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A novel bacterial ?-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities.

ABSTRACT: 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.

SUBMITTER: Zhang A

PROVIDER: S-EPMC7324980 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A novel bacterial β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities.

Zhang Alei A Mo Xiaofang X Zhou Ning N Wang Yingying Y Wei Guoguang G Chen Jie J Chen Kequan K Ouyang Pingkai P

Biotechnology for biofuels 20200629

<h4>Background</h4>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 ce ...[more]

PMID: 32612678

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Project description:BackgroundXylan, the major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-L-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes.ResultsHere, we cloned an acetyl xylan esterase (PcAxe) from Penicillium chrysogenum P33 and expressed it in Pichia pastoris GS115. The optimal pH and temperature of the recombinant PcAxe (rPcAxe) for 4-nitrophenyl acetate were 7.0 and 40 °C, respectively. rPcAxe is stable across a broad pH range, retaining 100% enzyme activity om pH 6-9 after a 1 h incubation. The enzyme tolerates the presence of a wide range of metal ions. Sequence alignment revealed a GH62 domain exhibiting α-L-arabinofuranosidase activity with pH and temperature optima of pH 7.0 and 50 °C, in addition to the expected esterase domain. rPcAxe displayed significant synergy with a recombinant xylanase, with a degree of synergy of 1.35 for the hydrolysis of delignified corn stover. Release of glucose was increased by 51% from delignified corn stover when 2 mg of a commercial cellulase was replaced by an equivalent amount of rPcAxe, indicating superior hydrolytic efficiency.ConclusionsThe novel bifunctional enzyme PcAxe was identified in P. chrysogenum P33. rPcAxe includes a carbohydrate esterase domain and a glycosyl hydrolase family 62 domain. This is the first detailed report on a novel bifunctional enzyme possessing acetyl xylan esterase and α-L-arabinofuranosidase activities. These findings expand our current knowledge of glycoside hydrolases and pave the way for the discovery of similar novel enzymes.

Project description:BackgroundN-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.ResultsA 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/μmol toward CC and 4-nitrophenol N,N'-diacetyl-β-d-chitobioside [p-NP-(GlcNAc)2], respectively. Analysis of the hydrolysis products revealed that CmChi1 exhibits exo-acting, endo-acting and N-acetyl-β-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%.ConclusionThe 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-β-d-glucosaminidase activities from Chitinolyticbacter species.

Dataset Information

A novel bacterial ?-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation and reverse hydrolysis activities.

Publications

A novel bacterial β-<i>N</i>-acetyl glucosaminidase from <i>Chitinolyticbacter meiyuanensis</i> possessing transglycosylation and reverse hydrolysis activities.

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