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Ryszard Brzezinski - One of the best experts on this subject based on the ideXlab platform.
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Uncoupling Chitosanase production from chitosan.
Bioengineered bugs, 2011Co-Authors: Ryszard BrzezinskiAbstract:There is a growing interest in Chitosanases as enzymatic tools to hydrolyze chitosan into bioactive forms: low molecular weight chitosan (LMWC) or chitosan oligosaccharides (CHOS). However Chitosanases are still expensive and methods of large-scale production of these enzymes are not yet established. The article reviews the approaches used for Chitosanase production in various bacterial hosts, pointing out the difficulties resulting from the necessity to include chitosan into the medium composition. A mutated Streptomyces host allows for the efficient production of several Chitosanases originating from actinobacteria in the absence of chitosan as inducer.
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Modification of genetic regulation of a heterologous Chitosanase gene in Streptomyces lividans TK24 leads to Chitosanase production in the absence of chitosan
Microbial cell factories, 2011Co-Authors: Marie-pierre Dubeau, Isabelle Guay, Ryszard BrzezinskiAbstract:Chitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and Chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression. This is the first study on the genetic regulation of a heterologous Chitosanase gene (csnN106) in Streptomyces lividans. Two S. lividans strains were used for induction experiments: the wild type strain and its mutant (ΔcsnR), harbouring an in-frame deletion of the csnR gene, encoding a negative transcriptional regulator. Comparison of Chitosanase levels in various media indicated that CsnR regulates negatively the expression of the heterologous Chitosanase gene csnN106. Using the ΔcsnR host and a mutated csnN106 gene with a modified transcription operator, substantial levels of Chitosanase could be produced in the absence of chitosan, using inexpensive medium components. Furthermore, Chitosanase production was of higher quality as lower levels of extracellular protease and protein contaminants were observed. This new Chitosanase production system is of interest for biotechnology as only common media components are used and enzyme of high degree of purity is obtained directly in the culture supernatant.
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Chitosanase from Streptomyces coelicolor A3(2): biochemical properties and role in protection against antibacterial effect of chitosan.
Biochemistry and Cell Biology, 2010Co-Authors: Mariana Gabriela Ghinet, Sébastien Roy, Dominic Poulin-laprade, Marie-Ève Lacombe-harvey, Rolf Morosoli, Ryszard BrzezinskiAbstract:Chitosan, an N-deacetylated derivative of chitin, has attracted much attention as an antimicrobial agent against fungi, bacteria, and viruses. Chitosanases, the glycoside hydrolases responsible for chitosan depolymerisation, are intensively studied as tools for biotechnological transformation of chitosan. The Chitosanase CsnA (SCO0677) from Streptomyces coelicolor A3(2) was purified and characterized. CsnA belongs to the GH46 family of glycoside hydrolases. However, it is secreted efficiently by the Tat translocation pathway despite its similarity to the well-studied Chitosanase from Streptomyces sp. N174 (CsnN174), which is preferentially secreted through the Sec pathway. Melting point determination, however, revealed substantial differences between these Chitosanases, both in the absence and in the presence of chitosan. We further assessed the role of CsnA as a potential protective enzyme against the antimicrobial effect of chitosan. A Streptomyces lividans TK24 strain in which the csnA gene was inactivated by gene disruption was more sensitive to chitosan than the wild-type strain or a Chitosanase-overproducing strain. This is the first genetic evidence for the involvement of Chitosanases in the protection of bacteria against the antimicrobial effect of chitosan.
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Mechanism of Chitosanase-oligosaccharide interaction: Subsite structure of Streptomyces sp. N174 Chitosanase and the role of Asp57 carboxylate
Journal of biochemistry, 2001Co-Authors: Hugo Tremblay, Tamo Fukamizo, Tsugihisa Yamaguchi, Ryszard BrzezinskiAbstract:We have investigated the mechanism of the interaction of Streptomyces sp. N174 Chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the Chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known Chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The Chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated Chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to Chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the Chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.
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High level expression of the Chitosanase from Streptomyces sp.N174 and study of its intramolecular interactions
2001Co-Authors: Josée Blanchard, Tamo Fukamizo, Isabelle Boucher, Ryszard BrzezinskiAbstract:Chitosan, a natural polymer derived from chitin possesses several properties depending on its degree of polymerization. The molecular weight of chitosan can be controlled by enzymatic hydrolysis using Chitosanases. Stable enzymes produced by efficient expression systems are needed to fulfill the needs of chitosan hydrolysis at an industrial scale. The knowledge on the structure-function relationships of the Chitosanases will open the way to rational approaches of improvement of enzyme thermostability. The article describes recent developments in Chitosanase studies, focusing on the properties of the Chitosanase from Streptomyces sp. N174.
Tamo Fukamizo - One of the best experts on this subject based on the ideXlab platform.
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Bacillus circulans MH-K1 Chitosanase: amino acid residues responsible for substrate binding.
Journal of biochemistry, 2005Co-Authors: Tamo Fukamizo, Junichi Saito, Yoshiho Nagata, Kunio Miki, Satoko Amano, Kei Yamaguchi, Tomoye Yoshikawa, Tomomi Katsumi, Michihiko Suzuki, Akikazu AndoAbstract:To identify the amino acids responsible for the substrate binding of Chitosanase from Bacillus circulans MH-K1 (MH-K1 Chitosanase), Tyr148 and Lys218 of the Chitosanase were mutated to serine and proline, respectively, and the mutated Chitosanases were characterized. The enzymatic activities of Y148S and K218P were found to be 12.5% and 0.16% of the wild type, respectively. When the (GlcN)3 binding ability to the Chitosanase was evaluated by fluorescence spectroscopy and thermal unfolding experiments, the binding abilities of both mutant enzymes were markedly reduced as compared with the wild type enzyme. The affinity of the enzyme for the trisaccharide decreased by 1.0 kcal/mol of binding free energy for Y148S, and 3.7 kcal/mol for K218P. The crystal structure of K218P revealed that Pro218 forms a cis-peptide bond and that the state of the flexible loop containing the 218th residue is considerably affected by the mutation. Thus, we conclude that the flexible loop containing Lys218 plays an important role in substrate binding, and that the role of Tyr148 is less critical, but still important, due to a stacking interaction or hydrogen bond.
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novel Chitosanase from streptomyces griseus hut 6037 with transglycosylation activity
Bioscience Biotechnology and Biochemistry, 2003Co-Authors: Toshiaki Tanabe, Tamo Fukamizo, Kazuko Morinaga, Masaru MitsutomiAbstract:Streptomyces griseus HUT 6037 inducibly produced two Chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, Chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60°C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the Chitosanases were obtained for identification of the anomeric form of the reaction products. Both Chitosanases produced the β-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of Chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding ...
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Novel Chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity.
Bioscience biotechnology and biochemistry, 2003Co-Authors: Toshiaki Tanabe, Tamo Fukamizo, Kazuko Morinaga, Masaru MitsutomiAbstract:Streptomyces griseus HUT 6037 inducibly produced two Chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, Chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60 degrees C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the Chitosanases were obtained for identification of the anomeric form of the reaction products. Both Chitosanases produced the beta-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of Chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding Chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 Chitosanase with transglycosylation activity.
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Mechanism of Chitosanase-oligosaccharide interaction: Subsite structure of Streptomyces sp. N174 Chitosanase and the role of Asp57 carboxylate
Journal of biochemistry, 2001Co-Authors: Hugo Tremblay, Tamo Fukamizo, Tsugihisa Yamaguchi, Ryszard BrzezinskiAbstract:We have investigated the mechanism of the interaction of Streptomyces sp. N174 Chitosanase with glucosamine hexasaccharide [(GlcN)(6)] by site-directed mutagenesis, thermal unfolding, and (GlcN)(6) digestion experiments, followed by theoretical calculations. From the energy-minimized model of the Chitosanase-(GlcN)(6) complex structure (Marcotte et al., 1996), Asp57, which is present in all known Chitosanases, was proposed to be one of the amino acid residues that interacts with the oligosaccharide substrate. The Chitosanase gene was mutated at Asp57 to Asn (D57N) and Ala (D57A), and the relative activities of the mutated Chitosanases were found to be 72 and 0.5% of that of the wild type, respectively. The increase in the transition temperature of thermal unfolding (T(m)), usually observed upon the addition of (GlcN)(n) to Chitosanase mutants unaffected in terms of substrate binding, was considerably suppressed in the D57A mutant. These data suggest that Asp57 is important for substrate binding. The experimental time-courses of [(GlcN)(6)] degradation were analyzed by a theoretical model in order to obtain the binding free energy values of the individual subsites of the Chitosanases. A (-3, -2, -1, +1, +2, +3) subsite model agreed best with the experimental data. This analysis also indicated that the mutation of Asp57 affects substrate affinity at subsite (-2), suggesting that Asp57 most likely participates in the substrate binding at this subsite.
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High level expression of the Chitosanase from Streptomyces sp.N174 and study of its intramolecular interactions
2001Co-Authors: Josée Blanchard, Tamo Fukamizo, Isabelle Boucher, Ryszard BrzezinskiAbstract:Chitosan, a natural polymer derived from chitin possesses several properties depending on its degree of polymerization. The molecular weight of chitosan can be controlled by enzymatic hydrolysis using Chitosanases. Stable enzymes produced by efficient expression systems are needed to fulfill the needs of chitosan hydrolysis at an industrial scale. The knowledge on the structure-function relationships of the Chitosanases will open the way to rational approaches of improvement of enzyme thermostability. The article describes recent developments in Chitosanase studies, focusing on the properties of the Chitosanase from Streptomyces sp. N174.
Isabelle Boucher - One of the best experts on this subject based on the ideXlab platform.
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High level expression of the Chitosanase from Streptomyces sp.N174 and study of its intramolecular interactions
2001Co-Authors: Josée Blanchard, Tamo Fukamizo, Isabelle Boucher, Ryszard BrzezinskiAbstract:Chitosan, a natural polymer derived from chitin possesses several properties depending on its degree of polymerization. The molecular weight of chitosan can be controlled by enzymatic hydrolysis using Chitosanases. Stable enzymes produced by efficient expression systems are needed to fulfill the needs of chitosan hydrolysis at an industrial scale. The knowledge on the structure-function relationships of the Chitosanases will open the way to rational approaches of improvement of enzyme thermostability. The article describes recent developments in Chitosanase studies, focusing on the properties of the Chitosanase from Streptomyces sp. N174.
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site directed mutagenesis of evolutionary conserved carboxylic amino acids in the Chitosanase from streptomyces sp n174 reveals two residues essential for catalysis
Journal of Biological Chemistry, 1995Co-Authors: Isabelle Boucher, Tamo Fukamizo, Yuji Honda, Gordon E Willick, Witold Neugebauer, Ryszard BrzezinskiAbstract:Abstract The comparison of four sequences of prokaryotic Chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the Chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and k, while K was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant Chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the Chitosanase and the other residues are not essential for catalytic activity.
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REACTION MECHANISM OF Chitosanase FROM STREPTOMYCES SP. N174
Biochemical Journal, 1995Co-Authors: Tamo Fukamizo, Sachio Goto, Isabelle Boucher, Yuji Honda, Ryszard BrzezinskiAbstract:Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified Chitosanase, and structures of the products were analysed by NMR spectroscopy. The Chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the Chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the Chitosanase were obtained in order to determine the anomeric form of the reaction products. The Chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the Chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The Chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the Chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.
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A new Chitosanase gene from a Nocardioides sp. is a third member of glycosyl hydrolase family 46
Microbiology, 1995Co-Authors: Jean-yves Masson, Isabelle Boucher, Witold Neugebauer, Dindial Ramotar, Ryszard BrzezinskiAbstract:Summary: Strain N106, a newly isolated soil actinomycete classified in the genus Nocardioides on the basis of its chemotaxonomy, produced an extracellular Chitosanase and was highly active in chitosan degradation. A gene library of Nocardioides sp. N106 was constructed in the shuttle vector pFD666 and recombinant plasmids carrying the Chitosanase gene (csnN106) were identified using the 5′-terminal portion of the Chitosanase gene from Streptomyces sp. N174 as a hybridization probe. One plasmid, pCSN106-2, was used to transform Streptomyces lividans TK24. The Chitosanase produced by S. lividans(pCSN106-2) is a protein of 29.5 kDa, with a pl 8.1, and hydrolyses chitosan by an endo-mechanism giving a mixture of dimers and trimers as end-products. N-terminal sequencing revealed that the mature Chitosanase is a mixture of two enzyme forms differing by one N-terminal amino acid. The csnN106 gene is 79.5% homologous to the csn gene from Streptomyces sp. N174. At the amino acid level, both Chitosanases are homologous at 74.4% and hydrophobic cluster analysis revealed a strict conservation of structural features. This Chitosanase is the third known member of family 46 of glycosyl hydrolases.
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Crystallization of a Chitosanase from Streptomyces N174.
Journal of molecular biology, 1993Co-Authors: Edward M. Marcotte, Isabelle Boucher, Ryszard Brzezinski, P. John Hart, Jon D. RobertusAbstract:Chitosanases are produced by many soil fungi and bacteria to degrade chitosan present in fungal cell walls. Here, we report the crystallization of a 29,500 dalton protein with chitosan endo-hydrolase activity isolated from Streptomyces N174. The crystals were grown by vapor diffusion. They are mechanically strong and diffract to at least 1.9 A resolution. The crystals belong to the monoclinic space group P2(1) with unit cell parameters a = 56.4 A, b = 59.6 A, c = 86.1 A and beta = 96.6 degrees. Cell parameters and crystal density are consistent with two Chitosanase molecules per asymmetric unit.
Ruyong Yao - One of the best experts on this subject based on the ideXlab platform.
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purification and characterization of a new cold adapted and thermo tolerant Chitosanase from marine bacterium pseudoalteromonas sp sy39
Molecules, 2019Co-Authors: Yu Zhou, Xuehong Chen, Yantao Han, Yanan Wang, Ruyong YaoAbstract:Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many Chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new Chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type Chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides.
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Purification and Characterization of A New Cold-Adapted and Thermo-Tolerant Chitosanase from Marine Bacterium Pseudoalteromonas sp. SY39
MDPI AG, 2019Co-Authors: Yu Zhou, Xuehong Chen, Yantao Han, Yanan Wang, Ruyong YaoAbstract:Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many Chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new Chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type Chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides
Yantao Han - One of the best experts on this subject based on the ideXlab platform.
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Design and Synthesis of a Chitodisaccharide-Based Affinity Resin for Chitosanases Purification.
Marine drugs, 2019Co-Authors: Linna Wang, Xuehong Chen, Mi Sun, Yantao HanAbstract:Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel Chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native Chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native Chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 Chitosanase. The three purified Chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free Chitosanase could be directly purified from supernatant of bacterial culture. The purified Chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10–50% yield. The novel purification methods established in this work can be applied to purify native Chitosanases in various scales, such as laboratory and industrial scales.
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purification and characterization of a new cold adapted and thermo tolerant Chitosanase from marine bacterium pseudoalteromonas sp sy39
Molecules, 2019Co-Authors: Yu Zhou, Xuehong Chen, Yantao Han, Yanan Wang, Ruyong YaoAbstract:Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many Chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new Chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type Chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides.
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Purification and Characterization of A New Cold-Adapted and Thermo-Tolerant Chitosanase from Marine Bacterium Pseudoalteromonas sp. SY39
MDPI AG, 2019Co-Authors: Yu Zhou, Xuehong Chen, Yantao Han, Yanan Wang, Ruyong YaoAbstract:Chitosanases play an important role in chitosan degradation, forming enzymatic degradation products with several biological activities. Although many Chitosanases have been discovered and studied, the enzymes with special characteristics are still rather rare. In this study, a new Chitosanase, CsnM, with an apparent molecular weight of 28 kDa was purified from the marine bacterium Pseudoalteromonas sp. SY39. CsnM is a cold-adapted enzyme, which shows highest activity at 40 °C and exhibits 30.6% and 49.4% of its maximal activity at 10 and 15 °C, respectively. CsnM is also a thermo-tolerant enzyme that recovers 95.2%, 89.1% and 88.1% of its initial activity after boiling for 5, 10 and 20 min, respectively. Additionally, CsnM is an endo-type Chitosanase that yields chitodisaccharide as the main product (69.9% of the total product). It’s cold-adaptation, thermo-tolerance and high chitodisaccharide yield make CsnM a superior candidate for biotechnological application to produce chitooligosaccharides