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Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-d-glucosamine production.

ABSTRACT: Background:N-acetyl-d-glucosamine (GlcNAc) possesses many bioactivities that have been used widely in many fields. The enzymatic production of GlcNAc is eco-friendly, with high yields and a mild production process compared with the traditional chemical process. Therefore, it is crucial to discover a better chitinase for GlcNAc production from chitin. Results:A novel chitinase gene (Cmchi1) cloned from Chitinolyticbacter meiyuanensis SYBC-H1 and expressed in Escherichia coli BL21(DE3) cells. The recombinant enzyme (CmChi1) contains a glycosyl hydrolase family 18 catalytic module that shows low identity (12-27%) with the corresponding domain of the well-characterized chitinases. CmChi1 was purified with a recovery yield of 89% by colloidal chitin affinity chromatography, whereupon it had a specific activity of up to 15.3 U/mg. CmChi1 had an approximate molecular mass of 70 kDa after the sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its optimum activity for colloidal chitin (CC) hydrolysis occurred at pH 5.2 and 50 °C. Furthermore, CmChi1 exhibited kcat/Km values of 7.8?±?0.11 mL/s/mg and 239.1?±?2.6 mL/s/?mol toward CC and 4-nitrophenol N,N'-diacetyl-?-d-chitobioside [p-NP-(GlcNAc)2], respectively. Analysis of the hydrolysis products revealed that CmChi1 exhibits exo-acting, endo-acting and N-acetyl-?-d-glucosaminidase activities toward N-acetyl chitooligosaccharides (N-acetyl CHOS) and CC substrates, behavior that makes it different from typical reported chitinases. As a result, GlcNAc could be produced by hydrolyzing CC using recombinant CmChi1 alone with a yield of nearly 100% and separated simply from the hydrolysate with a high purity of 98%. Conclusion:The hydrolytic properties and good environmental adaptions indicate that CmChi1 has excellent potential in commercial GlcNAc production. This is the first report on exo-acting, endo-acting and N-acetyl-?-d-glucosaminidase activities from Chitinolyticbacter species.

SUBMITTER: Zhang A

PROVIDER: S-EPMC6020246 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Molecular characterization of a novel chitinase <i>Cm</i>Chi1 from <i>Chitinolyticbacter meiyuanensis</i> SYBC-H1 and its use in <i>N</i>-acetyl-d-glucosamine production.

Zhang Alei A He Yumei Y Wei Guoguang G Zhou Jie J Dong Weiliang W Chen Kequan K Ouyang Pingkai P

Biotechnology for biofuels 20180626

<h4>Background</h4><i>N</i>-acetyl-d-glucosamine (GlcNAc) possesses many bioactivities that have been used widely in many fields. The enzymatic production of GlcNAc is eco-friendly, with high yields and a mild production process compared with the traditional chemical process. Therefore, it is crucial to discover a better chitinase for GlcNAc production from chitin.<h4>Results</h4>A novel chitinase gene (<i>Cmchi1</i>) cloned from <i>Chitinolyticbacter meiyuanensis</i> SYBC-H1 and expressed in <i ...[more]

PMID: 29983742

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Project description:Background:N-Acetyl glucosamine (GlcNAc) and N-Acetyl chitooligosaccharides (N-Acetyl COSs) exhibit many biological activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Particularly, higher N-Acetyl COSs with degree of polymerization from 4 to 7 ((GlcNAc)4-(GlcNAc)7) show good antitumor and antimicrobial activity, as well as possessing strong stimulating activity toward natural killer cells. Thus, it is of great significance to discover a ?-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs. Results:The gene encoding the novel ?-N-acetyl glucosaminidase, designated CmNAGase, was cloned from Chitinolyticbacter meiyuanensis SYBC-H1. The deduced amino acid sequence of CmNAGase contains a glycoside hydrolase family 20 catalytic module that shows low identity (12-35%) with the corresponding domain of most well-characterized NAGases. The CmNAGase gene was highly expressed with an active form in Escherichia coli BL21 (DE3) cells. The specific activity of purified CmNAGase toward p-nitrophenyl-N-acetyl glucosaminide (pNP-GlcNAc) was 4878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40 °C. The V max, K m, K cat, and K cat/K m of CmNAGase for pNP-GlcNAc were 16,666.67 ?mol min-1 mg-1, 0.50 ?mol mL-1, 25,555.56 s-1, and 51,111.12 mL ?mol-1 s-1, respectively. Analysis of the hydrolysis products of N-Acetyl COSs and colloidal chitin revealed that CmNAGase is a typical exo-acting NAGase. Particularly, CmNAGase can synthesize higher N-Acetyl COSs ((GlcNAc)3-(GlcNAc)7) from (GlcNAc)2-(GlcNAc)6, respectively, showed that it possesses transglycosylation activity. In addition, CmNAGase also has reverse hydrolysis activity toward GlcNAc, synthesizing various linked GlcNAc dimers. Conclusions:The observations recorded in this study that CmNAGase is a novel NAGase with exo-acting, transglycosylation, and reverse hydrolysis activities, suggest a possible application in the production of GlcNAc or higher N-Acetyl COSs.

Project description:The causative agent of the pandemic identified as SARS-CoV-2 leads to a severe respiratory illness similar to SARS and MERS with fever, cough, and shortness of breath symptoms and severe cases that can often be fatal. In our study, we report our findings based on molecular docking analysis which could be the new effective way for controlling the SARS-CoV-2 virus and additionally, another manipulative possibilities involving the mimicking of immune system as occurred during the bacterial cell recognition system. For this purpose, we performed molecular docking using computational biology techniques on several SARS-CoV-2 proteins that are responsible for its pathogenicity against N-acetyl-D-glucosamine. A similar molecular dynamics analysis has been carried out on both SARS-CoV-2 and anti-Staphylococcus aureus neutralizing antibodies to establish the potential of N-acetyl-D-glucosamine which likely induces the immune response against the virus. The results of molecular dynamic analysis have confirmed that SARS-CoV-2 spike receptor-binding domain (PDB: 6M0J), RNA-binding domain of nucleocapsid phosphoprotein (PDB: 6WKP), refusion SARS-CoV-2 S ectodomain trimer (PDB: 6X79), and main protease 3clpro at room temperature (PDB: 7JVZ) could bind with N-acetyl-D-glucosamine that these proteins play an important role in SARS-CoV-2's infection and evade the immune system. Moreover, our molecular docking analysis has supported a strong protein-ligand interaction of N-acetyl-D-glucosamine with these selected proteins. Furthermore, computational analysis against the D614G mutant of the virus has shown that N-acetyl-D-glucosamine affinity and its binding potential were not affected by the mutations occurring in the virus' receptor binding domain. The analysis on the affinity of N-acetyl-D-glucosamine towards human antibodies has shown that it could potentially bind to both SARS-CoV-2 proteins and antibodies based on our predictive modelling work. Our results confirmed that N-acetyl-D-glucosamine holds the potential to inhibit several SARS-CoV-2 proteins as well as induce an immune response against the virus in the host.

Dataset Information

Molecular characterization of a novel chitinase CmChi1 from Chitinolyticbacter meiyuanensis SYBC-H1 and its use in N-acetyl-d-glucosamine production.

Publications

Molecular characterization of a novel chitinase <i>Cm</i>Chi1 from <i>Chitinolyticbacter meiyuanensis</i> SYBC-H1 and its use in <i>N</i>-acetyl-d-glucosamine production.

Similar Datasets

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