DOCSLIB.ORG

Studies on a Purified Cellobiose Phosphorylase by James K

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Studies on a Purified Cellobiose Phosphorylase by James K Studies on a purified cellobiose phosphorylase by James K Alexander A THESIS Submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Bacteriology Montana State University © Copyright by James K Alexander (1959) Abstract: Cellobiose phosphorylase, obtained from cell-free extracts of Clostridium themocellum strain 65l, has been purified sufficiently to eliminate interfering reactions. The purified enzyme catalyzes the phosphorolysis of cellobiose with the formation of equimolar quantities of glucose-l-phosphate and glucose. The reaction is reversible for equimolar quantities of cellobiose and inorganic phosphate are formed from glucose and glucose—l-phosphate. At equilibrium the molar ratio of cellobiose and inorganic phosphate to glucose-l-phosphate and glucose is about 2 to 1. The equilibrium constant, K = (glucose-l-phosphate)(glucose)/(cellobiose)(inorganic phosphate), at 37 C and pH 7 is about 0.23. The optimum pH for the activity of cellobiose phosphorylase appears to be about 7.0. The temperature coefficient between 20 C and 40 C is about 2.4. The effect of various inhibitors has been determined and the results suggest that a sulfhydryl group is essential for the activity of the enzyme. The synthesis of glucosyl-D-xyloses glucosyl-L-xylose, glucosyl-D-deoxyglucose, and glucosyl-D-glucosamine apparently is catalyzed by cellobiose phosphorylase. The enzyme also catalyzes the arsenolysis of cellobiose. Beta-glucose—l-phosphate is unable to replace alpha-glucose-l-phosphate in the synthesis of cellobiose. A Walden inversion apparently occurs in the reaction. Cellobiose phosphorylase apparently does not catalyze the exchange between glucose-l-phosphate and arsenate or between cellobiose and D-xylose. These results suggest that a ' cellobiose-enzyme-phosphate complex is formed in the reaction. STUDIES OM A PURIFIED CELLOBIOSE PHOSPHORYLASE Dy JAMES K 0 ADEXAfJDER A THESIS Stibmitted to the Graduate Faculty ' . : ' in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Bacteriology at Montana State College . ’' * - - V - ' ■ ‘ ’ "I ’ - _ • , • . Approyeds Edad3 Major: Department Deah3, Graduat^Diwision ’ Bogdman3 Montana- ,, H7 .DB')? 2 TABLE OF CONTENTS ACKNOWLEDGEMENTS 6 ABSTRACT 7 INTRODUCTION 8 REVIEW OF THE LITERATURE IO Enzymes involved in the decomposition of cellulose 12 Phosphorylases MATERIALS AND METHODS 19 Enzyme source 19 Crude enzyme preparations 20 Purified enzyme preparations 21 Protein determinations 22 Determination of the products and reactants of the cellobiose phosphorylase reaction 22 a) Glucose determinations 23 b) Cellobiose determinations 2h c) Inorganic phosphate determinations 26 d) Glucose-l-phosnhate determinations 27 Phosphoglucomutase activity 27 Chranatography 27 Colorimetric determinations of reducing sugars 27 Kinase activity 28 Barbital-acetate buffer 28 RESULTS 29 A, Studies on the cellobiose phosphorylase reaction 29 Elimination of interfering reactions 29 Analyses of the products and reactants of cellobiose phosphorolysis 35 Analyses of the products and reactants of cellobiose synthesis 35 Equilibrium constant of the cellobiose phosphorylase reaction 39 Free energy calculations U2 B. Properties of cellobiose phosphorylase Ui The effect of hydrogen ion concentration on the activity of cellobiose phosphorylase Wi The effect of temperature on the activity of cellobiose phosphorylase Ii5 The effect of p-chloromercuribenzoate on the activity of cellobiose phosphorylase Ii7 The effect of AgNO^ on the activity of cellobiose phosphorylase 1|8 The effect of phlorizin on the activity of cellobiose phosphorylase 00 130563 3 - - ' : The effect Cf glucose tin cellobiose phosphorolysls Si The effect of NaF^ (EI^aSOitj ,:NaGls and on the activity of cellobiose:: phosphoiylase Sh Spetiificity of cellobiose phosphoiylase 56 1) The,.Synthesis of disacoharides ■ . 56 2) The phosphorolysls'of disaccharides 6l 3) '' Beta-glucose-fl^phosphate 6a 63 Go The•mechanism Of the■cellobiose phosphoiylase reaction 6? De Pht#ays of cellohitise '.titithbolisst -in '0» thermocellum 71 DISCUSSION 7h The specificity of cellobiose phosphorylase 81 The mechanism of the cellobiose phosphorylase reaction 87 SUBttiRT 9k REFERENCES 96 k FIGURES Figure I, Disappearance of inorganic phosphate during the . phosphorolysis of cellobiose, ■ 36 Figure Se The liberation of'inorganic phosphate during the ‘ ■ • synthesis of ‘ cellobiose= 38 Figure 3» The Activity of Cellbbiose phosphorylase at ■ different pH,values,' ■ Figure "he Chromatographs of' 'glucosyl^D-zylose and ' < glucosyl-Ir-sylose, 60 Figure The arsenolysis ■ of cellobiose with cellobiose ^ • phdsphbrylasev " ■' ■' 66 Figure' 6» . Chromatographs of reaction mixtures incubated with cellobiose and B-osylbse= 70 Figure 7« Evidence that cellobibkinase'is produced by ‘ Qlostridium thermocellum strain 631, _ 73 Figure Se Pathways of cellobiose utilization Iy Clostridium ' thermocellum^ 7h Figure 9o Biagramaticrepresentation of the Specificity of cellobiose phosphorylase, ' ' 53 Figure IOb The relationship of the Sucrose^, maltose^ and cellobiose phosphorylase reactions^ 89 Figure 11* The formation of" an inverted glucose-enzyme complex’from alpha-glucose-l-phosphate and sucrose phosphorylase* 1 90 Figure 12, The formation of sucrose and free sucrose phosphorylase from a glucose-enzyme complex and fructose* 90 Figure 13* ^n illustration of the single displacement mechanism. proposed for the maltose phosphorylase reaction* 90 Figure Ihb A proposed single displacement, mechanism for the Cellobiose phosphorylase reaction* 92 TABLES Table I The activity of enzyme preparations from Clostridium thermocellum strain 631 on glucdse-l-phosphatee 30 Table II Determination of cellobiase activity in enzyme pre^ parations from Clostridium 'thermocellum strain 631* 33 Table III Analyses'of the products and reactantsof Cellobiose phosphorolysis* 37 Table IF Analyses of the products and reactants of cellbbiose synthesis* 37 Table F Analyses of the products and reactants of a reaction with purified Cellobiose phosphorylase and approximately equilibrium amounts of cellobiose^ inorganic phosphate* glucose and glucose-l-phosphate* 1*0 Table FI Summary of equilibrium'data for the cellobiose phosphorylase reaction* hi Table FII The activity of cellobiose phosphorylase at different temperatures» h7 Table Fill The effect of p-ehloromercuribenzoate on the activity of cellobiose phosphorylase* U9 Table IX The effect of silver nitrate on the activity of Cellobiose phosphorylase* 2o 5' Table X The ,effect of phlorizin on the activity of eellobiose PhosphoryiaserO gl Table Xl The effect ,of'.glucose on eellobiose phoephorolysis= §3 ■Table' X H The effect.of .HaFs. HaGls (Wj4)2Soas and % S % on the activity of eellobiose phosphoryIase3 $$ ■Table ■XIII Liberation of inorganic phosphate when eellobiose phosphozylase was incubated'with glueose*?*l-phosphate and various other compounds* 58 Table XIV The change in Inorganic phosphate when./cellobiose phosphorylase was incubated "with 'inorganic phosphate and different disaccharides * '' ' 62 Table- XV Liberation of inorganic phosphate when eellobiose ,phosphorylase"was incubated with"glucose and beta- glucose-l-phosphate of alpha-glue os e-»l"phosphat e * 6h Table XVI The' disappe||fance of inorganic phosphate in a reaction mixture used as a control in the arsenolysis experiment*. 65 Table XVII The formation of free glucose when eellobiose phosphory­ lase was incubated with gluoose-l-phosphate and arsenate*68 Table XFIII A summary of the"information concerning the specificity of eellobiose phosphorylase* 83 j, ACOJOWLEBGEMEMTS The author wishes to thank Digie Richard Ho MeBee for his guidance and advice throughout the tiburse of this study* The author is also 1 ^ - ' indebted to Dr* Nels Me. Nelson and Dr* John E* Gander for their advice* The helpful suggestions of Dr* touis DS * Smith and Dr* William. G* Walter in writing this thesis are greatly appreciated* This investigation was supported in part by a research grant from the National institutes of Health* * 7 ABSTRACT Cellobiese phosphorylase s obtained from cell-free extracts of Clostridium themocellum strain 651, has been purified sufficiently to eliminate interfering reactions. The purified enzyme catalyzes the phosphorolysis of eellobibse with the formation of equimolar quantities of glucose—1—phosphate and glucose. The reaction is reversible for equimolar quantities of cellobiose and inorganic phosphate are formed from glucose and glucose—1—phosphateo At equilibrium the molar ratio of Cellobiosej and inorganic phosphate to glucose—I-phosphate and glucose is about 2 to 1«, The equilibrium constants K ” (glucose-l-phosphate)(glucose)/(cellobiose)(inorganic phosphate)> at 37 C and pH 7 is about 0*23. The optimum pH for the activity of cellobiose phosphorylase appears to be about 7» O0 The temperature coefficient between 20 C and Uo C is about 2 The effect of various inhibitors has been determined and the results suggest that a sulfhydryl group is essential for the activity of the enzyme. The synthesis of glueosyl-B-saylose,v gluqosyl-L-xylose , glucosyl- D-deoxyglucose, and glucosyl-D-glucosamine» apparently is catalyzed by cellobiose phosphorylase. The enzyme also catalgrzes the arsenolysis of cellobiose* Beta-glucose— 1-phosphate is unable to replace alpha-glucose-1- phosphate in the synthesis of cellobiose. A Walden inversion apparently occurs in the reaction* Cellobiose phosphorylase apparently does not catalyze
Load more

Recommended publications
  • Bacteria Belonging to Pseudomonas Typographi Sp. Nov. from the Bark Beetle Ips Typographus Have Genomic Potential to Aid in the Host Ecology
    insects Article Bacteria Belonging to Pseudomonas typographi sp. nov. from the Bark Beetle Ips typographus Have Genomic Potential to Aid in the Host Ecology Ezequiel Peral-Aranega 1,2 , Zaki Saati-Santamaría 1,2 , Miroslav Kolaˇrik 3,4, Raúl Rivas 1,2,5 and Paula García-Fraile 1,2,4,5,* 1 Microbiology and Genetics Department, University of Salamanca, 37007 Salamanca, Spain; [email protected] (E.P.-A.); [email protected] (Z.S.-S.); [email protected] (R.R.) 2 Spanish-Portuguese Institute for Agricultural Research (CIALE), 37185 Salamanca, Spain 3 Department of Botany, Faculty of Science, Charles University, Benátská 2, 128 01 Prague, Czech Republic; [email protected] 4 Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology of the Academy of Sciences of the Czech Republic, 142 20 Prague, Czech Republic 5 Associated Research Unit of Plant-Microorganism Interaction, University of Salamanca-IRNASA-CSIC, 37008 Salamanca, Spain * Correspondence: [email protected] Received: 4 July 2020; Accepted: 1 September 2020; Published: 3 September 2020 Simple Summary: European Bark Beetle (Ips typographus) is a pest that affects dead and weakened spruce trees. Under certain environmental conditions, it has massive outbreaks, resulting in attacks of healthy trees, becoming a forest pest. It has been proposed that the bark beetle’s microbiome plays a key role in the insect’s ecology, providing nutrients, inhibiting pathogens, and degrading tree defense compounds, among other probable traits. During a study of bacterial associates from I. typographus, we isolated three strains identified as Pseudomonas from different beetle life stages. In this work, we aimed to reveal the taxonomic status of these bacterial strains and to sequence and annotate their genomes to mine possible traits related to a role within the bark beetle holobiont.
    [Show full text]
  • A) and Co-Fermentation (B
    Simultaneous Co-Fermentation of Mixed Sugars: A Promising Strategy for Producing Cellulosic Biofuels and Chemicals Na Wei PI: Yong-Su Jin Energy Biosciences Institute /Institute for Genomic Biology University of Illinois at Urbana-Champaign Corn ethanol vs. Cellulosic ethanol Corn starch Cellulosic biomass Gelatinization Pretreatment + Cellulases Amylases Glucose + Xylose + Acetate Glucose + Fermentation inhibitors yeast yeast Ethanol + CO2 Ethanol + CO2 • Single sugar fermentation • Mixed sugar fermentation • No fermentation inhibitors • Fermentation inhibitors • Easy high loading • Difficulties in high loading 2 Saccharomyces cerevisiae: a workhorse strain for industrial ethanol production The most widely used yeast since ancient times in baking and brewing Osmotolerant and ethanol-tolerant Numerous genetic/genomic tools are available Overexpression / Knockout Expression of heterologous enzymes Cannot utilize xylose Not suitable for producing cellulosic biofuels 3 Basic strategy in metabolic engineering of xylose fermentation in S. cerevisiae Scheffersomyces stipitis Saccharomyces cerevisiae Xylose Xylose XYL1 Xylitol Xylitol XYL2 Xylulose Xylulose XYL3 X-5-P X-5-P PPP and Glycolysis PPP and Glycolysis Ethanol Ethanol . Natural xylose fermenting . High ethanol tolerance . Low ethanol tolerance . Amenable to metabolic engineering 4 Laboratory evolution of an engineered S. cerevisiae strain for further improvement DA24 n 16 Enrichment Single colony by serial culture isolation in 80 g/L of xylose Evaluation 5 Comparison of xylose fermentation capability between engineered S. cerevisiae and S. stipitis Engineered S. cerevisiae S. stipitis The engineered S. cerevisiae strain consumed xylose almost as fast as S. stipitis, the fastest xylose-fermenting yeast 6 Ha et al. PNAS, 108:504-509 Why we want to co-ferment cellobiose and xylose? Typical fermentation profile of glucose and xylose mixture Glucose Glycolysis Ethanol Pentose Phosphate Pathway CO2 Xylose 7 Engineered S.
    [Show full text]
  • Bioresources.Com
    PEER-REVIEWED ARTICLE bioresources.com ADSORPTION OF CELLOBIOSE-PENDANT POLYMERS TO A CELLULOSE MATRIX DETERMINED BY QUARTZ CRYSTAL MICROBALANCE ANALYSIS Shingo Yokota,† Takefumi Ohta, Takuya Kitaoka,* and Hiroyuki Wariishi Cellobiose-pendant polymers were synthesized by radical polymerization and their affinity for a cellulose matrix was investigated by quartz crystal microbalance (QCM). A 2-(methacryloyloxy)ethylureido cellobiose (MOU-Cel) macromer was synthesized by coupling cellobiosylamine with 2-(methacryloyloxy)ethyl isocyanate followed by polymerization in an aqueous radical reaction system. The interaction of the resulting poly(MOU-Cel) with a pure cellulose matrix in water was evaluated by QCM analysis. Poly(MOU-Cel) was strongly adsorbed to the cellulose substrate, whereas neither cellobiose nor MOU-Cel macromer exhibited an attractive interaction with cellulose. This specific interaction was not inhibited by the presence of ionic contaminants, suggesting that multiple cellobiopyranose moieties in each polymer molecule might cooperatively enhance its affinity for cellulose. Moderate insertion of acrylamide units into the polymer backbone improved the affinity for cellulose, possibly due to an increased mobility of sugar side chains. Polymers such as these, with a high affinity for cellulose, have potential applications for the surface functionalization of cellulose-based materials, including paper products. Keywords: Cellobiose; Cellulose; Sugar-pendant polymer; Non-electrostatic interaction; Quartz crystal microbalance Contact information: Department of Forest and Forest Products Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; †Present address: Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan; *Corresponding author: [email protected] INTRODUCTION Cellulose is the most abundant, renewable carbohydrate resource and has been widely used as a raw material in a variety of applications (Klemm et al.
    [Show full text]
  • Cellobiose 2-Epimerase, Process for Producing
    (19) TZZ ¥ZZ_T (11) EP 2 395 080 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 15/00 (2006.01) C12N 1/15 (2006.01) 06.08.2014 Bulletin 2014/32 C12N 1/19 (2006.01) C12N 1/21 (2006.01) C12N 5/10 (2006.01) C12N 9/90 (2006.01) (2006.01) (2006.01) (21) Application number: 10738433.1 C12N 15/09 C12P 19/00 (22) Date of filing: 25.01.2010 (86) International application number: PCT/JP2010/050928 (87) International publication number: WO 2010/090095 (12.08.2010 Gazette 2010/32) (54) CELLOBIOSE 2-EPIMERASE, PROCESS FOR PRODUCING SAME, AND USE OF SAME CELLOBIOSE 2-EPIMERASE, HERSTELLUNGSVERFAHREN DAFÜR UND VERWENDUNG CELLOBIOSE 2-ÉPIMÉRASE, PROCÉDÉ DE PRODUCTION DE CELLE-CI ET UTILISATION DE CELLE-CI (84) Designated Contracting States: (74) Representative: Daniels, Jeffrey Nicholas AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Page White & Farrer HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL Bedford House PT RO SE SI SK SM TR John Street London WC1N 2BF (GB) (30) Priority: 05.02.2009 JP 2009025070 (56) References cited: (43) Date of publication of application: WO-A1-2008/062555 14.12.2011 Bulletin 2011/50 • PARK CHANG-SU ET AL: "Characterization of a (73) Proprietor: Hayashibara Co., Ltd. recombinant cellobiose 2-epimerase from Okayama-shi, Okayama (JP) Caldicellulosiruptor saccharolyticus and its application in the production of mannose from (72) Inventors: glucose.", APPLIED MICROBIOLOGY AND • WATANABE Hikaru BIOTECHNOLOGY DEC 2011 LNKD- PUBMED: Okayama-shi 21691788,vol.
    [Show full text]
  • Enzymatic Glycosylation of Small Molecules
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by University of Groningen University of Groningen Enzymatic Glycosylation of Small Molecules Desmet, Tom; Soetaert, Wim; Bojarova, Pavla; Kren, Vladimir; Dijkhuizen, Lubbert; Eastwick- Field, Vanessa; Schiller, Alexander; Křen, Vladimir Published in: Chemistry : a European Journal DOI: 10.1002/chem.201103069 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2012 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Desmet, T., Soetaert, W., Bojarova, P., Kren, V., Dijkhuizen, L., Eastwick-Field, V., ... Křen, V. (2012). Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require Tailored Catalysts. Chemistry : a European Journal, 18(35), 10786-10801. https://doi.org/10.1002/chem.201103069 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum.
    [Show full text]
  • TO Thermostable Sucrose Phosphorylase 20120924
    Thermostable sucrose phosphorylase Ghent Bio-Energy Valley, a consortium of research laboratories of Ghent University, is seeking partners interested in the industrial and enzymatic production of glycosides or glucose-1-phosphate at temperatures of 60°C or higher using a thermostable phosphorylase. Introduction Sucrose phosphorylase catalyses the reversible phosphorolysis of sucrose into α-glucose-1-phosphate and fructose. Because of the broad acceptor specificity of the enzyme, it can further be used for the production of a number of glycosylated compounds. At the industrial scale, such production processes are preferably run at 60°C or higher, mainly to avoid microbial contamination. Unfortunately, no sucrose phosphorylases are known yet which perform well at such high temperatures. Technology Researchers at Ghent University have identified a thermostable sucrose phosphorylase derived from the prokaryote Bifidobacterium adolescentis which performs well at 60°C or higher. The enzyme is active for at least 16h at 60°C when it is in the continuous presence of its substrate, and/or, when it is mutated at specific residues, and/or, when it is immobilized on a carrier or when it is immobilized by cross-linking so that it is part of a so-called “cross- linked enzyme aggregate (CLEA)”. Applications Enzymatic and/or recombinant methods to produce high value glycosides at elevated temperatures. Advantages • being able to produce glycosylated compounds at high temperatures avoids microbial contamination; • recycling of the immobilized biocatalyst in consecutive reactions increases the commercial potential. Status of development The sucrose phosphorylase of B. adolescentis was recombinantly expressed in E. coli and further purified. In addition, the enzyme was immobilized on an epoxy- activated enzyme carrier (Sepabeads EC-HFA) or was incorporated in a CLEA or was mutated to obtain enzyme variants.
    [Show full text]
  • Rapid Method for the Estimation of Total Free Monosaccharide Content of Corn Stover Hydrolysate Using HPAE-PAD
    Application Note 225 Note Application Rapid Method for the Estimation of Total Free Monosaccharide Content of Corn Stover Hydrolysate Using HPAE-PAD Valoran Hanko and Jeff Rohrer Thermo Fisher Scientific, Sunnyvale, CA, USA Introduction The Thermo Scientific™ Dionex™ CarboPac™ PA1 Compositional carbohydrate analysis of biocrop feedstock anion-exchange column rapidly elutes corn stover is essential to the efficient production of cellulosic carbohydrates, resolving the monosacccharides from the ethanol.1 Corn stover is the leaf, husk, stalk, and cob unretained and undetected noncarbohydrate components remaining in the field after harvest and makes up about of acid-hydrolyzed corn stover. Under these conditions, half the yield of a crop of corn. It can also contain weeds disaccharides and trisaccharides are also resolved, with a and grasses collected during the harvest of corn stover. total elution time less than 10 min. Thus, the sum of all Along with other lignocellulosic biomass, corn stover the carbohydrate peaks is a useful measure of the energy provides 1.3 billion tons of raw material annually and is a production potential of acid-hydrolyzed corn stover. common feedstock for fermentation systems used in The carbohydrate content of acid-hydrolyzed corn stover biofuel production.2 can be very high, and for some carbohydrates can even Corn stover is acid-hydrolyzed to release a water-soluble approach their saturation concentration in an aqueous mixture of carbohydrates, typically consisting of arabi- solution. When used for saccharide determinations, PAD nose, glucose, galactose, mannose, xylose, fructose, and with a gold working electrode is a sensitive detection cellobiose among other noncarbohydrate substances in method, but like other detection techniques it does not 0.5 to 1.5% (w/w) sulfuric acid.3,4 The concentration of have an unlimited linear range.
    [Show full text]
  • Of Sucrose Phosphorylase: Combining Improved Stability with Altered Specificity
    Int. J. Mol. Sci. 2012, 13, 11333-11342; doi:10.3390/ijms130911333 OPEN ACCESS International Journal of Molecular Sciences ISSN 1422-0067 www.mdpi.com/journal/ijms Article An Imprinted Cross-Linked Enzyme Aggregate (iCLEA) of Sucrose Phosphorylase: Combining Improved Stability with Altered Specificity Karel De Winter, Wim Soetaert and Tom Desmet * Centre of Expertise for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Biosciences Engineering, Ghent University, Coupure Links 653, Ghent B-9000, Belgium; E-Mails: [email protected] (K.D.W.); [email protected] (W.S.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +32-9-2649-920; Fax: +32-9-2646-032. Received: 28 August 2012; in revised form: 5 September 2012 / Accepted: 5 September 2012 / Published: 11 September 2012 Abstract: The industrial use of sucrose phosphorylase (SP), an interesting biocatalyst for the selective transfer of α-glucosyl residues to various acceptor molecules, has been hampered by a lack of long-term stability and low activity towards alternative substrates. We have recently shown that the stability of the SP from Bifidobacterium adolescentis can be significantly improved by the formation of a cross-linked enzyme aggregate (CLEA). In this work, it is shown that the transglucosylation activity of such a CLEA can also be improved by molecular imprinting with a suitable substrate. To obtain proof of concept, SP was imprinted with α-glucosyl glycerol and subsequently cross-linked with glutaraldehyde. As a consequence, the enzyme’s specific activity towards glycerol as acceptor substrate was increased two-fold while simultaneously providing an exceptional stability at 60 °C.
    [Show full text]
  • New Computational Approaches for Investigating the Impact of Mutations on the Transglucosylation Activity of Sucrose Phosphorylase Enzyme
    Thesis submitted to the Université de la Réunion for award of Doctor of Philosophy in Sciences speciality in bioinformatics New computational approaches for investigating the impact of mutations on the transglucosylation activity of sucrose phosphorylase enzyme Mahesh VELUSAMY Thesis to be presented on 18th December 2018 in front of the jury composed of Mme Isabelle ANDRE, Directeur de Recherche CNRS, INSA de TOULOUSE, Rapporteur M. Manuel DAUCHEZ, Professeur, Université de Reims Champagne Ardennes, Rapporteur M. Richard DANIELLOU, Professeur, Université d'Orléans, Examinateur M. Yves-Henri SANEJOUAND, Directeur de Recherche CNRS, Université de Nantes, Examinateur Mme Irène MAFFUCCI, Maître de Conférences, Université de Technologie de Compiègne, Examinateur Mme Corinne MIRAL, Maître de Conférences HDR, Université de Nantes, Examinateur M. Frédéric CADET, Professeur, Université de La Réunion, Co-directeur de thèse M. Bernard OFFMANN, Professeur, Université de Nantes, Directeur de thèse ெ்்ந்ி ிைு்ூுத் ெ்யாம் ெ்த உதி்ு ையகு் ானகு் ஆ்ற் அிு. -ிு்ு் ுதி் அ்ப் ுுக், எனு த்ைத ேுாி, தா் க்ூி, அ்ண் ு்ு்ுமா், த்ைக பா்பா, ஆ்தா ுு்மா், ீனா, அ்ண் ்ுக், ெபிய்பா, ெபிய்மா ம்ு் உுுைணயா் இு்த அைண்ு ந்ப்கு்ு் எனு மனமா்்த ந்ி. இ்த ஆ்ி்ைக ுுைம அை3த்ு ுுுத்்காரண், எனு ஆ்ி்ைக இய்ுன் ேபராிிய் ெப்னா்் ஆஃ்ேம். ப்ேு துண்கி் நா் மனதாு், ெபாுளாதார அளிு் க்3்ி் இு்தேபாு, என்ு இ்ெனாு த்ைதயாகே இு்ு எ்ைன பா்்ு்ெகா்3ா். ுி்பாக, எனு ூ்றா் ஆ்ு இுிி், அ்்ு எ்ளோ தி்ப்3 க3ைமக் ம்ு் ிர்ிைனக் இு்தாு், அைத்ெபாு்பு்தாு, அ் என்ு ெ்த ெபாுளாதார உதி, ப்கைB்கழக பிு ம்ு் இதர ி்ாக ்ப்த்ப்3 உதிகு்ு எ்ன ைகமா்ு ெகாு்தாு் ஈ3ாகாு.
    [Show full text]
  • Converting Galactose Into the Rare Sugar Talose with Cellobiose 2-Epimerase As Biocatalyst
    molecules Article Converting Galactose into the Rare Sugar Talose with Cellobiose 2-Epimerase as Biocatalyst Stevie Van Overtveldt, Ophelia Gevaert, Martijn Cherlet, Koen Beerens and Tom Desmet * Centre for Synthetic Biology, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Gent, Belgium; [email protected] (S.V.O.); [email protected] (O.G.); [email protected] (M.C.); [email protected] (K.B.) * Correspondence: [email protected]; Tel.: +32-9264-9920 Academic Editors: Giorgia Oliviero and Nicola Borbone Received: 17 September 2018; Accepted: 29 September 2018; Published: 1 October 2018 Abstract: Cellobiose 2-epimerase from Rhodothermus marinus (RmCE) reversibly converts a glucose residue to a mannose residue at the reducing end of β-1,4-linked oligosaccharides. In this study, the monosaccharide specificity of RmCE has been mapped and the synthesis of D-talose from D-galactose was discovered, a reaction not yet known to occur in nature. Moreover, the conversion is industrially relevant, as talose and its derivatives have been reported to possess important antimicrobial and anti-inflammatory properties. As the enzyme also catalyzes the keto-aldo isomerization of galactose to tagatose as a minor side reaction, the purity of talose was found to decrease over time. After process optimization, 23 g/L of talose could be obtained with a product purity of 86% and a yield of 8.5% (starting from 4 g (24 mmol) of galactose). However, higher purities and concentrations can be reached by decreasing and increasing the reaction time, respectively. In addition, two engineering attempts have also been performed.
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Taxonomic Binning Approaches and Functional Characteristics of the Microbial Community During the Anaerobic Digestion of Hydrolyzed Corncob
    energies Article Taxonomic Binning Approaches and Functional Characteristics of the Microbial Community during the Anaerobic Digestion of Hydrolyzed Corncob Luz Breton-Deval 1 , Ilse Salinas-Peralta 1 , Jaime Santiago Alarcón Aguirre 2, Belkis Sulbarán-Rangel 2,* and Kelly Joel Gurubel Tun 2,* 1 Catedras Conacyt, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 62210, Mexico; [email protected] (L.B.-D.); [email protected] (I.S.-P.) 2 Department of Water and Energy, University of Guadalajara Campus Tonalá, Tonalá 45425, Mexico; [email protected] * Correspondence: [email protected] (B.S.-R.); [email protected] (K.J.G.T.); Tel.: +52-33-2000-2300 (K.J.G.T.) Abstract: Maize forms the basis of Mexican food. As a result, approximately six million tons of corncob are produced each year, which represents an environmental issue, as well as a potential feedstock for biogas production. This research aimed to analyze the taxonomic and functional shift in the microbiome of the fermenters using a whole metagenome shotgun approach. Two strategies were used to understand the microbial community at the beginning and the end of anaerobic digestion: (i) phylogenetic analysis to infer the presence and coverage of clade-specific markers to assign taxonomy and (ii) the recovery of the individual genomes from the samples using the binning of the assembled scaffolds. The results showed that anaerobic digestion brought some noticeable changes and the main microbial community was composed of Corynebacterium variable, Desulfovibrio desulfuricans, Vibrio furnissii, Shewanella spp., Actinoplanes spp., Pseudoxanthomonas spp., Saccharomonospora azurea, Citation: Breton-Deval, L.; Agromyces spp., Serinicoccus spp., Cellulomonas spp., Pseudonocardia spp., Rhodococcus rhodochrous, Salinas-Peralta, I.; Alarcón Aguirre, Sphingobacterium spp.
    [Show full text]