incubation temperature and medium pH. These factors affect the cost of both upstream as well as
59 downstream processing at industrial level. Hence, physical parameters were optimized in order to attain hyper-production of levansucrase from Z. mobilis KIBGE-IB14.
4.3.1.1 Effect of Incubation time
Fermentation time of an enzyme has a profound effect on cost of enzyme production and its product formation. The pattern of levansucrase production and bacterial growth with reference to fermentation time period was observed by incubating the Z. mobilis KIBGE-IB14 for different time intervals. In the present study maximum cell concentration and enzyme production was observed at 24 hours (Figure-9) which clearly demonstrated the direct relationship of time interval among microbial growth and enzyme synthesis. When the maximum enzyme production was achieved, a gradual decrease in microbial growth and enzyme synthesis was monitored. This declined might be due to the release of unwanted secondary metabolites which altered the pH of the medium. It has been reported that prolong incubation period also declined the enzyme level which might be due to the depletion of medium ingredients and allowed the microorganism to use enzymatic protein as nitrogen source during fermentation that ultimately decreased the enzyme level (Arnold et al., 1998; Ahmed,
2008). The result of present study is in contrast with the findings of levansucrase from
B. megaterium using solid-state fermentation where maximum enzyme production was obtained after 72 hours (Ahmed, 2008).
60
Figure-9: Levansucrase production from Z. mobilis KIBGE-IB14 at various time intervals (Mean ± S.E., n=3).
4.3.1.2 Effect of Temperature
Optimization of growth parameters has an important role in order to achieve high enzyme yield. Temperature is one of the crucial factor necessary for the growth of microorganism that ultimately lead to higher production of extracellular enzymes.
Moreover, optimum temperature is different for every microbial factory. In the current analysis, the optimum temperature for bacterial growth and levansucrase production was monitored by incubating Z. mobilis KIBGE-IB14 at different temperatures ranging from 20-60°C. It was observed that the maximum biomass yield and enzyme production was achieved at 30°C (Figure-10) which indicated the mesophilic nature of the organism. However, further increase in incubation temperature after reaching maxima resulted in lower cell growth and enzyme production. Previously it was observed that at high temperatures, large amount of heat was generated which resulted in inhibition in cell growth that in turn declined the
61 levansucrase formation (Babu and Satyanarayana, 1995). It was reported that the maximum enzyme production was achieved at 30°C for B. subtilis NRC33a and
Acetobacter diazotrophicus (Abdel-Fattah et al., 2005; Hernández et al., 1999). On the contrary, it was also reported that maximum levansucrase production by mutant strain of Z. mobilis and Bacillus sp. was found at 55°C and 7.0°C, respectively
(Belghith et al., 2012 ; Muro et al., 2000).
Figure-10: Production of levansucrase from Z. mobilis KIBGE-IB14 at different temperatures (Mean ± S.E., n=3).
4.3.1.3 Effect of pH
Another important cultivation parameter is pH of the medium, which directly affects microbial growth and enzyme production. This parameter is extremely critical as it regulates the nature of end product (acidic or alkaline) that eventually decides its directions in industrial applications. Previously, it has been demonstrated that alteration in pH of fermentation medium highly disturbs the enzymatic process by
62 changing the permeability of cell membrane along with the movement of particles across the microbial cell (Moon and Parulekar, 1991). Furthermore, the change in pH of medium affects the growth and nutritional availability of microbes by altering the ionization state of nutrients (Slonczewski et al., 2009). To observe the effect of pH on cell mass and levansucrase production, Z. mobilis KIBGE-IB14 was inoculated in fermentation medium having different acidic and alkaline pH ranging from 5.0 to 9.0.
It was found that maximum levansucrase production was achieved in medium having pH-6.5 (Figure-11). Several other researchers also reported pH-6.0 and pH-6.5 for the production of levansucrase from B. subtilis BB04 and Bacillus sp. respectively
(Vaidya and Parasad, 2012; Belghith et al., 2012). In a contradistinction to the current data, levansucrase production was also noticed at pH-5.0 (acidic) and pH-7.5
(alkaline) from Z. mobilis 113S and B. subtilis Natto CCT 7712 respectively (Vigants et al., 2013; Gonçalves et al., 2013).
Figure-11: Levansucrase production from Z. mobilis KIBGE-IB14 at various pH values (Mean ± S.E., n=3).
63 4.3.2 Influence of Carbon Source on Levansucrase Production
Carbon source is one of the integral component of production medium as it is associated with the production of cellular material as well as microbial growth (Dunn,
1985). In the current investigation, sucrose was used as a carbon source to accomplish necessary requirements of microorganism.
4.3.2.1 Effect of Sucrose
The effect of sucrose concentrations on final cell mass and levansucrase production was observed by incubating Z. mobilis KIBGE-IB14 in fermentation medium having different sucrose concentrations. It has been examined that increase in sucrose concentration resulted in an enhancement of enzyme secretion while caused reduction in microbial growth (Figure-12). The maximum enzyme production was observed at
15% of sucrose was used in the medium. Afterwards, increase in sucrose concentration showed unfavorable effect on enzyme synthesis. As it was suggested that below 10%, sucrose is mainly used for microbial growth and higher sucrose concentrations resulted in more enzyme production but cell growth inhibition (Chen and Liu, 1996). The current work disagrees with the findings of Belghith et al. (2012) where maximum levansucrase production from Bacillus sp. was observed with 30% of sucrose present in the medium.
64
Figure-12: Production of levansucrase from Z. mobilis KIBGE-IB14 at various substrate concentrations (Mean ± S.E., n=3).
4.3.3 Optimization of Nitrogen Sources for Levansucrase Production
Microorganisms require organic (amino acids and proteins) and inorganic
(ammonium salts) nitrogen sources during fermentation process for the production of extracellular enzymes. The enzyme yields as well as bacterial growth extremely affected by the type and concentration of nitrogen source used during culture processing method. In this study, two types of nitrogen sources, yeast extract and peptone were optimized for high levansucrase production.
4.3.3.1 Effect of Yeast extract
Yeast extract is frequently used in bacteriological media composition in the form of nitrogen source as it is rich in essential vitamins, minerals and nucleic acids. The effect of different concentrations of yeast extract on cell mass and enzyme production was revealed that both microbial growth and enzyme production was directly proportional to the yeast extract concentration up to 1.0%. In the present study,
65 maximum results were achieved with 1.0% of yeast extract incorporated in the medium (Figure-13). Moreover, further increase in yeast extract concentration caused reduction in enzyme production but increased cell growth. According to Fein et al.
(1983), the incorporation of yeast extract to the fermentation medium provides vitamins that are necessary for the growth of Z. mobilis. Zhang et al. (2014) reported the same findings for the production of levansucrase from B. methylotrophicus SK
21.002. On the other hand, some researchers observed an increase in levansucrase production by adding 2.0% and 5.0% of yeast extract for Z. mobilis and thermophilic
Bacillus sp. respectively (Gonçalves et al., 2013; Belghith et al., 2012).
Figure-13: Levansucrase production from Z. mobilis KIBGE-IB14 at different yeast extract concentrations (Mean ± S.E., n=3).
4.3.3.2 Effect of Peptone
Peptone is most widely used nitrogen source in culture media to grow microorganisms as it contains natural sources such as amino acids, peptides and proteins. Figure-14 demonstrated the influence of peptone on cell growth and levansucrase yield by using
66 medium containing different concentrations of peptone. These findings indicated that the maximum enzyme production was achieved at 0.2% of peptone. In agreement with present data, Jakob et al. (2012) reported the maximum levansucrase production at
0.2% of peptone from Gluconobacter species. On contrary, different studies noticed that 5.0% of peptone concentration showed maximal enzyme synthesis from B. methylotrophicus SK 21.002 (Zhang et al., 2014).
Figure-14: Levansucrase production from Z. mobilis KIBGE-IB14 at different peptone concentrations (Mean ± S.E., n=3).
4.3.4 Influence of Micronutrients for Levansucrase Production
The osmotic pressure of the culture propagation medium maintained by the incorporation of micronutrients or trace elements. It has been reported that the utilization of substrate by microorganisms can increased with the addition of appropriate concentration of salt (Gao et al., 2010). To observe the impact of micronutrients on levansucrase production, Z. mobilis KIBGE-IB14 was fermented in medium containing various concentrations of K2HPO4 and CaCl2.2H2O.
67 4.3.4.1 Effect of Di-Potassium Hydrogen Phosphate (K2HPO4)
Influence of different concentrations of di-potassium hydrogen phosphate in culture medium were observed on bacterial growth and enzyme production. The present data was revealed that the microbial growth as well as enzyme production increased with the increase in di-potassium hydrogen phosphate concentration in medium. It was found that 1.5% di-potassium hydrogen phosphate was the most favorable concentration for the bacterial growth and production of levansucrase (Figure-15). Di- potassium hydrogen phosphate provided potassium and phosphorous to the medium which acts as a cofactor for enzymes and constituent of nucleic acids, nucleotides and phospholipids respectively. In contrast to the present study, some researchers found
0.15% and 0.3% of di-potassium hydrogen phosphate for the maximum yield of levansucrase from Pediococcus acidilactici and Erwinia herbicola (Youssef et al.,
2014; Han and Clarke, 1990).
Figure-15: Production of levansucrase from Z. mobilis KIBGE-IB14 at various di-potassium hydrogen phosphate (K2HPO4) concentrations (Mean ± S.E., n=3).
68 4.3.4.2 Effect of Calcium Chloride (CaCl2.2H2O)
+2 The influence of different concentrations of diavalent ion (Ca ) on the growth of Z. mobilis KIBGE-IB14 and levansucrase production was examined. The present work represents the increase in the bacterial growth and enzyme production up to the 0.01% concentration of calcium chloride and further increased in calcium chloride concentration decreased cell mass and enzyme yield (Figure-16). It was reported that calcium chloride improved the stability of cellular membrane by regulating the entrance and exit of materials. Basically, calcium chloride enhances the permeability of membrane by making the endocellular pressure consistent with exocellular pressure
(Zeng, et al., 2010). On contrary, different studies indicated that as the concentration of calcium chloride increased, the biomass concentration was also increased (Bajpai and Margaritis, 1984).
Figure-16: Production of levansucrase from Z. mobilis KIBGE-IB14 at various Calcium chloride (CaCl2.2H2O) concentrations (Mean ± S.E., n=3).
69 4.4 Purification of Levansucrase
There are multiple methods available for the purification of enzymes by applying
various biotechnological techniques. However, the selection of an appropriate method
for purification of enzyme needs some important considerations (Mukhopadhayay,
2001). These include:
Prospective utilization of enzyme Stability of enzyme against selected method Location of enzyme (extracellular or intracellular) Nature of producer microorganism Cost of enzyme production
A multi-step scheme was designed for the purification of levansucrase from Z. mobilis
KIBGEIB-14 using various techniques (Figure-17). After each step of purification
process, the purity and percent recovery of levansucrase was calculated. This purified
levansucrase will be used in the collection of information regarding characterization
and kinetic analysis of enzyme. A number of protocols for the purification of
levansucrase from diverse microbial species are available in literature (Tian et al.,
2011; van Hijum et al., 2001; Hettwer et al., 1995; Yanase et al., 1992; Kuramitsu,
1975).
70
Figure-17: Purification scheme of levansucrase from Z. mobilis KIBGE-IB14.
71 4.4.1 Partial Purification of Levansucrase
There are various precipitating agents available for efficient purification of enzymes
depending upon the nature of enzyme. In current research, three different reported
precipitating agents were used to precipitate levansucrase including salt (ammonium
sulphate 40%), solvent (ethanol 60%) and polymer (PEG 4000 30%) (Youssef et al.,
2014; Tian et al., 2011; Ohtsuka et al., 1992). From the data obtained, maximum
levanuscrase precipitation was observed with ammonium sulphate (40%) having
specific levasucrase activity of 9,232 and fold purification of about 1.36 as compared
to crude enzyme (Table-10). While 1.07 and 1.21 fold purification was obtained as
compared to crude enzyme from ethanol and PEG 4000 respectively.
After the selection of precipitating agent, its appropriate concentration was
calculated by gradient precipitation of ammonium sulphate (Table-11). The
precipitation method invoves the harvesting of microbial cells after fermentation by centrifugation at 35000 g for 15 minutes at 4°C. As a result of centrifugation,
bacterial cells were separated leaving behind the cells free filtrate or supernatent. This
obtained supernatent was subjected to gradient precipitation by ammonium sulphate
ranging from 30% to 60%. After addition of salt at each step, the reaction mixture was
equilibrated for 18 hours at 4°C and then recovered precipitates were dissolved in
sodium phosphate buffer (100 mM, pH-6.0). In gradient precipitation process,
maximum levansucrase activity was noticed at 50% ammonium sulphate in which
levansucrase activity was increased up to 1.61 fold as compared to crude enzyme
(Table-11). At low concentration of salt, decreased enzyme activity was noted might
be due to the insufficient precipitation of enzyme. Wherever, increased concentration
of salt caused negative effect on the activity of levansucrase might be due to the denaturation of enzyme. In line with this agreement, Ohtsuka et al. (1992) used 50% 72 ammonium sulphate for the precipitation of levansucrase from Rahnella aquatilis
JCM-1683. In contrast, partial purification of levansucrase from Microbacterium laevaniformans ATCC 15953 was carried out by 60% ammonium sulphate resulted in
2.6 fold purification with 95% yield (Park et al., 2003). Tian et al. (2011) achieved the highest fractionation yield of 73% by using PEG 200 at a concentration of 30%.
Moreover, maximum levansucrase activity was revealed by fractional precipitation of levansucrase from Pediococcus acidilactici by using ethanol concentration of 65%
(Youssef et al., 2014).
73
Table-10: Partial purification of levansucrase from Z. mobilis KIBGE-IB14 with different precipitating agents of various concentrations.
Total Volume Total Enzyme Activity Total Protein Specific Activity Precipitating Agents -1 Fold Purification (ml) (U) (mg) (U mg )
Crude 500 1,118,000 1,646 6,754 1.0
Ammonium Sulphate 5 301,390 32.64 9,232 1.36 (40%)
Ethanol (60%) 5 243,445 33.40 7,288 1.07
PEG 4000 (30%) 5 271,190 33.02 8,212 1.21
74
Table-11: Partial purification of levansucrase from Z. mobilis KIBGE-IB14 with gradient precipitation of ammonium sulphate.
Total Volume Total Enzyme Activity Total Protein Specific Activity Precipitating Agents -1 Fold Purification (ml) (U) (mg) (U mg )
Crude 500 1,118,000 1,646 6,754 1.0
Ammonium Sulphate 5 278,950 34.265 8,140 1.20 (30%)
Ammonium Sulphate 5 301,390 32.64 9,232 1.36 (40%)
Ammonium Sulphate 5 346,625 31.9 10,865 1.61 (50%)
Ammonium Sulphate 5 286,430 29.465 9,721 1.43 (60%)
75
4.4.2 Desalting of Partially Purified Levansucrase
After precipitation of enzyme, the salt was removed from the partially purified sample of levansucrase by using desalting method. The pre-quilibrated desalting column PD-
10 packed with Sephadex G-25 was used for the removal of salt. After desalting, the specific activity and fold purification of levansucrase from Z. mobilis KIBGEIB-14
-1 -1 was incresaed from 10,865 U mg to 23,225 U mg and 1.61 to 3.44 fold respectively (Table-12). When the salt was removed from the sample, it was further
® concentrated by using centricon centrifugal filter device.
4.4.3 Gel Permeation Chromatography
It is an extremely critical stage during downstream process to purify enzyme from other contaminants and protein to such an extent that a single homogeneous active form of enzyme was retrieved. The cost of purification process depends on number of steps involve during purification. Hence, it is important to minimize these steps in order to reduce the overall expense associated with this process. In current analysis, the final stage of purification accomplished by the separation of partially purified levansucrase protein of Z. mobilis KIBGE-IB14 from other proteins through gel permeation or size exclusion chromatography. To achieve maximum protein separation and yield, several chromatographic runs were conducted throughout the process. For this purpose, the pre- equilibrated sample with sodium phosphate buffer (100.0 mM, pH-6.0) of partially purified enzyme was loaded on the column of Sepharose CL-6B (1.5 cm 50.0 cm).
-1 The same buffer was used to elute the sample. The flow rate of 0.5 ml min was selected for the collection of protein fractions. The enzyme activity and total protein of all fractions were estimated by Trinder (1969a; 1969b) and Lowry’s et al. (1951) respectively. The enzyme activity of levansucrase
76 from Z. mobilis KIBGE-IB14 was detected in the fraction numbers from 68 to 71
(Figure-18). These active fractions were pooled and then stored at -20°C for further characterization. Complete data for purification of levansucrase from Z. mobilis
KIBGE-IB14 is summarized in Table-12. The data validated that purified
-1 levansucrase showed specific activity of 81,983 U mg with 12.13 fold and 1.77% yield. The purity of sample was further confirmed by performing gel electrophoresis.
Different fold purification of levansucrase from diverse microbial origin has been shown by many previously reported researchers. Nakapong et al. (2013) purified levansucrase from B. licheniformis RN-01 using DEAE Toyopearl 650-M column followed by Butyl Toyopearl 650-M column resulted in the fold purification of 1528 with 69% yield. In another finding, Dahech et al. (2012) reported 177 fold purification of levansucrase from B. licheniformis using Sephacryl S-200 column. It has been observed that levansucrase from Z. mobilis was purified about 80 to 150 folds using
Sephacryl S-300 column whereas, it was reported earlier that the anion exchange chromatography purified the levansucrase from Z. mobilis up to 112 fold (Vigants et al., 2003; Vigants et al., 2001). Therefore, it has been inferred that purification method should adjust in a manner that it recovered highly purified enzyme under controlled conditions.
77
Table-12: Complete purification profile of levansucrase produced by Z. mobilis KIBGE-IB14.
Fold Total Volume Total Enzyme Activity Total Protein Specific Activity Sample -1 (ml) (U) (mg) (U mg ) Purification % Yield
Crude 500 1,118,000 1,646 6,754 1.0 100
Ammonium sulphate 5 346,625 31.9 10,865 1.61 31 (50%)
Desalting 5 382,280 16.46 23,225 3.44 34.20 PD-10
Sepharose CL-6B 1 19,840 0.242 81,983 12.13 1.77
78
Figure-18: Gel permeation chromatography of levansucrase from Z. mobilis KIBGE-IB14.
79 4.5 Biochemical Characterization of Purified Levansucrase
The success of multifarious industrial applications of any enzyme depends on the optimization of catalytic parameters. The main factors that must be considered to design an enzymatic assay include enzyme-substrate reaction time, pH, temperature and its kinetic evaluation as these parameters play central role in the alteration of enzymatic activities. Furthermore, stability (thermal and storage) profile of enzyme as well as proper concentrations of different activators, inhibitors and stabilizers are also very significant for better understanding of enzymatic phenomenon utilized in the application of these enzymes.
4.5.1 Enzyme-Substrate Reaction Time Profile
The reaction time required for catalytic activity of purified levansucrase was determined by reacting the enzyme with its substrate from 5.0 to 60.0 minutes.
Results showed that levansucrase exhibited maximum enzyme activity when the reaction mixture was kept for 5.0 minutes. Subsequently, a decline in enzyme activity was noticed as the reaction time increased (Figure-19). In agreement with these findings, Rehman et al. (2013) also observed the maximum pectinase activity from B. licheniformis KIBGE-IB21 in 5.0 minutes. On the other side, it was evident from observation that maximum levansucrase activity was achieved by reacting the enzyme for 60.0 minutes (Homann et al., 2007).
80
Figure-19: The effect of reaction time on catalytic activity of levansucrase from Z. mobilis KIBGE-IB14. (Mean ± S.E., n=3).
4.5.2 Temperature Activity Profile
The rate of enzyme-catalyzed reaction highly affected by temperature as in the case of other chemical reactions. When the temperature rises, rate of reaction also accelerates due to high collision and increase kinetic movement of molecules until an optima achieved. The more increase in kinetic motion of molecules after this point breaks hydrogen bonding that causes enzyme denaturation by constituting conformational changing of enzyme (Russell, 2008). The temperature dependence of levansucrase from Z. mobilis KIBGE-IB14 was revealed by keeping enzyme at various temperatures ranging from 25°C to 65°C. The findings of current research showed that maximum levansucrase activity attained at 35°C and enzyme activity decreased as the temperature increased beyond its optimum range (Figure-20). This decrease in catalytic activity of enzyme is might be due to the cleavage of peptide bonds as a result of high kinetic energy that ultimately transforms the conformation of active site
81
by modifying its secondary and tertiary structure (Rehman et al., 2014). On contrary
to this study, optimum temperature reported in literature of purified levansucrase from
Pediococcus acidilactici was 40°C (Youssef et al., 2014). Moreover, maximum
catalytic activity exhibited by levansucrase from Bacillus sp. TH4-2 was noted at
50°C (Ammar et al., 2002).
Figure-20: The effect of temperature on levansucrase activity from Z. mobilis KIBGE-IB14 (Mean ± S.E., n=3).
4.5.3 pH Profile and Selection of Buffer
pH is the most crucial factor for enzymatic activity as it interferes enzymes by
varying its electrical charges (-COOR and -NH2) which is present on posterior as well
as interior surface of enzyme molecule. This alteration of charges may vary the
conformation of active site of enzyme which in turn influence the binding of substrate
to enzyme. In different pH conditions, enzymes of diverse microbial sources perform
in a different manner due to its unique biological system. The pH sensitive enzymes require a buffering environment specific for the free catalytic activity deprived of any 82 hindrance. The foremost function of buffer is to resist variation in hydrogen ion concentration due to the influence of internal or external environmental conditions.
All buffers are able to restrained changes in hydrogen ion concentration over an optimal pH range. Therefore, buffer helps to stabilize and regulate the anticipated pH throughout the enzyme assay. The pH requirement for levansucrase activity was evaluated by using wide pH range of four different buffers (5.0-9.0) possessing same ionic strength. The observed results interpreted that the most favourable pH range for enzyme activity lies within 6.0-7.0 with maximum enzyme activity at pH-6.0 of sodium phosphate buffer (Figure-21). Similar results were reported by Gonçalves et al. (2013) in the case of optimization of levansucrase activity obtained from B. subtilis. It was noticed by Tian et al. (2011) that the maximum levansucrase activity from B. subtilis lies in the pH range of 6.0-6.5. In contrast, the optimal levansucrase activity from extracellular Z. mobilis KIBGE-IB14 was found at pH 5.0 (Vigants et al., 2013). After an extensive research study of levansucrase from Z. mobilis,
Goldman et al. (2008) noted that there are two forms of levansucrase i.e. dimeric and microfibrils which mainly depends upon the pH and ionic strength of environment provided. The dimeric form of enzyme noticed when the pH is higher than 7.0 whereas, enzyme is present in microfibrils at pH less than 6.0. Moreover, the production and kinetic characteristics of polysaccharides is highly influenced by pH change as the enzyme produces FOS at pH higher than 7.0 while levan synthesis was catalyzed by levansucrase at pH below 6.0.
83
Figure-21: The influence of pH dependence on catalytic activity of levansucrase from Z. mobilis KIBGE-IB14 (Mean ± S.E., n=3).
4.5.4 Selection of Ionic Strength of Buffer
The ionic strength of buffer is expected to be one of the essential parameter for the reaction and stabilization of enzyme. In enzyme substrate interaction, the movement of charged molecules exists on the active site of enzyme highly influenced its catalytic activity by disturbing the attachment of substrate to the enzyme. After the selection of sodium phosphate buffer (pH-6.0), its ionic strength has been evaluated by varying it from 25 mM to 200 mM (Figure-22). It was evident from the data that levansucrase from Z. mobilis KIBGE-IB14 showed its maximum activity at 100 mM of sodium phosphate buffer and then activity started to declined. Current investigation recommends the importance of molarity on the activity of levansucrase because a decline of catalytic activity was monitored with the increase of ionic concentration.
Same ionic strength of different buffers has previously been reported for levansucrase activity from Acetobacter diazotrophicus (Hernández et al., 1995).
84
Figure-22: The effect of ionic strength of sodium phosphate buffer (pH-6.0) on catalytic activity of levansucrase from Z. mobilis KIBGE-IB14 (Mean ± S.E., n=3).
4.5.5 Kinetic Analysis
The concentration of substrate is a crucial factor which directly influences the enzyme-catalyzed reactions. When the substrate concentration is low, less collision of enzyme with substrate is observed but the enzymatic reaction accelerates as the concentration of substrate increases. Although, when the maximum rate (Vmax) of enzyme attained, the rate of reaction does not escalate with the increase in substrate concentration and it becomes constant (Russell, 2008). In order to estimate the kinetics parameters such as Km (Michaelis-Menten constant) and Vmax (Maximum reaction rate) values of levansucrase, double reciprocal Lineweaver-Burk plot was used. Different substrate concentrations ranging from 0.025 M to 0.4 M were used for the determination of substrate saturation constant at constant pH and temperature. It was observed that the Vmax and Km values of levansucrase from Z. mobilis KIBGE-
85 -1 -1 IB14 were 21381 U mg and 0.02308 moles L respectively (Figure-23). Vmax and Km
-1 values of levansucrase from B. subtilis BB04 have been reported to be 16.8 mM L and
-1 127.2 U mg (Vaidya and Parasad, 2012). In another study, the values of Vmax and Km
-1 -1 were observed as 714 mol min mg and 50 mM respectively of levansucrase from Rahnella aquatilis JCM-1683 (Ohtsuka et al., 1992).
Figure-23: Lineweaver-Burk plot and Michaelis-Menten constant of levansucrase from Z. mobilis KIBGE-IB14.
4.5.6. Stability Studies
The ability of enzyme to tolerate numerous environmental conditions is necessary to execute various applications in any industry. The unstable enzymes cannot tolerate the harsh industrial situations as compared to stable enzymes. Therefore, stable enzymes can manage industrial conditions in a more improved manner. The residual activity is used to monitor the stability profile of an enzyme (Georis et al., 2000).
86 4.5.6.1 Thermal Stability
The thermal stability of an enzyme is represented by the resistance in its catalytic activity at elevated temperature for prolonged time period. It is the most vital parameter required at industrial level for successful commercialization of enzyme (Bhatti et al., 2006). The investigation of levansucrase with reference to the thermal stability was examined by incubating it at several temperatures (25-55°C) for different time periods ranging from
30-120 minutes (Figure-24). It was exhibited that levansucrase is quite stable at 25°C and
35°C as enzyme retained 83% and 92% activity for 120 minutes respectively. Conversely, levansucrase displayed thermal denaturation when it was exposed to 45°C and 55°C after
120 minutes. In the case of 45°C, about 23% catalytic activity of levansucrase was retained after 120 minutes. While complete deterioration of levansucrase activity was noticed after exposure of enzyme for 120 minutes at 55°C. The loss in the activity of levansucrase might be due to the rise in kinetic energy of enzymatic molecules. This results in the disruption of three dimensional structure of enzyme due to the cleavage of amino acid interactions that retains its native structure.
87
Figure-24: Thermal stability profile of levansucrase from Z. mobilis KIBGE- IB14 (Mean ± S.E., n=3).
4.5.6.2 Storage Stability
The stabilization of an enzyme on the basis of its biological structure is considered as paramount because it expressively decreases the operational budget (Wang et al., 2013).
The storage stability or shelf life of an enzyme is the ability of an enzyme to retain its catalytic activity for longer time period. It has been reported that the activity of enzyme gradually declined when the enzyme was stored for longer period of time. The change in protein structure takes place due to the heating and cooling from ambient temperature
(Cevik et al., 2011; de Queiroz et al., 2006). The storage stability of levansucrase suggested that it was stable at low temperature and retained about 94% activity for 180 days (4.0 months) at -80°C (Figure-25). This stability might be due to the less probability of protein denaturation at freezing temperature either by ceasing the biochemical reaction of enzyme or changing its three dimensional structure. At 4°C and
88
37°C, activity of levansucrase was found 72% and 50% respectively after 20 days
which decreased more as the storage time increased. In addition, complete loss of
levansucrase activity was observed at 37°C and 4°C after 60 and 120 days
respectively. It was reported that during storage of an enzyme, its activity deteriorated
which might be due to the autolysis, unfolding as well as denaturation of enzyme
(Lyer and Ananthanarayan, 2008). Reese and Avigad, (1966) reported that the
levansucrase from Aerobacter levanicum showed its activity up to 2.0 weeks at 4°C. It
has also been observed that levansucrase activity from B. megaterium showed its
activity for more than 6.0 months at -20°C (Homann et al., 2007).
Figure-25: Storage stability profile of levansucrase from Z. mobilis KIBGE-IB14
(Mean ± S.E., n=3).
4.5.7 Effect of Metal Ions
Metal ions are able to create interactions with the active site of enzyme which affects
the discipline of activation, inhibition and stabilization in enzyme-substrate reactions.
The biocatalysts can be represented as activators, inhibitors or stabilizers in the 89 presence of different metal ions having appropriate concentration. Current study assessed the effect of different metal ions (monovalent, divalent and trivalent ions) with various concentrations on catalytic performance of levansucrase (Table-13). The results exhibited
+2 +2 that Ni and Zn did not show any significant effect on the levansucrase activity at all
+ + + +2 +2 +2 +2 +2 concentrations. Whereas, K , Na , Cs , Ba , Ca , Cu , Mg and Mn acted as stimulators as they enhanced levansucrase activity by 5%, 2%, 1%, 6%, 25%, 2%, 4% and
2% respectively at 1 mM concentration which then decreased by increasing the
+2 +2 concentration of metal ions. Some divalent ions such as Co and Hg inhibited the activity of levansucrase by 24% and 92% at 1 mM concentration and the activity of enzyme was gradually decreased with the increasing concentration of these metal ions.
+2 While, Hg was found be a strong inhibitor in the case of levansucrase from Z. mobilis
KIBGE-IB14. It has been reported that enzyme becomes inactive in the presence of heavy metals due to the non-competitive inhibition in which metal binds with enzyme other than active site because of conformational changes in active site of enzyme (Shanmugam and
Sathishkumar, 2009; Singh, 2003). Most of similar results regarding the effect of metal ions (1 mM) on the activity of levansucrase has been documented by Kang et al. (2005).
In addition, it has been noticed that levansucrase activity from B. subtilis Natto CCT 7712
+ + + was increased by monovalent cations i.e. Na , K and Zn (4 mM to 8 mM) while partial
+2 +2 +2 +2 inhibition of enzyme activity was observed with divalent cations Ba , Ca , Mn , Fe
+2 +2 and Cu (2 mM to 8 mM) except Zn that first inhibited the activity at 2 mM and then again activated in other concentrations (Gonçalves et al., 2013). In contrast, different
+2 results have been reported by Ammar et al. (2002) that Zn (5 mM) showed inactivation
+2 of levansucrase from Bacillus sp. TH4 activity while Fe (5mM) acted as activator.
+3 +3 Moreover, trivalent ions such as Fe and Al showed
90 negative effect on levansucrase activity as they inhibited the enzyme activity by 12% and 18% respectively at 1 mM concentration. It has been reported that the activity of
+3 +3 levansucrase was inhibited by Al (88%) while Fe did not show any considerable effect at 5 mM concentration (Hettwer et al., 1995). Therefore, this study suggested the contradictory results related to the activation or inhibition of levansucrase activity due to differences in salt concentrations used as well as production of enzyme from diverse microbial sources.
Table-13: Effect of different metal ions on the catalytic activity of levansucrase from Z. mobilis KIBGE-IB14.
Relative Activity (%) Metal Ions 60.0 minutes
1mM 5mM 10mM Control 100 Monovalent ions + K 105 93 90
+ Na 102 95 92
+ Cs 101 94 88
Divalent ions
+2 106 99 87 Ba +2 Ca 125 133 136
+2 Co 76 68 54
+2 Cu 102 89 76
+2 8 4 2 Hg +2 Mg 104 96 88
+2 102 98 90 Mn +2 Ni 98 94 89
+2 Zn 97 92 90
Trivalent ions +3 Fe 88 80 73
+3 82 71 65 Al
*All the metal ions used were in the form of chloride salts.
91 4.5.8 Effect of Organic Solvents
The performance of enzyme is affected by organic solvents in a way that it causes alteration in active site of enzyme (Koskinen and Klibanov, 1996). It has been reported that solvents can also affect the enzyme activity by disturbing its interaction with substrate and product. This disturbance is due to the alteration of substrate and product concentration in the aqueous layer surrounding the enzyme that hinders the diffusion of product and permeation of substrate (Kawakami and Nakahara, 1994;
Yang and Robb, 1994). The stability and catalytic activity of levansucrase in water- miscible environment was investigated by incubating the enzyme with different organic solvents in various concentrations (Table-14). The study exhibited that all solvents did not show any significant effect on levansucrase activity at low concentrations (1 mM) except isopropanol and DMSO that acts as an activator and inhibitor of enzyme respectively. At 1 mM concentration of solvents, isopropanol stimulates the enzyme activity by 10% while DMSO showed 14% inhibition and this relative percent declined more as the concentration of organic solvents increased. At higher concentrations of solvents (5 mM and 10 mM), the enzyme activity remains almost stable in the case of ethanol and methanol while formaldehyde and chloroform found to be the inhibitors of enzyme. The inhibition of enzyme activity by organic solvents might be due to the penetration of solvents in the active site of enzyme where it causes electrostatic repulsion between enzyme and substrate that renders its interaction (Koskinen and Klibanov, 1996). Previously reported the effect of different organic solvents (1,4-Dioxane, Acetone, Acetonitrile and DMSO) on activity of levansucrase from B. subtilis (Castillo and López-Munguıa, 2004).
92
Table-14: Influence of different organic solvents on levansucrase activity obtained from Z. mobilis KIBGE-IB14.
Relative Activity (%) Organic 60.0 minutes Solvents
1mM 5mM 10mM
Control 100
DMSO 86 72 50 Ethanol 100 100 96
Methanol 100 98 94 Isopropanol 110 96 87 Formaldehyde 96 82 76 Chloroform 100 95 84
4.5.9 Effect of Surfactants
The surfactants are acknowledged as an important environmental factor that induce
their effect on the activity of enzyme. This study represents an attempt to find out the
effect of surfactants on the activity of levansucrase from Z. mobilis KIBGE-IB14. To
achieve this aim, enzyme was mixed with different anionic (SDS) and non-ionic
(Triton X-100, Tween-20 and Tween-80) surfactants at various concentrations (Table-
15). According to current investigation, non-ionic detergents i.e. Triton X-100,
Tween-20 and Tween-80 did not expressively produce any changes in the activity of
levansucrase at different concentrations. This might be due to the fact that these non-
ionic surfactants do not cause inhibition or inactivation of enzymes as they prevent
the aggregation of protein in solution and provide stabilization to protein activity.
Similar observation was found in pepsin where Triton X-100 and Tween-80 acted as
stabilizers (Guzman et al., 2016). In contrast, anionic detergent i.e. SDS showed
slight inhibition on levansucrase activity as it inhibited the enzyme by 11% at 1 mM
concentration which gradually increased as the concentration of surfactant was
93
increased. Hettwer et al. (1995) revealed that SDS slightly inhibited the levansucrase
activity by 73%. It has been reported that this inhibition was due to the ability of all
proteins to absorb anionic detergent that denatures its structure by disrupting bonds
which ultimately obstructs protein function (Bartnik, 1992). Zikmanis et al. (2005)
described the effect of different surfactants of various concentrations on levansucrase
activity from Z. mobilis.
Table-15: Impact of different surfactants on catalytic activity of levansucrase from Z. mobilis KIBGE-IB14.
Relative activity (%) Surfactants 60.0 minutes
1mM 5mM 10mM
Control 100
Tween-20 100 100 92
Triton-X100 98 93 88
SDS 89 80 75
Tween-80 100 98 90
4.6 Approximate Molecular Weight and Zymography of Purified Levansucrase
At each step of purification, the approximate molecular weight of purified
levansucrase from Z. mobilis KIBGE-IB14 as well as its purity analysis was assessed
by performing electrophoresis (SDS-PAGE). The purified sample was
electrophoresed using 12% resolving and 4.0% stacking gel in denaturizing SDS-
PAGE followed by zymographic analysis. In zymography, the gel was stained by
Periodic-Schiff’s staining method to confirm the presence of levansucrase band. The
molecular weight was estimated using molecular weight markers by comparing
94 relative mobility of sample stained with Coomassie brilliant blue R-250. The comparison of known molecular weight protein marker and SDS-PAGE of levansucrase represented the molecular weight of levansucrase from Z. mobilis
KIBGE-IB14 about 130 kDa. The pre-zymogram in which sample was treated with ethanol precipitation as well as zymogram of levansucrase confirmed the presence of purified band (Figure-26). Different extensive studies regarding levansucrase purification reported various molecular weight of levansucrase. Molecular weight of levansucrase KIBGE-IB14 has been found to close to the levansucrase produced by
Streptococcus salivarius ATCC 25975 on SDS-PAGE of about 125 kDa (Song and
Jacques, 1999). In contrast, the molecular weight of levansucrase from Z. mobilis found to be 96 kDa by size exclusion chromatography (Goldman et al., 2008). In another finding, the SDS-PAGE revealed a single protein band of 55 to 56 kDa of levansucrase from Z. mobilis (Sangiliyandi et al., 1998). The molecular weight of levansucrase from B. subtilis and B. amyloliquefaciens was reported to be 50 kDa and
52 kDa respectively (Mäntsälä and Puntala, 1982).
95
Lanes: M: (Marker) PageRuler Broad Range Unstained Protein Ladder 1: Partially purified levansucrase from Z. mobilis KIBGE-IB14. 2: Purified levansucrase from Z. mobilis KIBGE-IB14. 3: Pre-zymography (Ethanol precipitated) of purified levansucrase from Z. mobilis KIBGE-IB14. 4: Zymography of purified levansucrase from Z. mobilis KIBGE-IB14.
Figure-26: Electrophoretic pattern and zymogram of purified sample of levansucrase from Z. mobilis KIBGE-IB14.
96 4.7 Amino Acid Analysis of Levansucrase
The composition of amino acids of levansucrase from Z. mobilis KIBGE-IB14 was analyzed after purification. The unique distribution of amino acids in levansucrase of
Z. mobilis KIBGE-IB14 is expressed in term of mole percentage (mole %) in Table-
16. It was revealed from the data that levansucrase of KIBE-IB14 is composed of non-polar (Proline, Alanine and Valine), polar (Glycine and Tryptophan) as well as basic amino acids (Lysine and Arginine) (Figure-27). While other amino acids
(Aspartic acid, Asparagine, Cysteine, Glutamic acid, Glutamine, Glycine, Histidine,
Isoleucine, Leucine, Methionine, Phenylalanine, Serine, Threonine and Tyrosine) were not detected. Among all residues, Proline, Lysine and Arginine showed highest relative percentage i.e. 47.06%, 37.05% and 14.34% respectively. It has been observed that Proline is a distinctive cyclic amino acid that provides stability to proteins at extremely high temperatures because it loses lower conformational entropy upon folding of proteins as compared to other amino acids. Thus, it is mostly present in thermostable proteins (Szpak, 2011). Rathsam et al. (1993) reported that a unique region presents at C-terminal of the FTF of S. salivarius composed of proline residues. It has also been observed that putative nucleotide-binding protein (LsdE) of levansucrase from Gluconacetobacter diazotrophicus consists of a domain which is rich in Glycine and Lysine residues (Arrieta et al., 2004). The molecular structure of levansucrase from B. subtilis suggested that the side chain of Arginine is present adjacent to the central pocket of active site which plays an important role in transfructosylation process (Li et al., 2008).
97 Table-16: Amino acid composition of levansucrase produced by Z. mobilis
KIBGE-IB14.
1 Letter Z. mobilis 3 Letter Amino Acids Abbreviation KIBGE-IB14 Abbreviation Mole %
Alanine Ala A 0.19 Arginine Arg R 14.34 Aspartic Acid Asp D -
Asparagine Asn N - Cysteine Cys C - Glutamic acid Glu E - Glutamine Gln Q - Glycine Gly G 0.26 Histidine His H - Isoleucine Ile I - Leucine Leu L - Lysine Lys K 37.05 Methionine Met M - Phenylalanine Phe F - Proline Pro P 47.06 Serine Ser S - Thrionine Thr T - Tryptophan Trp W 0.28 Tosine Tyr Y -
Valine Val V 0.08
98
Figure-27: Amino acid profile of levansucrase produced by Z. mobilis KIBGE-IB14.
99
Chapter 5 Conclusions
The industrial processes have been modified to a greater extent by the catalytic action
of extracellular levansucrase originating from microbial sources. The cost efficient
enzyme production requires the screening of media components and fermentation
conditions by using submerged fermentation technology. Therefore, the focus of
current study was to optimize the production parameters of levansucrase from Z.
mobilis KIBGE-IB14 as well as the determination of its enzymatic properties by
characterization and purification strategies.
Different microbial strains were screened for levansucrase production and the maximum production of levansucrase was noticed by the strain identified as Z. mobilis KIBGE-IB14 [GenBank: HM102366]. This study has established the potential of newly isolated strain of Z. mobilis KIBGE-IB14 for maximum production of levansucrase under the optimized fermentation conditions of sucrose (15%) at 30°C, pH-6.5 after 24 hours of
cultivation period. Levansucrase was partially purified after ammonium sulphate gradient precipitation (50%) with the fold purification of 1.61 and 31% yield. The fold purification of desalted levansucrase was found to be 3.44 with percent recovery of 34.20%. The desalted sample was subjected to sepharose CL-6B chromatographic column to acquire the highest purity of levansucrase which was 12.13 fold with 1.77% yield. Kinetic analysis of levansucrase revealed that the enzyme performed its maximum activity in 100.0 mM sodium phosphate buffer (pH-6.0) at 35°C after 5.0 minutes of enzyme substrate reaction time period.
100
Hydrolytic activity (Km) and maximum velocity (Vmax) were calculated using Lineweaver-Burk plot by measuring enzyme-substrate reaction in various concentration of sucrose (substrate). The stability profile of levansucrase exhibited that the enzyme showed stability against thermal denaturation at 35°C for 120 minutes. In addition, it was also noted that the levansucrase showed its storage stability at -80°C and 4°C for
longer time period as compared to 37°C for 180 days. The characterization of levansucrase was also determined by investigating its enzyme activity in the presence of metal salts, organic solvents and surfactants. The approximate molecular weight of purified levansucrase was found to be 130.0 kDa by SDS-PAGE which was further confirmed by its zymographic analysis. Amino acid profile of levansucrase expressed that levansucrase of KIBE-IB14
consists of non-polar (Proline, Alanine and Valine), polar (Glycine and
Tryptophan) as well as basic amino acids (Lysine and Arginine).
The essence of the current study is that the reported bacterium exhibited unique
properties such as mild growth conditions, non-pathogenic and extracellular
levansucrase producer makes it a promising candidate to be used at industrial level. It
is also anticipated that purified levansucrase allows it to use in different industries
where high enzyme purity is required for many applications.
In future, the N-terminal sequencing, genome annotation and crystallographic
structure of protein could help in the determination of its three dimensional structure
for better understanding of functions and kinetic mechanisms for the action of
levansucrase.
101
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Chapter 7 APPENDICES
APPENDIX-I
Research Instruments
The instruments applied during research work are enlisted below:
Amino Acid Analyser LC 10A/C-R7A, Shimadzu, USA
Analytical balance EP64C, Explorer Pro, Chaus Corp., New Jersey USA
Spectrophotometer UVS-2700, Dual Beam and Autocell, Labomed Inc., USA
Chromatography System Bio-Rad, California, USA
Refrigerated Centrifuge 5810 R, Eppendorf AG, Hamburg, Germany
Water bath Julabo SW23, Germany
Autoclave Nuve OT 4060, Turkey
Incubators Memmert, Germany
Electrophoresis unit Bio-Rad Laboratories, California, USA
Gel Documentation unit GelDoc 2000, Bio-Rad Laboratories, USA pH meter AD1000, Adwa, Europe
Ultra Centrifuge L-100XP, Beckman-Coulter Inc., USA
Magnetic stirrer RCT Basic, Fisher Scientific, USA
Protein Sequencer ABI Procise 491 Protein Sequencer, USA
Vortex mixer Jeio Tech, USA
Nanophotometer P-Class IMPLEN, Germeny
120 APPENDIX-II
Reagents Index
The list of chemicals used in the current study is described as follows:
Dextrose Sigma-Aldrich
Sucrose Sigma-Aldrich
Nutrient broth Oxide
Calcium chloride Sigma-Aldrich
Dipotassium hydrogen phosphate Serva
Potassium dihydrogen phosphate Serva
Agar-agar Research Organics
TEMED Serva
Ammonium persulphate Serva
Coomassie brilliant blue R-250 Sigma-Aldrich
Copper sulfate Sigma-Aldrich
Ethanol Lab-Scan
Ethylenediaminetetraacetic acid MP Biomedicals
Ferrous sulphate Sigma-Aldrich
Folin-Ciocalteu reagent Merck
Formaldehyde Sigma-Aldrich
Glycerol Serva
Methanol Sigma-Aldrich
Sodium chloride Sigma-Aldrich
Congo red dye Serva
Sodium dodecyl sulphate Serva
Sodium hydroxide Sigma-Aldrich
Sodium potassium tartrate Sigma-Aldrich
121 Tris Serva
Tween 80 Sigma-Aldrich
Urea Sigma-Aldrich
Yeast extract Oxide
Mercury chloride Sigma-Aldrich
Manganese chloride Serva
Sodium acetate Serva
Sodium citrate MP Biomedicals, LLC
Hydrochloric acid Sigma-Aldrich
Nickel chloride Serva
Barium chloride Serva
Potassium chloride Sigma-Aldrich
Acetic acid Riedel De Haen
Citric acid Merck
Copper chloride Sigma-Aldrich
Cesium chloride Serva
Triton X-100 Sigma-Aldrich
DMSO Serva
Zinc chloride Sigma-Aldrich
Ethidium bromide Serva
Schiff’s reagent Sigma-Aldrich
Broad range unstained protein ladder Thermo
122 APPENDIX-III
Gram’s Staining
Preparation of Reagents
Reagents prepared for Gram’s staining of culture prior to start of experiment are as
follows:
Crystal Violet 1.0 g crystal violet dissolved in 10.0 ml of 95% ethanol. 0.4 g ammonium oxalate dissolved in 40.0 ml double deionized water
For crystal violet reagent, the above mentioned chemical solutions mixed together.
Gram’s Iodine Iodine crystal (1.0 g) Potassium iodide (2.0 g)
The solution of iodine crystal and potassium iodide were added in 300.0 ml double
deionized water. Then, this prepared reagent left overnight to make a homogenous
solution.
Safranin Solution
Stock Solution (10×) 2.5 g safranin was dissolved in 100.0 ml of 95% ethanol.
Working Solution (1×)
Stock solution of 10.0 ml was mixed with 90.0 ml double deionized water to prepare a working solution of 1×.
Decolorizing Solution
95% ethanol was used as a decolorizing solution.
123 Method
1. Crystal violet solution was flooded on heat fixed culture smear and left for one
minute.
2. The slide was washed with tap water after 1.0 minute.
3. Then, the smear was flooded with Gram’s iodine solution for one minute and
then washed with tap water.
4. For the purpose of destaining, 95% ethanol was poured onto the smear for
10.0 seconds and immediately washed with tap water.
5. A solution of safranin was poured on the smear for 30 seconds and then again
washed with tap water.
6. Slide was allowed to air dry.
7. Microscopy of stained smear was performed by using microscope under oil
immersion lens.
124 APPENDIX-IV
Spore Staining
Reagent Preparation
Following reagents were prepared prior to start the experiment for staining of spores.
Malachite Green
7.6 g malachite green was dissolved in 100.0 ml double deionized water.
Safranin
The solution was prepared according to the above mentioned concentration and procedure.
Method
1. In heat fixing method, a thin smear was prepared by passing 5.0 to 6.0 times over flame.
2. Malachite green was poured on smear and kept for 10.0 minutes.
3. Slide was rinsed with tap water
4. A counter stain safranin was used to stain the smear for 15.0 seconds.
5. This counter dye was removed by washing with tap water gently.
6. At last, slide was observed under oil immersion lens of microscope.
125 APPENDIX-V
Buffer Preparation
The buffers used in experiment of research work includes 100.0 mM of each acetate, citrate and sodium phosphate.
Citrate Buffer (100.0 mM, pH: 5.0-6.0)
The preparation of citric acid buffer requires a weak acid (100.0 mM citric acid solution) and a conjugate base (100.0 mM sodium citrate solution). In 100.0 mM of citric acid solution, 1.9213 g citric acid was dissolved in double deionized water with the final volume was adjusted up to 100.0 ml in a volumetric flask. Similarly, 100.0 ml of sodium citrate di-hydrate solution was formed by adding its 2.94 g in double deionized water with adjustment of its final volume. Various buffers with different pH ranging from 5.0 to 6.0 was prepared by slowly mixing weak acid and conjugate base until the desired pH was achieved. Sodium Phosphate Buffer (100 mM, pH: 6.0-8.0)
For the preparation of sodium phosphate buffer, di-sodium hydrogen phosphate
(1.779 gm) and sodium di-hydrogen phosphate (1.56 gm) were separately added in double deionized water adjusting the final volume of 100.0 ml by volumetric flask.
The required pH was obtained through digital pH meter by mixing these solutions. Acetate Buffer (100.0 mM, pH: 7.0-8.0)
Acetate buffer was prepared by the mixing of sodium acetate and acetic solutions. In
100.0 mM sodium acetate solution, its 0.82 g was mixed in deionized water adjusting the final volume of 100.0 ml by using volumetric cylinder. Whereas, a separate solution of acetic acid (100.0 mM) was prepared by adding 0.572 ml of 100% pure acetic acid in double deionized water making its final volume up to 100.0 ml. Then,
126 the desired pH of buffer was attained by gradual mixing of both sodium acetate and acetic acid solutions. Glycine-Sodium Hydroxide Buffer (100.0 mM, pH: 8.0-9.0)
Sodium hydroxide (0.4 g) and glycine (0.75 g) were prepared separately in 100.0 ml double deionized water and mixed until desired pH value achieved.
127 APPENDIX-VI
Preparation of Reagents for SDS-PAGE
Solution A (Acrylamide Bis-acrylamide) 30.0 g Acrylamide 0.8 g Bis-acrylamide
The polymer and cross linker were dissolved in 80.0 ml of double deionized water ® with the final volume made up to 100.0 ml. Whatman filter paper No.1 was used to
filter the sample and then stored at 4°C for further use.
Solution B (1.5 M Tris-HCl; pH-8.8) 36.3 g Tris-HCl
About 1.5 M concentration of solution was achieved by dissolving salt in 200.0 ml
double deionized water while attaining its pH up to 8.8 by slow incorporation of 1.0 N
hydrochloric acid (HCl). The prepared solution was kept at 4°C.
Solution C (0.5 M Tris-HCl; pH-6.8) 6.05 g Tris-HCl
In order to make 100.0 ml of 0.5 M Tris-HCl solution, the salt was dissolved in
double deionized water. The pH of solution C was adjusted up to 6.8 by gradual
addition of 1.0 N hydrochloric acid (HCl) which was then stored at 4°C.
Solution E (Ammonium persulfate; APS, 2.0%)
In APS solution, 10.0 g salt was dissolved in 100.0 ml double deionized water. The
reagent should be freshly prepared just before use and must be placed on ice bath
during preparation.
TEMED (N, N, N', N'-Tetramethylethylenediamine)
During this process, a commercially prepared TEMED was used which must be stored
at 4°C.
128 Reservoir Buffer (0.025 M Tris-glycine buffer; pH-8.5): 3.025 g Tris 14.4 g Glycine
To achieve a pH value 8.5, both chemicals were dissolved in 1000.0 ml of double
deionized water. Prepared buffer was kept at 4°C.
Sample Diluting Buffer (2.0 ×, Tris-HCl; pH-6.8): 10% Glycerol 0.5 M Tris-HCl; pH-6.8 (Solution C)
-1 1000 gL SDS 2-mercaptoethanol Bromophenol blue (Tracking dye)
In this reagent, 2.5 ml solution C and 2.0 ml glycerol was thoroughly mixed. Then,
add few crystals of bromophenol blue dye and 1.0 ml of 2-mercaptoethanol. The final
volume was made up to 10.0 ml using double deionized water. About 1.0 ml aliquots
from this buffer was prepared and stored at −20°C.
Staining Solution (Coomassie brilliant blue R-250): Coomassie brilliant blue R-250 Methanol Acetic acid
Staining solution was prepared by mixing of 0.1 g of Coomassie brilliant blue R-250
with 40.0 ml of methanol and 10.0 ml of glacial acetic acid in 100.0 ml of double
® deionized water. Whatman filter paper No.1 was used to filter the staining solution.
For further applications, prepared dye was stored at room temperature.
129 Destaining Solution Methanol Glacial acetic acid
The destaining solution requires a mixture of 1:4 ratio prepared from glacial acetic
acid and methanol respectively. Its final volume was adjusted up to 1000.0 ml with
double deionized water. This reagent was kept at room temperature.
130 APPENDIX-VII
Preparation of Reagents for Enzyme Assay Sample
Purified levansucrase Buffer
100 mM sodium phosphate buffer, pH-6.0 Substrate
Sucrose (250.0 mM) Reagent Kit
Glucose oxidase peroxidase (GOD-PAP) kit (Randox Laboratories Ltd., UK) Standard
-1 Glucose (10.0 g L )
131 APPENDIX-VIII
Reagents Preparation for Total Protein Assay Reagent-A Sodium hydroxide (4.0 g) Sodium carbonate (20.0 g)
The pellets of sodium hydroxide were dissolved in double deionized water (900.0 ml).
Then, in the same solution, sodium carbonate was incorporated. With the help of
double deionized water, the final volume of reagent was made up to 1000.0 ml. This
prepared reagent was stored at 4°C.
Reagent-B1 Copper sulphate (1.0 g)
Copper sulfate was dissolved in 90.0 ml double deionized water. After complete
mixing of compound, the final volume of solution was adjusted up to 100.0 ml and
then kept at room temperature.
Reagent-B2 Sodium potassium tartrate (2.0 g)
This compound was mixed in double deionized water of 100.0 ml. The prepared
reagent-B2 kept at room temperature.
Reagent-C
Reagent A, B1 and B2 were mixed together in the ratio of 100:1:1 respectively. Fresh
solution of reagent-C must be prepared prior to use.
Reagent-E
Commercially available Folin-Ciocalteu reagent was used by making the dilution of
reagent in the ratio of 1:1 with double deionized water. Before any usage, diluted
Reagent-E was freshly prepared.
132 Standard
-1 Stock Solution (1.0 mg ml )
About 0.1 gm of bovine serum albumin was dissolved in double deionized water
(100.0 ml) for the preparation of stock solution. It should be placed at 4°C.
-1 Working Solution (0.250 mg ml )
Stock solution of 0.1 ml was diluted up to 4.0 ml with double deionized water in the ratio of 1:3.
133 APPENDIX-IX
List of Publications
Research Articles:
1. Shaheen, S., Aman, A. and Siddiqui, N. N. (2017). Influence of Metal ions, Surfactants and Organic Solvents on the Catalytic Performance of Levansucrase from Zymomonas mobilis KIBGE-IB14. Journal of Basic and Applied Sciences, 13: 41-46.
2. Shaheen, S., Aman, A., Qader, S. A. U. and Siddiqui, N. N. (2017). Hyper- production of levansucrase from Zymomonas mobilis KIBGE-IB14 using submerged fermentation technique. Pakistan Journal of Pharmaceutical Sciences. (Accepted).
GenBank Deposition:
GenBank Accession # KF905652 (2013)
Qaiser,S., Shahid,F., Shaheen,S., Nawaz,A., Hameed,A., Aman,A., Ajmal,M., Siddiqui,N.N. and Ul Qader,S.A. Aspergillus versicolor strain KIBGE-IB37 18S ribosomal RNA gene, partial sequence; mitochondrial.
Paper/ Poster Presentation in Conference
2016 Nadir Naveed Siddiqui. Sidra Shaheen, Zahida Shabbir, Afsheen Aman, (Oral) Shah Ali Ul Qader “Mutagenesis studies of glucosyltransferase for the production of bioloymer from Leuconostoc mesenteroides KIBGE-IB22M20” 15th International symposium on Biopolymers (ISBB 2016), September 26 –29, 2016, Madrid Spain. 2016 Nadir Naveed Siddiqui. Sidra Shaheen, Zahida Shabbir, Afsheen Aman, (Poster) Shah Ali Ul Qader “Effect of UV irradiation on production of glucosyltransferase from Leuconostoc mesenteroides” 13th Biennial Conference on “Recent Advances & Challenges in Molecular
Biology, Biochemistry and Biotechnology”, August 25-27, 2016 being hosted by Center for Advanced Drug Research, COMSATS, Abottabad. 2015 Sidra Shaheen, Nadir Naveed Siddiqui, and Shah Ali Ul Qader. (Oral) “Microbial isolation and production optimization for high yield of levansucrase from Zymomonas mobilis KIBGE-IB 14” 3rd International Conference on “Environmental Horizon; Nurture Nature: Serve to Conserve” Jointly organized by Department of chemistry and International Centre for Chemical and Biological Sciences (ICCBS), University of Karachi, Karachi, Pakistan on January 9-11, 2015.
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