DOCSLIB.ORG

Iluud Promotor: Prof.Dr .N.C.M .Laan E Voormalighoogleraa R Ind Ebiochemi E

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

EXPLORINGTH E LIMITSO F VANILLYL-ALCOHOL OXIDASE iLuUD Promotor: Prof.dr .N.C.M .Laan e Voormalighoogleraa r ind eBiochemi e Co-promotor: Dr. W.J.H. vanBerke l Universitair docent, DepartementAgrotechnologi ee n Voedingswetenschappen, Laboratoriumvoo r Biochemie, Wageningen Universiteit Promotiecommissie: Prof.dr .A.J.R .Heck ,Universitei t vanUtrech t Prof.dr .W.M .d eVos ,Wageninge n Universiteit Prof.dr .R . Wever,Universitei t vanAmsterda m Dr.A .Mattevi ,Universit a degli Studid iPavi a t.)o<S2o\ .Z-^fc) /J RobertH.H .va nde n Heuvel EXPLORINGTH E LIMITSO F VANILLYL-ALCOHOL OXIDASE Proefschrift ter verkrijging van degraa d vandocto r opgeza gva nd erecto r magnificus vanWageninge n Universiteit, prof.dr . ir.L .Speelman , inhe t openbaar teverdedige n opvrijda g 6apri l200 1 desnamiddag st ehal ftwe e ind eAul a Contents Chapter1 Introduction: Biocatalytic production of natural vanillin andopticall ypur e compounds Chapter2 Enzymatic synthesis ofnatura l vanillin 11 Chapter3 Regio-an dstereospecifi c conversion of4-alkylphenol s 23 byth ecovalen t flavoprotein vanillyl-alcohol oxidase Chapter4 Tuning the reactivity of enzyme-bound /j-quinone 39 methidesb ymediu m engineering Chapter5 Direction of the reactivity of vanillyl-alcohol oxidase 47 with 4-alkylphenols Chapter6 Asp17 0 is crucial for the redox properties of vanillyl- 55 alcohol oxidase Chapter 7 Inversion of stereospecificity of vanillyl-alcohol 75 oxidase Chapter8 Covalent flavinylation is essential for efficient redox 89 catalysis invanillyl-alcoho l oxidase Chapter9 Detection of intact megaDalton protein assemblies of 107 vanillyl-alcohol oxidaseb ymas s spectrometry References 115 Summary 129 Samenvatting 135 Nawoord 141 Curriculumvita e 143 Listo f publications 145 ^oVW {-2.16D Stellingen behorende bij het proefschrift Exploring the limits ofvanillyl-alcoho l oxidase 1. Er is geen fundamentele reden waarom enzymen minder actief zouden zijn in niet-waterige mediada n inwaterig emedia . Klibanov,A .M .Improvin genzyme sb yusin gthe mi norgani csolvents .Nature 409 , 241-246(2001). 2. Om een winnaar te zijn moet een atleet niet alleen pijn accepteren, maar ook lereno merme et e levene nzelf s lereno me rnaa rui t te kijken. 3. Net zoals een mens het 'recht' heeft om te leven heeft een mens ook het 'recht' omt esterven . 4. Tussen humaan en Escherichia coli methylenetetrahydrofolate reductase bestaat 30% sequentie identiteit. Daarom is voorzichtigheid geboden wanneer men de interpretatie van de kristalstructuur van het Escherichiacoli enzym wil extrapoleren naarhe thuman eenzym . Guenther, B.D. et al. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia.Nat. Struct. Biol. 6,359-36 5(1999) . 5. Dewereldbevolkin g valtt everdele n invluchtelinge n entoeristen . DeNapolitaans e schilder FrancescoClemente . 6. Kleuren leiden alleen maaraf , het gaat omd e contrasten. 7. Men zou vaker bij zichzelf te rade moeten gaan in plaats van verantwoordelijkheden aft ewende n naar overheidsinstanties. 8. Gezien het feit dat een verbetering van de enantiomere ratio van 1{e.e. = 0) naar 1000 (e.e. > 99%) overeenkomt met een AAGvoo r katalyse van slechts 17kJ*mor 1 is het niet verassend dat een of enkele aminozuurmutaties in een enzymgrot e effecten kunnenhebbe n opd e stereospecificiteit. May, O., Nguyen, P.T. & Arnold, F.H. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of L-methionine. Nat. Biotechnol. 18,317-32 0 (2000). van den Heuvel, R.H.H., Fraaije, M.W., Ferrer, M., Mattevi,A . & van Berkel,W.J.H . Inversion of stereospecificity of vanillyl-alcohol oxidase. Proc. Natl.Acad. Sci. U.S. A. 97, 9455-9460 (2000). 9. Sporten resulteert inhog e dosesgeestelijk e enlichamelijk e energie. Chapter1 BIOCATALYTIC PRODUCTION OF NATURAL VANILLIN AND OPTICALLY PURE COMPOUNDS Chapter1 Introduction The research described in this thesis was performed within the framework of the Innovation Oriented Research Program (IOP) Catalysis of the Dutch Ministry of Economy Affairs. Together with six other projects, we were active in the Enzymatic Oxidation cluster. The aim within this cluster was to evaluate the potential of oxidative enzymes for thebiocatalyti c production ofindustriall y relevant compounds. In recent years, the interest for enzymatic processes has increased because of the rising demand for natural and environmentally friendly ingredients and products that have improved functionalities. The key advantage of enzymes is that they catalyze reactions specifically, often regio- and/or stereospecifically, and under mild conditions,thereb y saving energy. Chemical processes are often less selective and less environmentally friendly. Another important factor that has triggered the use of enzymes for the production of valuable compounds such as pharmaceuticals, fine- chemicals, and flavors and fragrances is the enormous progress made in genetic, medium, and protein engineering. These techniques provide us with the tools to overproduce and redesign enzymes, paving the way to form large amounts of the product of interest. In this chapter, we discuss the biocatalytic methods for the production of the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) (1) and the production of optically pure aromatic compounds. Next, we introduce the enzyme vanillyl-alcohol oxidase as being a promising candidate to produce these compounds. Production of naturalvanilli n Vanillin iswidel y used asa flavorin g compound inth e food industry.Vanilli n has also some potential medical applications, since in recent years, itha s become apparent that vanillin is an antioxidant and may have the potential to prevent oxidative damage in mammalian cells ''2. Moreover, it has been reported that vanillin is a potential agent for thetreatmen t of sickle cell anemia3. Natural vanillin is produced from glucovanillin (2) when the beans of the orchid Vanillaplanifolia are submitted to a multi-step curing procedure 4 (Fig. 1). After this curingprocess ,vanilli n isth emos t abundant component ofth e bean at aleve l of about 2-3% by weight 5'6. Other important aromatic components in the bean are 4- hydroxybenzaldehyde, 4-hydroxybenzylalcohol, and vanillic acid (4-hydroxy-3- methoxybenzoic acid). At present, only 0.4% of the world flavor market (5*10 4 kg/year out of 12*106kg/year ) isoriginatin g from V. planifolia with synthetic vanillin providing the remainder 7. Synthetic vanillin can be obtained from a number of chemical procedures using the feedstocks coniferin (glucoside of coniferyl alcohol (4- hydroxy-3-methoxycinnamyl alcohol), eugenol (4-allyl-2-methoxyphenol), lignin, or Introduction guaiacol (2-methoxyphenol) 4, and is by far cheaper than the natural product from vanillabeans . With the increasing interest in natural products alternative processes are being developed to produce natural vanillin. This is possible since the European Union legislation allows the origin of natural products deriving from living cells or their components, including incubations in which only isolated enzymes are applied 8.Th e Vanilla plant is not ideal for biotechnological purposes, as the vanillin pathway is not very actively expressed and theplan t is slowly growing 4'9. Therefore, much effort has been put in the production of vanillin metabolites using microorganisms. Microbes can be selected for their ability to grow on a precursor of vanillin as sole energy and carbon source andth e expression levels canb eraise d bymolecula r genetics. Among the potential feedstocks suggested in literature, eugenol (3), the main component of clove oil, and ferulic acid (4-hydroxy-3-methoxycinnamic acid) (4), an abundant cell-wall material, are the most promising (for recent reviews see 4'10). Eugenol catabolism to vanillin via ferulic acid hasbee n characterized inPseudomonas strains n'12 andSerratia marcescens 13(Fig . 1).I ti sinterestin g tomentio n heretha tth e first reaction inth epathwa y from eugenolt o ferulic acid, thehydroxylatio n of eugenol to coniferyl alcohol, is also catalyzed by vanillyl-alcohol oxidase from Penicillium simplicissimum l4. Vanillic acid (5) is the major product obtained from the catabolism of ferulic acid by several microorganisms like Pseudomonas 15, Streptomyces 16, and Rhodotorula 1? species. Both carboxylic acids can be used as starting material for the production of natural vanillin. It has been reported that a Nocardia strain produces vanillin from vanillic acid 18. Substantially higher levels of vanillin have been obtained with Amycolatopsis 19 and Streptomycessetonii l6. Lesage-Meessen et al. 8'20 reported a two-step bioconversion process for the production of vanillin from ferulic acid. First, ferulic acid iscatabolize d byAspergillus nigert ovanilli c acid. Second, vanillic acid is reduced byPycnoporus cinnabarinus intovanilli n (Fig. 1) Next to microbes, isolated enzymes form an attractive alternative source for the production of natural vanillin. The pungent principal of red pepper, capsaicin (8- methyl-W-vanillyl-6-nonenamide) (6) is a precursor of vanillin and can be easily obtained at relatively low cost. In the first step of the conversion of capsaicin to vanillin, capsaicin is enzymically or chemically hydrolyzed to form vanillylamine (4- hydroxy-3-methoxybenzylamine) (7) 21. This latter compound can be easily oxidized byamin e oxidases 22an dvanillyl-alcoho l oxidase 23yieldin g vanillin (Fig. 1). Chapter 1 COOH J^* N"S» D OCH3 OCH R =C4H6 —CH=CH—CH(CH
Recommended publications
  • Biotransformation of Vanillin Into Vanillyl Alcohol by a Novel Strain of Cystobasidium Laryngis Isolated from Decaying Wood

    Biotransformation of Vanillin Into Vanillyl Alcohol by a Novel Strain of Cystobasidium Laryngis Isolated from Decaying Wood

    Rönnander et al. AMB Expr (2018) 8:137 https://doi.org/10.1186/s13568-018-0666-4 ORIGINAL ARTICLE Open Access Biotransformation of vanillin into vanillyl alcohol by a novel strain of Cystobasidium laryngis isolated from decaying wood Jonas Rönnander1* , Joel Ljunggren2 , Erik Hedenström2 and Sandra Ann Ingela Wright1* Abstract Vanillin is an aromatic aldehyde found as a component of lignocellulosic material, and in the cured pods of orchi- daceae plants. Like other phenolic substances, vanillin has antimicrobial activity and can be extracted from lignin either by a thermo-chemical process or through microbial degradation. Vanillin, can serve as a model monomer in biodegradation studies of lignin. In the present study, a yeast isolated from decaying wood on the Faroe Islands, was identifed as Cystobasidium laryngis strain FMYD002, based on internal transcribed spacer sequence analysis. It dem- onstrated the ability to convert vanillin to vanillyl alcohol, as detected by ultra-high performance liquid chromatogra- phy–quadrupole-time-of-fight. Structural analysis of vanillyl alcohol was carried out by using gas chromatography– mass spectrometry and 1H NMR spectroscopy, and further verifed by synthesis. The reduction of vanillin to vanillyl alcohol has been documented for only a few species of fungi. However, to our knowledge, this biotransformation has not yet been reported for basidiomycetous yeast species, nor for any representative of the subphylum Pucciniomyco- tina. The biotransformation capability of the present strain might prove useful in the industrial utilisation of lignocel- lulosic residues. Keywords: Vanillin, Cystobasidium, Bioconversion, Biodegradation, Cystobasidiomycetes, Rhodotorula Introduction an aromatic aldehyde derived from the guaiacyl subunit Lignin is an abundant polymer in nature, a natural aro- of lignin (Brebu and Vasile 2010; Fache et al.
  • Vanillyl Alcohol Dehydrogenase) from Rhodopseudomonas Acidophila M402 Purification, Identification of Reaction Product and Substrate Specificity

    Vanillyl Alcohol Dehydrogenase) from Rhodopseudomonas Acidophila M402 Purification, Identification of Reaction Product and Substrate Specificity

    Agric. Biol Chem., 47 (10), 2173~2183, 1983 2173 A NewDye-Linked Alcohol Dehydrogenase (Vanillyl Alcohol Dehydrogenase) from Rhodopseudomonas acidophila M402 Purification, Identification of Reaction Product and Substrate Specificity Kei Yamanaka and Yasutaka Tsuyuki Institute of Applied Biochemistry, and Graduate School of Master's Program in Environmental Sciences, The University of Tsukuba, Sakura-mura, Niihari-gun, Ibaraki 305, Japan Received October 19, 1982 A new dye-linked alcohol dehydrogenase (vanillyl alcohol dehydrogenase) was purified to homogeneity from cells of Rhodopseudomonas acidophila strain M402 grown aerobically on vanillyl alcohol. The reaction product from vanillyl alcohol was identified as vanillin as judged by its melting point, elemental analysis and IR, mass and NMR spectra. The molecular weight of the enzyme was estimated to be approximately 72,000 as determined by gel filtration and the isoelectric point was pH 6.01. The most characteristic feature of this enzyme is its wide substrate specificity range. The enzyme catalyzes the dehydrogenation of various aromatic and aliphatic alcohols and aldehydes with phenazine methosulfate as electron acceptor. The active substrates of this enzyme are as follows: Vanillyl alcohol, benzyl alcohol, cinnamyl alcohol, 2-phenylethanol, 2-phenoxyethanol, aliphatic alcohols of C2 to G8, rr<m?-cinnamaldehyde, formaldehyde, propionaldehyde and butyraldehyde. The highest activities were obtained with vanillyl alcohol, rc-propanol and ^-butanol at the same level. The apparent Kmvalues were as follows: 112/zm for vanillyl alcohol, 7/jm for benzyl alcohol, 180/im for «-propanol, 14^m for #-butanol, 20/zm for 2-phenoxyethanol, 12/iM for 2-phenylethanol and 105 /zm for butyraldehyde. These activities were confirmed to be catalyzed by a single enzyme by activity staining on polyacrylamide gels.
  • Precursor of Ether Phospholipids Is Synthesized by a Flavoenzyme

    Precursor of Ether Phospholipids Is Synthesized by a Flavoenzyme

    Precursor of ether phospholipids is synthesized by a flavoenzyme through covalent catalysis Simone Nencia, Valentina Pianoa, Sara Rosatib, Alessandro Alivertic, Vittorio Pandinic, Marco W. Fraaijed, Albert J. R. Heckb, Dale E. Edmondsone, and Andrea Mattevia,1 aDepartment of Biology and Biotechnology, University of Pavia, 27100 Pavia, Italy; bBiomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University and Netherlands Proteomics Centre, 3584 CH Utrecht, The Netherlands; cDepartment of Biosciences, University of Milan, 20133 Milan, Italy; dMolecular Enzymology Group, University of Groningen, 9747 AG Groningen, The Netherlands; and eDepartment of Biochemistry, Emory University, Atlanta, GA 30322 Edited by Emil F. Pai, Ontario Cancer Institute/Princess Margaret Hospital, Toronto, ON, Canada, and accepted by the Editorial Board October 5, 2012 (received for review August 31, 2012) The precursor of the essential ether phospholipids is synthesized ADPS enzymes, whereas type 1 arises from mutations in PEX7, by a peroxisomal enzyme that uses a flavin cofactor to catalyze the protein mediating the peroxisomal import of ADPS (9–12). a reaction that does not alter the redox state of the substrates. Here we report a structural and mechanistic investigation of The enzyme crystal structure reveals a V-shaped active site with mammalian ADPS in WT and mutated forms. The biochemical a narrow constriction in front of the prosthetic group. Mutations hallmark of the enzyme is that it uses a redox cofactor, flavin causing inborn ether phospholipid deficiency, a very severe adenine dinucleotide (FAD), to catalyze a reaction that does not genetic disease, target residues that are part of the catalytic alter the redox state of the substrates (10, 13).
  • 2010 Physical Biosciences Research Meeting

    2010 Physical Biosciences Research Meeting

    2010 Physical Biosciences Research Meeting Sheraton Inner Harbor Hotel Baltimore, MD October 17-20, 2010 Office of Basic Energy Sciences Chemical Sciences, Geosciences & Biosciences Division 2010 Physical Biosciences Research Meeting Program and Abstracts Sheraton Inner Harbor Hotel Baltimore, MD October 17-20, 2010 Chemical Sciences, Geosciences, and Biosciences Division Office of Basic Energy Sciences Office of Science U.S. Department of Energy i Cover art is taken from the public domain and can be found at: http://commons.wikimedia.org/wiki/File:Blue_crab_on_market_in_Piraeus_-_Callinectes_sapidus_Rathbun_20020819- 317.jpg This document was produced under contract number DE-AC05-060R23100 between the U.S. Department of Energy and Oak Ridge Associated Universities. The research grants and contracts described in this document are, unless specifically labeled otherwise, supported by the U.S. DOE Office of Science, Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division. ii Foreword This volume provides a record of the 2nd biennial meeting of the Principal Investigators (PIs) funded by the Physical Biosciences program, and is sponsored by the Chemical Sciences, Geosciences, and Biosciences Division of the Office of Basic Energy Sciences (BES) in the U.S. Department of Energy (DOE). Within DOE-BES there are two programs that fund basic research in energy-relevant biological sciences, Physical Biosciences and Photosynthetic Systems. These two Biosciences programs, along with a strong program in Solar Photochemistry, comprise the current Photo- and Bio- Chemistry Team. This meeting specifically brings together under one roof all of the PIs funded by the Physical Biosciences program, along with Program Managers and staff not only from DOE-BES, but also other offices within DOE, the national labs, and even other federal funding agencies.
  • Hydrodeoxygenation of Lignin Model Compounds Over a Copper Chromite

    Hydrodeoxygenation of Lignin Model Compounds Over a Copper Chromite

    Applied Catalysis A: General 447–448 (2012) 144–150 Contents lists available at SciVerse ScienceDirect Applied Catalysis A: General jo urnal homepage: www.elsevier.com/locate/apcata Hydrodeoxygenation of lignin model compounds over a copper chromite catalyst ∗ Keenan L. Deutsch, Brent H. Shanks Department of Chemical & Biological Engineering, Iowa State University, Ames, IA 50011, USA a r t i c l e i n f o a b s t r a c t Article history: The hydrodeoxygenation of benzyl alcohol, phenol, anisole, o-cresol, catechol, guaiacol, and vanillyl alco- ◦ Received 19 April 2012 hol were carried out from 150 to 275 C at 50 bar H2 with a CuCr2O4·CuO catalyst in a decalin solvent. Received in revised form 5 September 2012 The hydroxymethyl group of benzyl alcohol was found to be highly reactive towards hydrogenolysis Accepted 13 September 2012 to form toluene. Demethoxylation of anisole to form benzene was found to be the primary reaction Available online 8 October 2012 pathway in contrast to demethylation and transalkylation reactions, which are more prevalent for con- ventional hydrotreating catalysts. The hydroxyl group of phenol strongly activated the aromatic ring Keywords: towards hydrogenation forming cyclohexanol which was subsequently dehydrated and hydrogenated Hydrogenolysis Hydrodeoxygenation to form cyclohexane. Reaction networks of increasing complexity were devised for the major functional groups and integrated to describe the most complex molecule studied, vanillyl alcohol. Copper catalyst Lignin © 2012 Elsevier B.V. All rights reserved. Bio-oil upgrading Copper chromite 1. Introduction liquid product from fast pyrolysis [6]. The lignin-derived compo- nents of biomass are commonly used as model compounds for The utilization of biomass to produce fuels and chemicals is a HDO because they possess the aromaticity that is important to topic of increasing importance as petroleum prices rise and reserves maintain to minimize hydrogen consumption [2].
  • The Institute of Paper Chemistry

    The Institute of Paper Chemistry

    The Institute of Paper Chemistry Appleton, Wisconsin Doctor's Dissertation Reaction Products of Lignin Model Compounds and Sodium Hydrosulfide Thomas G. Zentner June, 1953 A STUDY OF THE REACTION PRODUCTS OF LIGNIN MODEL COMPOUNDS AND SODIUM HYDROSULFIDE A thesis submitted by Thomas G. Zentner B.S. 1948, Texas A & M College M.S. 1950, Lawrence College in partial fulfillment of the requirements of The Institute of Paper Chemistry for the degree of Doctor of Philosophy from Lawrence College, Appleton, Wisconsin June, 1952 TABLE OF CONTENTS INTRODUCTION 1 HISTORICAL REVIEW 2 PRESENTATION OF THE PROBLEM 8 EXPERIMENTAL PROCEDURES 10 Synthesis of Compounds 10 Synthesis of 1-(4-Hydroxy-3-methoxyphenyl)-l-propanol 10 Synthesis of 1-(4-Benzoxy-3-methoxyphenyl)-l-propanol 11 Reaction of 1-(4-Benzoxy-3-methoxyphenyl) l-propanol with Benzyl Chloride 12 Synthesis of Propiovanillone 14 Synthesis of (-(4-Acetyl-2-methoxyphenoxy)acetovanillone 17 Attempted Synthesis of a-(2-Methoxy-4-methylphehoxy)- propiovanillone 17 Attempted Synthesis of 4-[l-(2-Methoxy-4-methylphenoxy)- l-propyl]guaiacol 21 Synthesis of 2t,4-Dihydroxy-3-methoxychalcone 23 Synthesis of 4,4'-Dihydroxy-3,3 -dimethoxychalcone 24 Synthesis of 4-Propionylpyrocatechol 24 Synthesis of Bis[l-(4-hydroxy-3-methoxyphenyl)-1- propyl] Disulfide 26 Reaction of Isolated Native Lignin with Potassium Hydrosulfide 27 Sodium Hydrosulfide Cooks 28 Cooking Liquor 28 General Procedures 30 Propiovanillone' 32 iii 2 ,4-Dihydroxy-3-methoxychalcone 34 4,4'-Dihydroxy-3,3 '-methoxychalcohe 37 4'-Hydroxy-3t-methoxyflavanone 39 2-Vanillylidene-3-coumaranone 41 Vanillin 44 G- (4-Acetyl-2-methoxyphenoxy)acetovanillone 45 1-(4-Hydroxy-3-methoxyphenyl)-1-propanol 49 DISCUSSION 58 SUMMARY AND CONCLUSIONS 69 LITERATURE CITED 71 INTRODUCTION Although the kraft process has been in use for many years, there is no sound explanation of the role played by the sulfide ion in the cook.
  • Wo 2010/045415 A2

    Wo 2010/045415 A2

    (12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 22 April 2010 (22.04.2010) WO 2010/045415 A2 (51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every A61K 31/196 (2006.01) A61P 29/00 (2006.01) kind of national protection available): AE, AG, AL, AM, A61K 31/015 (2006.01) A61P 19/02 (2006.01) AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, A61K 31/165 (2006.01) A61K 45/06 (2006.01) CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (21) International Application Number: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, PCT/US2009/060768 KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, (22) International Filing Date: ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, 15 October 2009 (15.10.2009) NO, NZ, OM, PE, PG, PH, PL, PT, RO, RS, RU, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TJ, TM, TN, TR, TT, (25) Filing Language: English TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (26) Publication Language: English (84) Designated States (unless otherwise indicated, for every (30) Priority Data: kind of regional protection available): ARIPO (BW, GH, 12/288,085 16 October 2008 (16.10.2008) US GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, (71) Applicant (for all designated States except US): NO- TM), European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, VARTIS AG [CH/CH]; Lichstrasse 35, CH-4056 Basel ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, (CH).
  • Page 1 of 35 RSC Advances

    Page 1 of 35 RSC Advances

    RSC Advances This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. This Accepted Manuscript will be replaced by the edited, formatted and paginated article as soon as this is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. www.rsc.org/advances Page 1 of 35 RSC Advances Performance of cobalt titanate towards H 2O2 based catalytic oxidation of lignin model compound Mariom Shilpy, Muhammad Ali Ehsan, Tammar Hussein Ali *, Sharifah Bee Abd Hamid *, Md. Eaqub Ali a Nanotechnology and Catalysis Research Center (NANOCAT), University Malaya, Kuala Lumpur 50603, Malaysia. E-mail: [email protected], [email protected] Abstract Manuscript Mixed metal cobalt titanium oxide (CoTiO 3) prepared by solution phase method has been evaluated for the liquid phase catalytic oxidation of vainly alcohol to vanillin using H2O2 as an oxygen source. The morphology, phase composition and crystal structure of the freshly prepared Accepted and reused CoTiO 3 catalyst was studied by SEM, EDX, XRD, XPS and Raman spectroscopy.
  • Biochemical Characterization of 2-Nitropropane Dioxygenase from Hansenula MRAKII

    Biochemical Characterization of 2-Nitropropane Dioxygenase from Hansenula MRAKII

    Georgia State University ScholarWorks @ Georgia State University Chemistry Theses Department of Chemistry 4-22-2008 Biochemical Characterization of 2-Nitropropane Dioxygenase from Hansenula MRAKII Slavica Mijatovic Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_theses Recommended Citation Mijatovic, Slavica, "Biochemical Characterization of 2-Nitropropane Dioxygenase from Hansenula MRAKII." Thesis, Georgia State University, 2008. https://scholarworks.gsu.edu/chemistry_theses/8 This Thesis is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Theses by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. BIOCHEMICAL CHARACTERIZATION OF 2-NITROPROPANE DIOXYGENASE FROM HANSENULA MRAKII by SLAVICA MIJATOVIC Under the Direction of Dr. Giovanni Gadda ABSTRACT 2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2- nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.
  • Isotope Effects As Probes for Enzyme Catalyzed Hydrogen-Transfer Reactions

    Isotope Effects As Probes for Enzyme Catalyzed Hydrogen-Transfer Reactions

    Molecules 2013, 18, 5543-5567; doi:10.3390/molecules18055543 OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Review Isotope Effects as Probes for Enzyme Catalyzed Hydrogen-Transfer Reactions Daniel Roston †, Zahidul Islam † and Amnon Kohen * Department of Chemistry, The University of Iowa, Iowa City, IA 52242, USA † These authors contributed equally to this work. * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-319-335-0234. Received: 11 April 2013; in revised form: 30 April 2013 / Accepted: 3 May 2013 / Published: 14 May 2013 Abstract: Kinetic Isotope effects (KIEs) have long served as a probe for the mechanisms of both enzymatic and solution reactions. Here, we discuss various models for the physical sources of KIEs, how experimentalists can use those models to interpret their data, and how the focus of traditional models has grown to a model that includes motion of the enzyme and quantum mechanical nuclear tunneling. We then present two case studies of enzymes, thymidylate synthase and alcohol dehydrogenase, and discuss how KIEs have shed light on the C-H bond cleavages those enzymes catalyze. We will show how the combination of both experimental and computational studies has changed our notion of how these enzymes exert their catalytic powers. Keywords: kinetic isotope effects; Marcus-like models; alcohol dehydrogenase; thymidylate synthase; hydrogen tunneling 1. Introduction One of the most powerful tools in the chemist’s arsenal for studying reaction mechanisms and transition state structures is to measure kinetic isotope effects (KIEs). KIEs have become a pivotal source of information on enzymatic reactions, and a number of recent reviews have described the successes of KIEs in enzymology, from determining molecular mechanisms and transition state Molecules 2013, 18 5544 structures that are useful for drug design [1], to answering questions on the roles of nuclear quantum tunneling [2,3], electrostatics [4], and dynamic motions [5–8] in enzyme catalyzed reactions.
  • A Xylenol Orange-Based Screening Assay for the Substrate Specificity

    A Xylenol Orange-Based Screening Assay for the Substrate Specificity

    molecules Article A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases Tom A. Ewing 1, Aster van Noord 1, Caroline E. Paul 2 ID and Willem J. H. van Berkel 1,* ID 1 Laboratory of Biochemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands; [email protected] (T.A.E.); [email protected] (A.v.N.) 2 Laboratory of Organic Chemistry, Wageningen University & Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands; [email protected] * Correspondence: [email protected]; Tel.: +31-317-482861 Received: 15 December 2017; Accepted: 11 January 2018; Published: 14 January 2018 Abstract: Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para-substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para-phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol.
  • Polyethylene Glycol Dehydrogenating Activity Demonstrated By

    Polyethylene Glycol Dehydrogenating Activity Demonstrated By

    Agric. Biol. Chem., 55 (3), 837-844, 1991 837 Polyethylene Glycol Dehydrogenating Activity Demonstrated by Dye-linked Alcohol Dehydrogenase of Rhodopseudomonas acidophila M402 Kei Yamanaka Department of Applied Chemistry, Faculty of Engineering, OkayamaUniversity of Science, Ridai-cho, Okayama 700, Japan Received October 18, 1990 Ethylene glycols and polyethylene glycols inhibited the growth of a photosynthetic bacterium, Rhodopseudomonasacidophila, strain M402 under aerobic-dark and anaerobic-light culture conditions. However, polyethylene glycol dehydrogenating activity was demonstrated with phenazine methosulfate (PMS) as an electron acceptor in parallel with the PMS-linked aromatic alcohol dehydrogenase activity. Addition of ethylene glycols or polyethylene glycols to the culture neither induced aromatic alcohol dehydrogenase, nor accelerated polyethylene glycol dehydrogenating activity. Purified PMS-linked aromatic alcohol dehydrogenase had high affinities toward diethylene glycol and polyethylene glycols. This indicated that the dye-linked aromatic alcohol dehydrogenase is capable of oxidizing these xenobiotic synthetic polymer alcohols, polyethylene glycols. There is no evidence on the existence of a specific polyethylene glycol dehydrogenase in this bacterium. Broad substrate specificity is a well-known vanillyl alcohol in R. acidophila under property of alcohol and aldehyde dehydroge- aerobic-dark conditions can be postulated as nases, either dye-linked or NAD+-dependent, follows: of methylotrophs and some non-methylo- Vanillyl alcohol->vanillin-åºvanillic acid trophic bacteria.1~7) Their broad substrate specificities, however, vary depending on the The first oxidation step of vanillyl alcohol organism. The range of width of specificity was to vanillin is catalyzed by the dye-linked usually decided by the researchers' interests, vanillyl alcohol dehydrogenase, and vanillin is mainly in compounds structurally related to further dehydrogenated by the dye-linked the main substrate molecule.