DOCSLIB.ORG

Purification and Characterization of the Bacillus Subtilis Levanase

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Purification and Characterization of the Bacillus Subtilis Levanase APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1995, p. 1953–1958 Vol. 61, No. 5 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology Purification and Characterization of the Bacillus subtilis Levanase Produced in Escherichia coli 1 2 1 ERICH WANKER, † ANTON HUBER, AND HELMUT SCHWAB * Institut fu¨r Biotechnologie, Arbeitsgruppe Genetik, Technische Universita¨t,1 and Institut fu¨r Physikalische Chemie, Karl-Franzens-Universita¨t,2 A-8010 Graz, Austria Received 11 August 1994/Accepted 2 March 1995 The enzyme levanase encoded by the sacC gene from Bacillus subtilis was overexpressed in Escherichia coli with the strong, inducible tac promoter. The enzyme was purified from crude E. coli cell lysates by salting out with ammonium sulfate and chromatography on DEAE–Sepharose CL-6B, S-Sepharose, and MonoQ-Sepha- rose. The purified protein had an apparent molecular mass of 75,000 Da in sodium dodecyl sulfate-polyac- rylamide gel electrophoresis, which is in agreement with that expected from the nucleotide sequence. Levanase was active on levan, inulin, and sucrose with Km values of 1.2 mM, 6.8 mM, and 65 mM, respectively. The pH optimum of the enzyme acting on inulin was 5.5, and the temperature optimum was 55&C. Levanase was rapidly inactivated at 60&C, but activity could be retained for longer times by adding fructose or glycerol. The enzyme activity was completely inactivated by Ag1 and Hg21 ions, indicating that a sulfhydryl group is involved. A ratio of sucrase to inulinase activity of 1.2 was found for the purified enzyme with substrate concentrations of 50 mg/ml. The mechanism of enzyme action was investigated. No liberation of fructo-oligomers from inulin and levan could be observed by thin-layer chromatography and size exclusion chromatography–low-angle laser light scattering–interferometric differential refractive index techniques. This indicates that levanase is an exoenzyme acting by the single-chain mode. Enzymes involved in the hydrolysis of polyfructans are of Arthrobacter ureafaciens (41) splits levan and inulin, but no interest both for fundamental studies and for industrial appli- sucrose-hydrolyzing activity could be detected. However, un- cations. Especially inulin is of growing interest as a renewable specific b-fructosidases which are active on levan and sucrose carbohydrate raw material for biotechnology. Two aspects are but not on inulin have not been described yet. of main importance: (i) production of pure fructose syrups, Most inulinases and levanases from microorganisms which so-called high-fructose inulin syrups, by enzymatic hydrolysis were investigated in more detail have been found to be exoen- of inulin (43) and (ii) direct fermentation of inulin by employ- zymes (11). They attack the inulin or levan molecules from the ing inulinase-producing microbes in order to synthesize various fructose end and liberate fructose as the sole reaction product. products such as ethanol or aceton-butanol (20, 21). Exoinulinases or exolevanases are incapable of hydrolyzing b-D-Fructofuranosidases are usually classified upon their melezitose (3-O-a-D-glucopyranosyl-b-D-fructofuranosyl-a-D- ability to hydrolyze levan (levanases), inulin (inulinases), and glucopyranoside), a trisaccharide with the same terminal con- also the disaccharide sucrose (sucrases and invertases). How- figuration as inulin (36). In melezitose the centrally located ever, many of these enzymes are capable of hydrolyzing more fructose is protected from terminal hydrolysis by the second than one type of these substrates. Inulinases (or inulases) glycosyl residue. Only endoinulinases are capable of hydrolyz- which are specific for inulin have been isolated only from ing melezitose. Most enzymes of this type have been isolated Jerusalem artichoke tubers (7, 12), whereas levanases which and characterized from fungi, among them the endoinulinases are specific for levan have been isolated from bacteria only. from Aspergillus ficuum (10) and the endoinulinase from C. Examples are the levanases of Streptococcus salivarius KTA-19 pannorum (47). (38) and Actinomyces viscosus ATCC 19246 (16). Conversely, a Most microbial levanases and inulinases which have been variety of nonspecific -D-fructofuranosidases have been found b purified and characterized in more detail were isolated from in bacteria, yeasts, and fungi. For example, inulinases and yeasts (31, 32, 35, 36) and filamentous fungi (10, 11, 26, 27, 46, levanases which are capable of hydrolyzing inulin, levan, and 47), whereas only a few bacterial enzymes have been purified sucrose have been isolated from Bacillus subtilis (17), Actino- and characterized so far (5, 16). myces viscosus ATCC 15987 (24), Streptococcus mutans (5), The enzyme levanase from B. subtilis is a b-D-fructofurano- Kluyveromyces fragilis (35), Chrysosporium pannorum (46, 47) sidase capable of hydrolyzing levan, inulin, and sucrose (17, 22, and Penicillium sp. strain (27). Enzymes active on inulin and 23, 33, 34). B. subtilis levanase was first described by Kunst et sucrose but not on levan have been found in filamentous fungi, al. (17) and assigned as levanase because specific activity on among them the -fructofuranosidases (I to III) from Aspergil- b levan, inulin, sucrose, and raffinose was observed. However, lus niger (42), the F2 inulinase from C. pannorum (47), and the according to the Avigad and Bauer classification (2), it should PII inulinase from Aspergillus niger (26). The inulinase II from be assigned as a nonspecific b-fructofuranosidase to distin- guish it from true levanases (specific 2 3 6 activity). Levanase has been partially purified (22) and characterized with crude * Corresponding author. Mailing address: Institut fu¨r Biotechnolo- gie, Arbeitsgruppe Genetik, Technische Universita¨t, Petersgasse 12, protein extracts (17). Levanase expression in B. subtilis is A-8010 Graz, Austria. Phone: (43) 316-873-8418. Fax: (43) 316-811- tightly regulated (23), and detectable amounts of enzyme are 050. Electronic mail address: [email protected]. found only with regulatory mutants (sacL mutants). The struc- † Present address: Department of Biological Chemistry, UCLA tural gene coding for levanase has been cloned in Escherichia School of Medicine, Los Angeles, CA 90024-1737. coli (13), sequenced, and characterized in detail (22, 33, 34). 1953 1954 WANKER ET AL. APPL.ENVIRON.MICROBIOL. The cloned gene has been overexpressed in E. coli, where a levanase protein of about 75 kDa is found mainly intracellu- larly, despite the presence of a secretion signal (44). In this report we describe the purification and detailed char- acterization, including the mechanism of action, of the B. sub- tilis levanase overexpressed in E. coli, focusing on the inulin- degrading activity of this enzyme. MATERIALS AND METHODS Bacterial strain and culture conditions. E. coli HB101 [ATCC 33649; F2 2 2 hsdS20(rB mB ) supE44 araA2 rpsL20(strR) xyl-5 mtl-I recA13] harboring pESI7HE (44) was used in all experiments. The bacterial cells were routinely grown at 378C in LB medium (1% Bacto Tryptone, 0.5% Bacto Yeast Extract, 0.5% NaCl) or M9 mineral salts medium (25), supplemented with essential FIG. 1. DEAE–Sepharose CL-6B chromatography of intracellular levanase amino acids (20 mg/liter) and with thiamine (1 mg/liter), containing 0.5% glucose produced in E. coli HB101. Ammonium sulfate-precipitated protein was applied or sucrose. For plasmid selection, ampicillin (100 mg/liter) was added. For on a DEAE–Sepharose CL-6B column, and elution was performed witha0to0.4 induction of the tac promoter, E. coli cultures were supplemented with 1 mM M NaCl gradient. The gradient was obtained by mixing a 25 mM Tris (pH 8) isopropyl-b-D-thiogalactopyranoside (IPTG) at inoculation or in the middle of bufferwitha1MNaCl solution. the exponential phase. The cells were harvested by centrifugation after an incu- bation of 16 to 24 h at a final optical density of about 0.5 (at 600 nm; Beckmann DU 50 photometer) when grown in M9 mineral salts medium or of 1.5 to 2 when grown in LB medium. Protein determination and enzymatic assays. The protein concentration was by heating samples to 958C. The fructose concentration in the samples was determined by the method of Bradford (4) by using a commercial reagent kit determined enzymatically (test combination). (Bio-Rad, Richmond, Calif.) as recommended by the supplier. Bovine serum Purification of levanase. Recombinant E. coli cells were grown in shake flask albumin (Bio-Rad) was used as the standard. cultures (300 ml) with glucose M9 mineral salts medium containing ampicillin. Unless otherwise noted, all enzymatic assays were carried out in inulin reac- For induction of levanase expression, 1 mM IPTG was added at the time of tion buffer (0.2 M Na2HPO4, 0.2 M sodium acetate, pH 5.5) with dissolved (3 min inoculation and cultures were incubated for 16 h at 378C (150 rpm). Cells were at 958C) or suspended (room temperature) inulin (Sigma, St. Louis, Mo.; average harvested, washed with 0.9% NaCl, resuspended in 25 mM Tris-HCl buffer (pH molecular weight, 5,000), dissolved levan (Sigma; average molecular weight, 107), 8), and disrupted by sonication (Braun Labsonic 2000) with four pulses of 30 s or sucrose (Sigma) as a substrate. each on ice. The cell debris was removed by centrifugation (65,500 3 g)for1h. For determination of inulinase activity, inulin was dissolved or suspended in The supernatant was stored at 2208C until used for the following purification inulin reaction buffer to a final concentration of 50 mg/ml, and following addition steps. of enzyme (appropriate dilution) the mixture was incubated at 37 or 558C for 30 (i) Ammonium sulfate precipitation. The crude lysate was first brought to 20% min. The levanase and sucrase activities were measured by using substrate con- saturation by addition of solid ammonium sulfate, and after incubation for 1 h centrations of 25 and 50 mg/ml, respectively. Incubation was performed at 558C the precipitated protein was removed by centrifugation and discarded.
Load more

Recommended publications
  • 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.
    [Show full text]
  • Highly Thermostable and Alkaline Α-Amylase from a Halotolerant- Alkaliphilic Bacillus Sp. Ab68
    Brazilian Journal of Microbiology (2008) 39:547-553 ISSN 1517-8382 HIGHLY THERMOSTABLE AND ALKALINE α-AMYLASE FROM A HALOTOLERANT- ALKALIPHILIC BACILLUS SP. AB68 Ashabil Aygan1*; Burhan Arikan2; Hatice Korkmaz2; Sadik Dinçer2; Ömer Çolak2 1Kahramanmaras Sutcu Imam University, Faculty of Science and Letters, Department of Biology, K. Maras, Turkey; 2Cukurova University, Faculty of Science and Letters, Department of Biology, Molecular Biology Laboratory, Adana, Turkey Submitted: August 13, 2007; Returned to authors for corrections: October 22, 2007; Approved: July 16, 2008. ABSTRACT An alkaliphilic and highly thermostable α-amylase producing Bacillus sp. was isolated from Van soda lake. Enzyme synthesis occurred at temperatures between 25ºC and 40ºC. Analysis of the enzyme by SDS-PAGE revealed a single band which was estimated to be 66 kDa. The enzyme was active in a broad temperature range, between 20ºC and 90ºC, with an optimum at 50ºC; and maximum activity was at pH 10.5. The enzyme was almost completely stable up to 80ºC with a remaining activity over 90% after 30 min pre-incubation. Thermostability was not increased in the presence of Ca2+. An average of 75% and 60ºC of remaining activity was observed when the enzyme was incubated between pH 5 and 9 for 1 h and for 2 h, respectively. The activity of the enzyme was inhibited by SDS and EDTA by 38% and 34%, respectively. Key words: Bacillus sp., α-amylase, Alkaliphilic, Thermostable, Enzyme. INTRODUCTION unique, buffered haloalkaline habitat appropriate for a stable development of obligately (halo)alkaliphilic microorganisms Amylases are one of the most important industrial enzymes. growing optimally at pH around 10 (39).
    [Show full text]
  • (12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall Et Al
    USOO9689046B2 (12) United States Patent (10) Patent No.: US 9,689,046 B2 Mayall et al. (45) Date of Patent: Jun. 27, 2017 (54) SYSTEM AND METHODS FOR THE FOREIGN PATENT DOCUMENTS DETECTION OF MULTIPLE CHEMICAL WO O125472 A1 4/2001 COMPOUNDS WO O169245 A2 9, 2001 (71) Applicants: Robert Matthew Mayall, Calgary (CA); Emily Candice Hicks, Calgary OTHER PUBLICATIONS (CA); Margaret Mary-Flora Bebeselea, A. et al., “Electrochemical Degradation and Determina Renaud-Young, Calgary (CA); David tion of 4-Nitrophenol Using Multiple Pulsed Amperometry at Christopher Lloyd, Calgary (CA); Lisa Graphite Based Electrodes', Chem. Bull. “Politehnica” Univ. Kara Oberding, Calgary (CA); Iain (Timisoara), vol. 53(67), 1-2, 2008. Fraser Scotney George, Calgary (CA) Ben-Yoav. H. et al., “A whole cell electrochemical biosensor for water genotoxicity bio-detection”. Electrochimica Acta, 2009, 54(25), 6113-6118. (72) Inventors: Robert Matthew Mayall, Calgary Biran, I. et al., “On-line monitoring of gene expression'. Microbi (CA); Emily Candice Hicks, Calgary ology (Reading, England), 1999, 145 (Pt 8), 2129-2133. (CA); Margaret Mary-Flora Da Silva, P.S. et al., “Electrochemical Behavior of Hydroquinone Renaud-Young, Calgary (CA); David and Catechol at a Silsesquioxane-Modified Carbon Paste Elec trode'. J. Braz. Chem. Soc., vol. 24, No. 4, 695-699, 2013. Christopher Lloyd, Calgary (CA); Lisa Enache, T. A. & Oliveira-Brett, A. M., "Phenol and Para-Substituted Kara Oberding, Calgary (CA); Iain Phenols Electrochemical Oxidation Pathways”, Journal of Fraser Scotney George, Calgary (CA) Electroanalytical Chemistry, 2011, 1-35. Etesami, M. et al., “Electrooxidation of hydroquinone on simply prepared Au-Pt bimetallic nanoparticles'. Science China, Chem (73) Assignee: FREDSENSE TECHNOLOGIES istry, vol.
    [Show full text]
  • Studies Into the Structural Basis of the DNA Uridine Endonuclease Activity of Exonuclease III Homolog Mth212
    Studies into the structural basis of the DNA uridine endonuclease activity of exonuclease III homolog Mth212 Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultäten der Georg-August Universität zu Göttingen Vorgelegt von Khaliun Tseden aus Greifswald, Deutschland Göttingen 2011 D7 Referent: Prof. Dr. Hans-Joachim Fritz Korreferent: PD Dr. Wilfried Kramer Tag der mündlichen Prüfung: 02. Mai 2011 TABLE OF CONTENTS Table of Contents 1 Introduction ………………………………………………………………….. 1 1.1. Background to the study ………………………………………………… 1 1.1.1. Necessity of mutation avoidance………………………………… 1 1.1.2. Mutations arising in DNA during replication …………………… 1 1.1.3. Exogenous sources of DNA damage …………………………….. 2 1.1.4. Endogenous sources of DNA damage …………………………… 2 1.1.4.1. Hydrolytic DNA deamination ........……………………… 4 1.1.5. Repair of uracil in DNA …………………………………………. 5 1.1.5.1. Uracil-initiated base excision repair …………………...... 5 1.1.5.2. Uracil-initiated nucleotide incision repair ……………… 7 1.2. Objective and methodology of the study ………………………………… 9 1.2.1. Objective of the study.…………………………………………… 9 1.2.2. Methodology of the study ………………………………………. 9 1.2.2.1. Necessity of screening or selection methodology in 11 directed evolution of enzymes …………………………………………… 1.2.2.2. Selection of a protein with acquired DNA uridine 11 endonuclease activity ………………………………………………….... 2. Materials and Methods ………………………………………………………. 13 2.1. Materials …………………………………………………………………. 13 2.1.1. Bacterial strains ………………………………………………….. 13 2.1.1.1. Escherichia coli …………………………………………. 13 2.1.1.2. Bacillus subtilis ………………………………………… 14 2.1.2. Bacteriophage strains …………………………………………….. 14 2.1.3. Plasmid vectors ………………………………………………… 15 2.1.4. 2’ Desoxyriboseoligonucleotides ………………………………. 17 2.1.5. Molecular ladders and markers ………………………………….. 21 2.1.6.
    [Show full text]
  • How Nature Can Exploit Nonspecific Catalytic and Carbohydrate Binding
    How nature can exploit nonspecific catalytic and carbohydrate binding modules to create enzymatic specificity Fiona Cuskina,b,1, James E. Flinta,1, Tracey M. Glosterc,1,2, Carl Morlanda, Arnaud Basléa, Bernard Henrissatd, Pedro M. Coutinhod, Andrea Strazzullie, Alexandra S. Solovyovaa, Gideon J. Daviesc, and Harry J. Gilberta,b,3 aInstitute for Cell and Molecular Biosciences, The Medical School, Newcastle University, Newcastle upon Tyne NE2 4HH, United Kingdom; bThe Complex Carbohydrate Research Center, The University of Georgia, Athens, GA 30602; cStructural Biology Laboratory, Department of Chemistry, University of York, York YO10 5DD, United Kingdom; dArchitecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7257, 13288 Marseille Cedex 9, France; and eInstitute of Protein Biochemistry, Consiglio Nazionale delle Ricerche, 80131 Naples, Italy Edited by Arnold L. Demain, Drew University, Madison, NJ, and approved October 30, 2012 (received for review July 16, 2012) Noncatalytic carbohydrate binding modules (CBMs) are components CBMs enhance the activity of their cognate enzymes, and though of glycoside hydrolases that attack generally inaccessible substrates. the mechanism(s) by which this occurs remains uncertain, CBMs CBMs mediate a two- to fivefold elevation in the activity of endo- most likely fulfill a targeting function by increasing the effective acting enzymes, likely through increasing the concentration of the concentration of the appended enzymes, in the vicinity of the appended enzymes in the vicinity of the substrate. The function of substrate, thereby enhancing catalytic efficiency (8, 9). CBMs appended to exo-acting glycoside hydrolases is unclear Fructans, such as inulin and levan (polymers of predominantly because their typical endo-binding mode would not fulfill a target- β-2,1– or β-2,6–linked fructose units, respectively) are common ing role.
    [Show full text]
  • 154581A0.Pdf
    No. 3914, NOVEMBER 4, 1944 NATURE 581 Alleged Role of Fructofuranose in the ture of fructofuranoses and fructopyranoses. The recent demonstration that glucose-1-phosphate (Cori Synthesis of Levan ester) and fructose form a dynamic equilibrium with THE view was long widely entertained that cells sucrose and phosphoric acid in the presence of a. synthesize macromolecules of polysaccharides and specific enzyme8 corroborates this view. Addition of proteins by a reversion .of the process of hydrolysis. fructose to sucrose does not inhibit levan production It has been suggested accordingly that the synthesis from the latter, yet fructose itself, although it pre­ of the polyfructoside levan specifically from aldo­ sumably contains ready fructofuranose, is not con­ side< >fructofuranosides (sucrose, raffinose) involves verted into levan by levansucrase7 • Similarly, levan­ two distinct steps : first, hydrolysis of the substrate ; sucrase fails to form levan from reaction mixtures secondly, polymerization of fructofuranose b;r a con­ in which fructofuranose is sustainedly liberated in densation involving removal of water1• Bacteria statu nascendi, for example, in reaction mixtures of which form levan from sucrose do so also from methyl gamma fructoside + yeast invertase, and of raffinose•. This polymerative type of sucrose de­ inulin inulase. gradation is concurrent with an ordinary hydrolytic (c) Extracts of an Aerobacter, although they pro­ inversions·'· The same banteria ferment !evans. duce levan from sucrose and hydro'yse the latter as Investigators might be tempted by these correlations well, do not hydrolyse levan. Thus they contain to consider the enzyme system, levansucrase, to be levansucrase and invertase but no polyfructosidase but a mixture of invertase and polyfructosidase.
    [Show full text]
  • First Crystal Structure of an Endo- Levanase – the BT1760 from A
    www.nature.com/scientificreports Corrected: Author Correction OPEN First crystal structure of an endo- levanase – the BT1760 from a human gut commensal Bacteroides Received: 5 April 2019 Accepted: 24 May 2019 thetaiotaomicron Published online: 11 June 2019 Karin Ernits 1, Priit Eek 2, Tiit Lukk 2, Triinu Visnapuu 1 & Tiina Alamäe 1 The endo-levanase BT1760 of a human gut commensal Bacteroides thetaiotaomicron randomly cuts a β-2,6-linked fructan, levan, into fructo-oligosaccharides providing a prebiotic substrate for gut microbiota. Here we introduce the crystal structure of BT1760 at resolution of 1.65 Å. The fold of the enzyme is typical for GH32 family proteins: a catalytic N-terminal fve-bladed β-propeller connected with a C-terminal β-sandwich domain. The levantetraose-bound structure of catalytically inactive mutant E221A at 1.90-Å resolution reveals diferences in substrate binding between the endo-acting fructanases. A shallow substrate-binding pocket of the endo-inulinase INU2 of Aspergillus fcuum binds at least three fructose residues at its fat bottom. In the levantetraose-soaked crystal of the endo-levanase E221A mutant the ligand was bent into the pond-like substrate pocket with its fructose residues making contacts at −3, −2, −1 and + 1 subsites residing at several pocket depths. Binding of levantetraose to the β-sandwich domain was not detected. The N- and C-terminal modules of BT1760 did not bind levan if expressed separately, the catalytic domain lost its activity and both modules tended to precipitate. We gather that endo-levanase BT1760 requires both domains for correct folding, solubility and stability of the protein.
    [Show full text]
  • Substrate Recognition by Two Members of Glycoside Hydrolase Family 32 Involved in Fructo-Oligosaccharide Metabolism in Lactobacillus Acidophilus NCFM
    Substrate recognition of glycoside hydrolase family 32 protein Substrate Recognition by Two Members of Glycoside Hydrolase Family 32 Involved in Fructo-oligosaccharide Metabolism in Lactobacillus acidophilus NCFM Hiroyuki NAKAI1,2*, Birte SVENSSON3, and Ken'ichi OHTSUBO1,2 (Received Decsmber ６, 2011) Summary Two fructo-oligosaccharide active enzymes, BfrA and ScrB, of glycoside hydrolase family 32 are found in Lactobacillus acidophilus NCFM and belong to phylogenetic clusters of bacterial β-fructosidase and sucrose 6-phosphate hydrolase, respectively, involved in the intracellular metabolism of kesto-oligosaccharides [β-D-fructofuranosyl-(2,1)] n-β-D-fructofuranosyl-(2,1)-α-D-glucopyranose (n, 1-3) and sucrose. Recombinant ScrB, produced in Escherichia coli, showed 20-1500-fold higher catalytic efficiency (kcat/Km) toward sucrose than raffinose [α-D-galactopyranosyl-(1,6)-α -D-glucopyranosyl-(1,2)-β-D-fructofuranose] and kesto-oligosaccharides. At high concentration of sucrose (0.4-1.0 M) ScrB catalysed formation of 1-kestose [β-D-fructofuranosyl-(2,1)-β-D-fructofuranosyl-(2,1)-α-D-glucopyranose] in 2.3-15% yield by transglycosylation. Recombinant BfrA in contrast efficiently hydrolysed kesto-oligosaccharides, in particular 1-kestose with 70- 300-fold higher kcat/Km than for sucrose and raffinose and thecat k /Km values decreased slightly with increasing degree of the polymerization of the kesto-oligosaccharides. Neither BfrA nor ScrB degraded the β-(2,1)- and β-(2,6)-linked fructans, inulin and levan. The outcome of the present detailed
    [Show full text]
  • Characterization of the Streptococcus Mutans GS-5Frua Gene Encoding
    INFECTION AND IMMUNITY, Nov. 1992, p. 4621-4632 Vol. 60, No. 11 0019-9567/92/114621-12$02.00/0 Copyright © 1992, American Society for Microbiology Characterization of the Streptococcus mutans GS-5 fruA Gene Encoding Exo-,1-D-Fructosidase ROBERT A. BURNEl 2* AnD JANA E. C. PENDERS' Departments ofDental Research' and ofMicrobiology and Immunology, 2 University ofRochester Medical Center, Rochester, New York 14642 Received 8 June 1992/Accepted 3 September 1992 The complete nucleotide sequence (5,010 bp) of the fructanase gene (fruA) and flanking regions of the chromosome of Streptococcus mutans GS-5 was determined. The fruA gene appears to be the sole transcript arising from a proximal promoter. The presumed precursor of the secreted FruA protein consists of 1,423 amino acids, and it has an Mr of 158,656 and a pI of 4.82. The N terminus of FruA has characteristics in common with signal peptides of gram-positive organisms. The C terminus consists of a serine- and threonine-rich region, followed by the peptide LPDTGD, 4 charged amino acids, 21 amino acids with a strongly hydrophobic character, and a charged pentapeptide tail, which are proposed to correspond to the wall- spanning region, the LPXTGX consensus sequence, and the membrane-spanning domains ofsurface-associated proteins of gram-positive cocci. The FruA protein has significant homology with the BaciUus subtilis levanase (SacC), the Bacteroidesfragilis levanase (ScrL), yeast invertases, and a number ofother 1-fructosidases but not with fructosyltransferase, glucosyltransferases, or glucan-binding proteins of oral streptococci. Genes with homology tofiuA were detected in S. mutans serotype c, e, and f strains, Streptococcus ratus, Streptococcus salivarius, and Streptococcus sanguis.
    [Show full text]
  • Evidence of Cellulose Metabolism by the Giant Panda Gut Microbiome
    Evidence of cellulose metabolism by the giant panda gut microbiome Lifeng Zhua,1,QiWua,1, Jiayin Daia, Shanning Zhangb, and Fuwen Weia,2 aKey Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China and bChina Wildlife Conservation Association, Beijing 100714, China Edited by Rita R. Colwell, University of Maryland, College Park, MD, and approved September 7, 2011 (received for review December 2, 2010) The giant panda genome codes for all necessary enzymes associ- cellulose digestion (10). Although the giant panda can use non- ated with a carnivorous digestive system but lacks genes for cellulosic material from the bamboo diet using enzymes coded in enzymes needed to digest cellulose, the principal component of its own genome, digestion of cellulose and hemicellulose is im- their bamboo diet. It has been posited that this iconic species must possible based on the panda’s genetic composition, and must be therefore possess microbial symbionts capable of metabolizing dependent on gut microbiome. However, previous research using cellulose, but these symbionts have remained undetected. Here we culture methods and small-scale sequencing identified three examined 5,522 prokaryotic ribosomal RNA gene sequences in wild predominant bacteria from the panda gut—Escherichia coli, and captive giant panda fecal samples. We found lower species Streptococcus, and Enterobacteriaceae—none of which aids in richness of the panda microbiome than of mammalian microbiomes cellulose digestion (11–13). Thus, an incomplete understanding for herbivores and nonherbivorous carnivores. We detected 13 of the gut microbial ecosystem in this interesting and high-profile operational taxonomic units closely related to Clostridium groups I species remains because of restrictions in methodology and past and XIVa, both of which contain taxa known to digest cellulose.
    [Show full text]
  • Supplemental Table S1: Comparison of the Deleted Genes in the Genome-Reduced Strains
    Supplemental Table S1: Comparison of the deleted genes in the genome-reduced strains Legend 1 Locus tag according to the reference genome sequence of B. subtilis 168 (NC_000964) Genes highlighted in blue have been deleted from the respective strains Genes highlighted in green have been inserted into the indicated strain, they are present in all following strains Regions highlighted in red could not be deleted as a unit Regions highlighted in orange were not deleted in the genome-reduced strains since their deletion resulted in severe growth defects Gene BSU_number 1 Function ∆6 IIG-Bs27-47-24 PG10 PS38 dnaA BSU00010 replication initiation protein dnaN BSU00020 DNA polymerase III (beta subunit), beta clamp yaaA BSU00030 unknown recF BSU00040 repair, recombination remB BSU00050 involved in the activation of biofilm matrix biosynthetic operons gyrB BSU00060 DNA-Gyrase (subunit B) gyrA BSU00070 DNA-Gyrase (subunit A) rrnO-16S- trnO-Ala- trnO-Ile- rrnO-23S- rrnO-5S yaaC BSU00080 unknown guaB BSU00090 IMP dehydrogenase dacA BSU00100 penicillin-binding protein 5*, D-alanyl-D-alanine carboxypeptidase pdxS BSU00110 pyridoxal-5'-phosphate synthase (synthase domain) pdxT BSU00120 pyridoxal-5'-phosphate synthase (glutaminase domain) serS BSU00130 seryl-tRNA-synthetase trnSL-Ser1 dck BSU00140 deoxyadenosin/deoxycytidine kinase dgk BSU00150 deoxyguanosine kinase yaaH BSU00160 general stress protein, survival of ethanol stress, SafA-dependent spore coat yaaI BSU00170 general stress protein, similar to isochorismatase yaaJ BSU00180 tRNA specific adenosine
    [Show full text]
  • Cazymes Catalogue 2021
    cazymes 2021 Visit the Online Store www.nzytech.com Newsletter NZYWallet Instantaneous Quotes Subscribe our newsletter to receive NZYWallet is a prepaid account Do you need an urgent quote? Just awesome news and promotions. that oers the flexibility you need add your products to Cart, proceed to focus on your research. With to Checkout, select quote and it’s NZYWallet you can buy any done! product from our Online Store, check your up-to-date balance and track your latest orders. Contact our customer service at [email protected] or your local sales representative for more information. Follow us: 2021 NZYTech NZYTech 2 Visit the Online Store www.nzytech.com Newsletter NZYWallet Instantaneous Quotes Subscribe our newsletter to receive NZYWallet is a prepaid account Do you need an urgent quote? Just awesome news and promotions. that oers the flexibility you need add your products to Cart, proceed to focus on your research. With to Checkout, select quote and it’s NZYWallet you can buy any done! product from our Online Store, check your up-to-date balance and track your latest orders. Contact our customer service at [email protected] or your local sales representative for more information. Follow us: cazymes 3 4 2021 NZYTech NZYTech OVERVIEW 8 GLYCOSIDE HYDROLASES 10 Acetylgalactosaminidases Acetylglucosaminidases Agarases Amylases @ a glance Amylomaltases Arabinanases Arabinofuranosidases Arabinopyranosidases Arabinoxylanases Carrageenases Cellobiohydrolases Cellodextrinases Cellulases Chitinases Chitosanases Dextranases Fructanases Fructofuranosidases
    [Show full text]