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Lubbert Dijkhuizen - One of the best experts on this subject based on the ideXlab platform.
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reaction kinetics and galactooligosaccharide product profiles of the β galactosidases from Bacillus Circulans kluyveromyces lactis and aspergillus oryzae
Food Chemistry, 2017Co-Authors: Huifang Yin, Lubbert Dijkhuizen, Jelle B Bultema, Sander S Van LeeuwenAbstract:β-Galactosidase enzymes are used in the dairy industry to convert lactose into galactooligosaccharides (GOS) that are added to infant formula to mimic the molecular sizes and prebiotic functions of human milk oligosaccharides. Here we report a detailed analysis of the clearly different GOS profiles of the commercial β-galactosidases from Bacillus Circulans, Kluyveromyces lactis and Aspergillus oryzae. Also the GOS yields of these enzymes differed, varying from 48.3% (B. Circulans) to 34.9% (K. lactis), and 19.5% (A. oryzae). Their incubation with lactose plus the monosaccharides Gal or Glc resulted in altered GOS profiles. Experiments with 13C6 labelled Gal and Glc showed that both monosaccharides act as acceptor substrates in the transgalactosylation reactions. The data shows that the lactose isomers β-d-Galp-(1→2)-d-Glcp, β-d-Galp-(1→3)-d-Glcp and β-d-Galp-(1→6)-d-Glcp are formed from acceptor reactions with free Glc and not by rearrangement of Glc in the active site.
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biochemical characterization of mutants in the active site residues of the β galactosidase enzyme of Bacillus Circulans atcc 31382
FEBS Open Bio, 2014Co-Authors: Jelle B Bultema, Bas J H Kuipers, Lubbert DijkhuizenAbstract:The Bacillus Circulans ATCC 31382 β-galactosidase (BgaD) is a retaining-type glycosidase of glycoside hydrolase family 2 (GH2). Its commercial enzyme preparation, Biolacta N5, is used for commercial-scale production of galacto-oligosaccharides (GOS). The BgaD active site and catalytic amino acid residues have not been studied. Using bioinformatic routines we identified two putative catalytic glutamates and two highly conserved active site histidines. The site-directed mutants E447N, E532Q, and H345F, H379F had lost (almost) all catalytic activity. This confirmed their essential role in catalysis, as general acid/base catalyst (E447) and nucleophile (E532), and as transition state stabilizers (H345, H379), respectively.
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rational design of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 to increase alpha cyclodextrin production
Journal of Molecular Biology, 2000Co-Authors: Bart A Van Der Veen, D. Penninga, Joost C M Uitdehaag, Gertjan W M Van Alebeek, Loraine M Smith, Bauke W Dijkstra, Lubbert DijkhuizenAbstract:Cyclodextrin glycosyltransferases (CGTase) (EC;2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus Circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 Angstrom resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity. (C) 2000 Academic Press.
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site directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 affect activity and product specificity
Biochemistry, 1995Co-Authors: D. Penninga, Jack Bergsma, Catherine L Lawson, Bauke W Dijkstra, Boris Strokopytov, H J Rozeboom, Lubbert DijkhuizenAbstract:Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus Circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 Angstrom resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins. This again points to an important role for the residue at position 195 in the formation of cyclic products.
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nucleotide sequence and x ray structure of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 in a maltose dependent crystal form
Journal of Molecular Biology, 1994Co-Authors: Catherine L Lawson, Lubbert Dijkhuizen, D. Penninga, R Van Montfort, Boris Strokopytov, H J Rozeboom, Kor H Kalk, G De Vries, Bauke W DijkstraAbstract:The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus Circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. Circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. Circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.
Bauke W Dijkstra - One of the best experts on this subject based on the ideXlab platform.
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rational design of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 to increase alpha cyclodextrin production
Journal of Molecular Biology, 2000Co-Authors: Bart A Van Der Veen, D. Penninga, Joost C M Uitdehaag, Gertjan W M Van Alebeek, Loraine M Smith, Bauke W Dijkstra, Lubbert DijkhuizenAbstract:Cyclodextrin glycosyltransferases (CGTase) (EC;2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus Circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 Angstrom resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity. (C) 2000 Academic Press.
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site directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 affect activity and product specificity
Biochemistry, 1995Co-Authors: D. Penninga, Jack Bergsma, Catherine L Lawson, Bauke W Dijkstra, Boris Strokopytov, H J Rozeboom, Lubbert DijkhuizenAbstract:Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus Circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 Angstrom resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins. This again points to an important role for the residue at position 195 in the formation of cyclic products.
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nucleotide sequence and x ray structure of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 in a maltose dependent crystal form
Journal of Molecular Biology, 1994Co-Authors: Catherine L Lawson, Lubbert Dijkhuizen, D. Penninga, R Van Montfort, Boris Strokopytov, H J Rozeboom, Kor H Kalk, G De Vries, Bauke W DijkstraAbstract:The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus Circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. Circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. Circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.
Warren W. Wakarchuk - One of the best experts on this subject based on the ideXlab platform.
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characterization of a buried neutral histidine residue in Bacillus Circulans xylanase nmr assignments ph titration and hydrogen exchange
Protein Science, 1996Co-Authors: Leigh A Plesniak, Warren W. Wakarchuk, Gregory P Connelly, Lawrence P McintoshAbstract:Bacillus Circulans xylanase contains two histidines, one of which (His 156) is solvent exposed, whereas the other (His 149) is buried within its hydrophobic core. His 149 is involved in a network of hydrogen bonds with an internal water and Ser 130, as well as a potential weak aromatic-aromatic interaction with Tyr 105. These three residues, and their network of interactions with the bound water, are conserved in four homologous xylanases. To probe the structural role played by His 149, NMR spectroscopy was used to characterize the histidines in BCX. Complete assignments of the 'H, I3C, and I5N resonances and tautomeric forms of the imidazole rings were obtained from two-dimensional heteronuclear correlation experiments. An unusual spectroscopic feature of BCX is a peak near 12 ppm arising from the nitrogen bonded 'HE' of His 149. Due to its solvent inaccessibility and hydrogen bonding to an internal water molecule, the exchange rate of this proton (4.0 X low5 s-' at pH* 7.04 and 30 "C) is retarded by > 106-fold relative to an exposed histidine. The pKa of His 156 is unperturbed at -6.5, as measured from the pH dependence of the "N- and 'H-NMR spectra of BCX. In contrast, His 149 has a pKa < 2.3, existing in the neutral N'*H tautomeric state under all conditions examined. BCX unfolds at low pH and 30 "C, and thus His 149 is never protonated significantly in the context of the native enzyme. The structural importance of this buried histidine is confirmed by the destablizing effect of substituting a phenylalanine or glutamine at position 149 in BCX.
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the pka of the general acid base carboxyl group of a glycosidase cycles during catalysis a 13c nmr study of Bacillus Circulans xylanase
Biochemistry, 1996Co-Authors: Lawrence P Mcintosh, Warren W. Wakarchuk, Greg Hand, Philip E Johnson, Manish D Joshi, Michael Korner, Leigh A Plesniak, Lothar Ziser, Stephen G WithersAbstract:The 20 kDa xylanase from Bacillus Circulans carries out hydrolysis of xylan via a two-step mechanism involving a covalent glycosyl−enzyme intermediate. In this double-displacement reaction, Glu78 f...
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abnormally high pka of an active site glutamic acid residue in Bacillus Circulans xylanase
FEBS Journal, 1995Co-Authors: Jamshid Davoodi, Warren W. Wakarchuk, Robert L. Campbell, Paul R. Carey, Witold K. SurewiczAbstract:The active site of Bacillus Circulans xylanase (1,4-β-D-xylanohydrolase, EC 3.2.1.8) contains two glutamic acid residues, Glu78 and Glu172, which are crucial for the catalytic activity of the enzyme. Fourier-transform infrared spectroscopy was used to determine the ionization state of these residues as a function of pH. For the wild-type enzyme, titration of one of the carboxylate groups occurs at pH 6.8. This titration is absent in the Glu78Gln and Glu172Gln variants of the enzyme. This, together with crystallographic data, indicates that Glu172 has an abnormally high pKa of 6.8, caused largely by electrostatic interactions of this residue with the proximal Glu78. Differential scanning calorimetry experiments with the wild-type xylanase and a number of its mutants have shown that the presence of two nearby carboxyl groups results in a pH-dependent destabilization of the protein structure.
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mutational and crystallographic analyses of the active site residues of the Bacillus Circulans xylanase
Protein Science, 1994Co-Authors: Warren W. Wakarchuk, Jamshid Davoodi, Robert L. Campbell, Wing L Sung, Makoto YaguchiAbstract:Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus Circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.
D. Penninga - One of the best experts on this subject based on the ideXlab platform.
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rational design of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 to increase alpha cyclodextrin production
Journal of Molecular Biology, 2000Co-Authors: Bart A Van Der Veen, D. Penninga, Joost C M Uitdehaag, Gertjan W M Van Alebeek, Loraine M Smith, Bauke W Dijkstra, Lubbert DijkhuizenAbstract:Cyclodextrin glycosyltransferases (CGTase) (EC;2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus Circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 Angstrom resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity. (C) 2000 Academic Press.
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site directed mutations in tyrosine 195 of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 affect activity and product specificity
Biochemistry, 1995Co-Authors: D. Penninga, Jack Bergsma, Catherine L Lawson, Bauke W Dijkstra, Boris Strokopytov, H J Rozeboom, Lubbert DijkhuizenAbstract:Tyrosine 195 is located in the center of the active site cleft of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus Circulans strain 251. Alignment of amino acid sequences of CGTases and alpha-amylases, and the analysis of the binding mode of the substrate analogue acarbose in the active site cleft [Strokopytov, B., et al. (1995) Biochemistry 34, (in press)], suggested that Tyr195 plays an important role in cyclization of oligosaccharides. Tyr195 therefore was replaced with Phe (Y195F), Trp (Y195W), Leu (Y195L), and Gly (Y195G). Mutant proteins were purified and crystallized, and their X-ray structures were determined at 2.5-2.6 Angstrom resolution, allowing a detailed comparison of their biochemical properties and three-dimensional structures with those of the wild-type CGTase protein. The mutant proteins possessed significantly reduced cyclodextrin forming and coupling activities but were not negatively affected in the disproportionation and saccharifying reactions. Also under production process conditions, after a 45 h incubation with a 10% starch solution, the Y195W, Y195L, and Y195G mutants showed a lower overall conversion of starch into cyclodextrins. These mutants produced a considerable amount of linear maltooligosaccharides. The presence of aromatic amino acids (Tyr or Phe) at the Tyr195 position thus appears to be of crucial importance for an efficient cyclization reaction, virtually preventing the formation of linear products. Mass spectrometry of the Y195L reaction mixture, but not that of the other mutants and the wild type, revealed a shift toward the synthesis (in low yields) of larger products, especially of beta- and gamma- (but no alpha-) cyclodextrins and minor amounts of delta-, epsilon-, zeta- and eta-cyclodextrins. This again points to an important role for the residue at position 195 in the formation of cyclic products.
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nucleotide sequence and x ray structure of cyclodextrin glycosyltransferase from Bacillus Circulans strain 251 in a maltose dependent crystal form
Journal of Molecular Biology, 1994Co-Authors: Catherine L Lawson, Lubbert Dijkhuizen, D. Penninga, R Van Montfort, Boris Strokopytov, H J Rozeboom, Kor H Kalk, G De Vries, Bauke W DijkstraAbstract:The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus Circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, showing 75% identity to the CGTase from B. Circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 A resolution. The structure of the enzyme is nearly identical to the CGTase from B. Circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.
Takeshi Watanabe - One of the best experts on this subject based on the ideXlab platform.
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a bacterial chitinase acts as catalyst for synthesis of the n linked oligosaccharide core trisaccharide by employing a sugar oxazoline substrate
Journal of Carbohydrate Chemistry, 2006Co-Authors: Atsushi Kobayashi, Takeshi Watanabe, Hideyuki Kuwata, Michinari Kohri, Ryuko Izumi, Shinichiro ShodaAbstract:A chitinolytic enzyme, chitinase A1 from Bacillus Circulans WL‐12, was found to catalyze a glycosyl‐transferring reaction to form the N‐linked oligosaccharide core structure, Man(β1‐4)‐GlcNAc(β1‐4)‐GlcNAc, by employing Man(β1‐4)‐GlcNAc‐oxazoline as glycosyl donor. When the reaction was carried out in the presence of 20 v/v% acetone, the trisaccharide was obtained in 32% yield. It has been shown for the first time that a chitinase behaves like an endo‐β‐N‐acetylglucosaminidase in spite of low structural similarity between them.
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chitosanase activity of the enzyme previously reported as β 1 3 1 4 glucanase from Bacillus Circulans wl 12
Bioscience Biotechnology and Biochemistry, 1998Co-Authors: Masaru Mitsutomi, Makoto Isono, Asako Uchiyama, Naoki Nikaidou, Taiji Ikegami, Takeshi WatanabeAbstract:Chitosanases 33 kDa and 40 kDa in size were detected in the culture supernatant of Bacillus Circulans WL-12. One of the two chitosanases, chitosanase 40 (40-kDa chitosanase) has been shown to be identical to the enzyme which has been reported previously as a β-1,3-1,4-glucanase by Bueno et al.1) The enzyme has been classified into family 8 glycosyl hydrolases together with the enzymes formally known as cellulase family D. This enzyme named chitosanase 40/β-1,3-1,4-glucanase hydrolyzed both chitosan and β-1,3-1,4-glucan with similar efficiency. However, the production of the enzyme was induced with chitosan but not by β-1,3-1,4-glucan. Therefore, it seems possible that the major substrate of this enzyme is chitosan rather than β-1,3-1,4-glucan. Analysis of degradation products generated from partially N-acetylated chitosan showed that chitosanase 40/β-1,3-1,4-glucanase hydrolyzes GlcN-GlcN and GlcN-GlcNAc linkages but not GlcNAc-GlcNAc nor GlcNAc-GlcN. The specificity for hydrolyzing linkages of this enzym...
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identification of glutamic acid 204 and aspartic acid 200 in chitinase a1 of Bacillus Circulans wl 12 as essential residues for chitinase activity
Journal of Biological Chemistry, 1993Co-Authors: Takeshi Watanabe, K Kobori, K Miyashita, T Fujii, H Sakai, M Uchida, H TanakaAbstract:Abstract Prokaryotic chitinases, class III plant chitinases, yeast chitinases, and endo-beta-N-acetylglucosaminidases share weak amino acid sequence similarities at the certain region of each enzyme. These regions have been assumed to be important for catalytic activities of the enzymes. To verify this assumption, three amino acid residues (Ser-160, Asp-200, Glu-204) in chitinase A1 of Bacillus Circulans WL-12 were chosen, based on the amino acid sequence alignment of the regions sharing sequence similarity, and were replaced by site-directed mutagenesis. Kinetic parameters for 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and seven mutant chitinases. Chitinases with Glu-204-->Gln mutation and Glu-204-->Asp mutation were essentially inactive and kcat values of these chitinases were approximately 1/5,000 and 1/17,000 of that of wild-type chitinase, respectively. Asp-200-->Asn mutation decreased the kcat value to approximately 1/350 of that of the wild-type enzyme, while the Km value decreased only slightly. On the other hand, neither the kcat value nor the Km value was affected by Asp-200-->Glu mutation. Thus, it appeared that Glu-204 and Asp-200 are directly involved in the catalytic events of chitinase A1. The role of the carboxyl group of Asp-200 can be fully substituted by that of Glu residue. The Ser-160-->Ala mutant retained 10% activity of the wild-type chitinase indicating that the hydroxyl group of Ser-160 is not absolutely required for the catalytic activity. These results indicate a lysozyme-type catalytic mechanism of the chitinase.
