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Takeshi Watanabe - One of the best experts on this subject based on the ideXlab platform.
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Identification and characterization of chitinolytic bacteria isolated from a freshwater lake
2018Co-Authors: Dinh Minh Tran, Takeshi Watanabe, Hayuki Sugimoto, Dzung Anh Nguyen, Kazushi SuzukiAbstract:To develop a novel type of biocontrol agent, we focus on bacteria that are characterized by both Chitinase activity and biofilm development. Chitinolytic bacteria were isolated from sediments and chitin flakes immersed in the water of a sand dune lake, Sakata, in Niigata, Japan. Thirty-one isolates from more than 5100 isolated strains were examined Chitinase activity and biofilm formation. Phylogenetic analysis of these isolates based on the 16S rRNA gene sequences revealed that most isolates belonged to the family Aeromonadaceae, followed by Paenibacillaceae, Enterobacteriaceae, and Neisseriaceae. The specific activity of Chitinase of four selected strains was higher than that of a reference strain. The molecular size of one Chitinase produced by Andreprevotia was greater than that of typical bacterial Chitinases. The dialyzed culture supernatant containing Chitinases of the four strains suppressed hyphal growth of Trichoderma reesei. These results indicate that these four strains are good candidates for biocontrol agents. The dialyzed culture supernatant containing Chitinases of the four isolated strains suppressed hyphal growth of Trichoderma reesei.
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comparison of enzymatic and antifungal properties between family 18 and 19 Chitinases from s coelicolor a3 2
Bioscience Biotechnology and Biochemistry, 2006Co-Authors: Tomokazu Kawase, Shinya Yokokawa, Akihiro Saito, Takeshi Fujii, Naoki Nikaidou, Kiyotaka Miyashita, Takeshi WatanabeAbstract:Streptomyces coelicolor A3(2) has 13 Chitinase genes encoding 11 family 18 and two family 19 Chitinases. To compare enzymatic properties of family 19 Chitinase and family 18 Chitinases produced by the same organism, the four Chitinases (Chi18bA, Chi18aC, Chi18aD, and Chi19F), whose genes are expressed at high levels in the presence of chitin, were produced in Eschericha coli and purified. The effect of pH on the hydrolytic activity was very different not only among the four Chitinases but also among the substrates. The hydrolytic activity of Chi19F, family 19 Chitinase, against soluble substrates was remarkably high as compared with three family 18 Chitinases, but was the lowest against crystalline substrates among the four Chitinases. On the contrary, Chi18aC, a family 18-subfamily A Chitinase, showed highest activity against crystalline substrates. Only Chi19F exhibited significant antifungal activity. Based on these observations, the roles of family 19 Chitinases are discussed.
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the third Chitinase gene chic of serratia marcescens 2170 and the relationship of its product to other bacterial Chitinases
Biochemical Journal, 1999Co-Authors: Kazushi Suzuki, Naoki Nikaidou, Mayumi Taiyoji, Noriko Sugawara, Bernard Henrissat, Takeshi WatanabeAbstract:The third Chitinase gene (chiC) of Serratia marcescens 2170, specifying Chitinases C1 and C2, was identified. Chitinase C1 lacks a signal sequence and consists of a catalytic domain belonging to glycoside hydrolase family 18, a fibronectin type III-like domain (Fn3 domain) and a C-terminal chitin-binding domain (ChBD). Chitinase C2 corresponds to the catalytic domain of C1 and is probably generated by proteolytic removal of the Fn3 and ChBDs. The loss of the C-terminal portion reduced the hydrolytic activity towards powdered chitin and regenerated chitin, but not towards colloidal chitin and glycol chitin, illustrating the importance of the ChBD for the efficient hydrolysis of crystalline chitin. Phylogenetic analysis showed that bacterial family 18 Chitinases can be clustered in three subfamilies which have diverged at an early stage of bacterial Chitinase evolution. Ser. marcescens Chitinase C1 is found in one subfamily, whereas Chitinases A and B of the same bacterium belong to another subfamily. Chitinase C1 is the only Ser. marcescens Chitinase that has an Fn3 domain. The presence of multiple, divergent, Chitinases in a single chitinolytic bacterium is perhaps necessary for efficient synergistic degradation of chitin.
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a modular family 19 Chitinase found in the prokaryotic organism streptomyces griseus hut 6037
Journal of Bacteriology, 1996Co-Authors: T Ohno, Naoki Nikaidou, Bernard Henrissat, Sylvie Armand, T Hata, M Mitsutomi, Takeshi WatanabeAbstract:The specificity of Chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial Chitinases. In order to study the primary structure of this unique Chitinase, the chiC gene specifying Chitinase C-1 was cloned and its nucleotide sequence was determined. The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa. Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV Chitinases which form glycosyl hydrolase family 19. The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial Chitinases and cellulases but not to the chitin-binding domains of plant Chitinases. The previously purified Chitinase C-1 from S. griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the Chitinase encoded by the chiC gene. High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that Chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I Chitinases. This is the first report of a family 19 Chitinase found in an organism other than higher plants.
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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.
Jeenkuan Chen - One of the best experts on this subject based on the ideXlab platform.
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colloid chitin azure is a dispersible low cost substrate for Chitinase measurements in a sensitive fast reproducible assay
Journal of Biomolecular Screening, 2010Co-Authors: Chiarui Shen, Yusheng Chen, Chingjen Yang, Jeenkuan ChenAbstract:Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of Chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost Chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining Chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure Chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant Chitinase) and AS1 (a chitinolytic bacterium with low levels of Chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1...
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colloid chitin azure is a dispersible low cost substrate for Chitinase measurements in a sensitive fast reproducible assay
Journal of Biomolecular Screening, 2010Co-Authors: Chiarui Shen, Yusheng Chen, Chingjen Yang, Jeenkuan Chen, Chaolin LiuAbstract:Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of Chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost Chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining Chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure Chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant Chitinase) and AS1 (a chitinolytic bacterium with low levels of Chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of Chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of Chitinase activity.
Chiarui Shen - One of the best experts on this subject based on the ideXlab platform.
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colloid chitin azure is a dispersible low cost substrate for Chitinase measurements in a sensitive fast reproducible assay
Journal of Biomolecular Screening, 2010Co-Authors: Chiarui Shen, Yusheng Chen, Chingjen Yang, Jeenkuan ChenAbstract:Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of Chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost Chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining Chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure Chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant Chitinase) and AS1 (a chitinolytic bacterium with low levels of Chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1...
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colloid chitin azure is a dispersible low cost substrate for Chitinase measurements in a sensitive fast reproducible assay
Journal of Biomolecular Screening, 2010Co-Authors: Chiarui Shen, Yusheng Chen, Chingjen Yang, Jeenkuan Chen, Chaolin LiuAbstract:Chitin and its derivatives are widely used as biomedical materials because of their versatility and biocompatibility. Chitinases are enzymes that produce chito-oligosaccharides from chitin. The assay of Chitinase activity is difficult because few appropriate substrates are available. In this study, the authors developed an efficient and low-cost Chitinase assay using colloidal chitin azure. The assay feasibility is evaluated and compared with traditional assays employing colloidal chitin and chitin azure. The authors found that the optimum pH for determining Chitinase activity using colloid chitin azure was pH 5 or 8. The method was sensitive, and the assay was complete within 30 min. When the assay was used to measure Chitinase activities produced by 2 strains of chitinolytic bacteria, BCTS (an Escherichia coli BL21 [DE3] expressing a secretory recombinant Chitinase) and AS1 (a chitinolytic bacterium with low levels of Chitinase), it was shown that cultivation in Bushnell-Haas selection medium caused AS1 to secrete a higher level of Chitinase than was secreted when the bacterium grew in other media. In summary, colloid chitin azure is a sensitive, feasible, reproducible, and low-cost substrate for the assay of Chitinase activity.
Krishna Bisetty - One of the best experts on this subject based on the ideXlab platform.
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Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications
Extremophiles, 2015Co-Authors: Faez Iqbal Khan, Krishna Bisetty, Kugen Permaul, Suren Singh, Md Imtaiyaz HassanAbstract:Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, Chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of Chitinases, of which Chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on Chitinases from different sources, mainly fungal Chitinase of T. lanuginosus and its industrial application.
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thermostable Chitinase ii from thermomyces lanuginosus ssbp cloning structure prediction and molecular dynamics simulations
Journal of Theoretical Biology, 2015Co-Authors: Faez Iqbal Khan, Kugen Permaul, Suren Singh, Algasan Govender, Krishna BisettyAbstract:Abstract Thermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable Chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable Chitinase II. Sequence analysis revealed that Chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that Chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of Chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.
Faez Iqbal Khan - One of the best experts on this subject based on the ideXlab platform.
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Chitinase from Thermomyces lanuginosus SSBP and its biotechnological applications
Extremophiles, 2015Co-Authors: Faez Iqbal Khan, Krishna Bisetty, Kugen Permaul, Suren Singh, Md Imtaiyaz HassanAbstract:Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, Chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability for temperature and pH. Thermomyces lanuginosus produced a large number of Chitinases, of which Chitinase I and II are successfully cloned and purified recently. Molecular dynamic simulations revealed that the stability of these enzymes are maintained even at higher temperature. In this review article we have focused on Chitinases from different sources, mainly fungal Chitinase of T. lanuginosus and its industrial application.
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thermostable Chitinase ii from thermomyces lanuginosus ssbp cloning structure prediction and molecular dynamics simulations
Journal of Theoretical Biology, 2015Co-Authors: Faez Iqbal Khan, Kugen Permaul, Suren Singh, Algasan Govender, Krishna BisettyAbstract:Abstract Thermomyces lanuginosus is a thermophilic fungus that produces large number of industrially-significant enzymes owing to their inherent stability at high temperatures and wide range of pH optima, including thermostable Chitinases that have not been fully characterized. Here, we report cloning, characterization and structure prediction of a gene encoding thermostable Chitinase II. Sequence analysis revealed that Chitinase II gene encodes a 343 amino acid protein of molecular weight 36.65 kDa. Our study reports that Chitinase II exhibits a well-defined TIM-barrel topology with an eight-stranded α/β domain. Structural analysis and molecular docking studies suggested that Glu176 is essential for enzyme activity. Folding studies of Chitinase II using molecular dynamics simulations clearly demonstrated that the stability of the protein was evenly distributed at 350 K.