A β-N-acetylhexosaminidase from Symbiobacterium thermophilum; gene cloning, overexpression, purification and characterization

Ogawa, Masahiro; Kitagawa, Masatoshi; Tanaka, Hideharu; Uéda, Kenji; Watsuji, Tomo‐o; Beppu, Teruhiko; Kondo, Akihiro; Kawachi, Ryu; Oku, Tatsuo; Nishio, Takeshi

doi:10.1016/j.enzmictec.2005.07.009

Cited by 21 publications

(21 citation statements)

References 19 publications

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“…However, CmNAGase showed good activities toward (GlcNAc) 2−6 with the highest activity for (GlcNAc) 2 , followed by (GlcNAc) 3 , (GlcNAc) 4 , (GlcNAc) 5 , and (GlcNAc) 6 . The results show that specific activity of CmNAGase toward N-acetyl COSs decreased when the degree of polymerization increased, which is similar with other reports [42]. The catalytic efficiency of GH20 LeHex20A from Lentinula edodes for (GlcNAc) 6 was greater than for (GlcNAc) 2 [21].…”

Section: Effect Of Metal Ions On Activity Of Cmnagasesupporting

confidence: 91%

A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

Zhang

Zhou

et al. 2020

Preprint

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Abstract Background: N-acetyl glucosamine (GlcNAc) and N-acetyl chitooligosaccharides (N-acetyl COSs) exhibit antitumor and antimicrobial activities, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries. Thus, it is crucial to discover a NAGase that can both synthesize GlcNAc and N-acetyl COSs. Results: The gene encoding the novel β-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 with the corresponding domain of the well-characterized NAGases. CmNAGase gene was highly expressed with soluble form in Escherichia coli BL21 (DE3) cells, whereupon it had a specific activity of 4,878.6 U/mg of protein toward p-nitrophenyl-N-acetyl glucosaminide. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, and Kcat of CmNAGase were 833.33 μmol·L-1 ·min-1, 10.9 mmol, and 6.37 ´ 108 mM·mg-1, respectively. Analysis of the hydrolysis products of N-acetyl chitooligosaccharides and colloidal chitin revealed that CmNAGase exhibits exo-acting activities. Particularly, it possesses transglycosylation activity, which can synthesize (GlcNAc)n+1 from (GlcNAc)n (n=1−6), respectively. In addition, CmNAGase also can catalyze GlcNAc to its dimers with various linked forms. Conclusions: The observations recorded in this study that CmNAGase is an exo NAGase with unique transglycosylation activity, suggests a possible application in the production of long-chain N-acetyl CHOs. This is first report of a bacterial NAGase, which can produce long-chain N-acetyl COSs via transglycosylation activity.

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Section: Effect Of Metal Ions On Activity Of Cmnagasesupporting

confidence: 91%

A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

Zhang

Zhou

et al. 2020

Preprint

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“…However, CmNAGase showed good activities toward (GlcNAc) 2 −(GlcNAc) 6 with the highest activity for (GlcNAc) 2 , followed by (GlcNAc) 3 , (GlcNAc) 4 , (GlcNAc) 5 , and (GlcNAc) 6 . These results show that speci c activity of CmNAGase toward Nacetyl COSs decreased when the degree of polymerization increased, which is similar with other reports [59]. The catalytic e ciency of GH20 LeHex20A from Lentinula edodes for (GlcNAc) 6 was greater than for (GlcNAc) 2 [60].…”

Section: The Substrate Speci City Of Cmnagasesupporting

confidence: 91%

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

Zhang

Zhou

et al. 2020

Preprint

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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 towards natural killer cells. Thus, it is of great significance to discover a β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-acetyl COSs. Results: The gene encoding the novel β-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 4,878.6 U/mg of protein. CmNAGase had a molecular mass of 92 kDa, and its optimum activity was at pH 5.4 and 40ºC. The Vmax, Km, Kcat, and Kcat/Km of CmNAGase for pNP-GlcNAc were 16,666.67 μmol min-1 mg-1, 0.50 μmol mL-1, 25,555.56 s-1, and 51,111.12 mL μmol-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, suggests a possible application in the production of GlcNAc or higher N-acetyl COSs.

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“…Furthermore, the molecular mass of HJ5Nag (55.9 kDa) is similar to that of some GlcNAcases, such as the GH 20 GlcNAcases from a soil-derived uncultured organism (53.4 kDa) [44], C. fimi (54.2 kDa) [45], Paenibacillus sp. (57.5 kDa) [25] and S. thermoviolaceus (58.3 kDa) [46], and the GH 3 GlcNAcase from S. thermophilum (55.9 kDa) [47]. …”

Section: Discussionmentioning

confidence: 99%

Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

et al. 2017

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BackgroundEnzymatic degradation of chitin has attracted substantial attention because chitin is an abundant renewable natural resource, second only to lignocellulose, and because of the promising applications of N-acetylglucosamine in the bioethanol, food and pharmaceutical industries. However, the low activity and poor tolerance to salts and N-acetylglucosamine of most reported β-N-acetylglucosaminidases limit their applications. Mining for novel enzymes from new microorganisms is one way to address this problem.ResultsA glycoside hydrolase family 20 (GH 20) β-N-acetylglucosaminidase (GlcNAcase) was identified from Microbacterium sp. HJ5 harboured in the saline soil of an abandoned salt mine and was expressed in Escherichia coli. The purified recombinant enzyme showed specific activities of 1773.1 ± 1.1 and 481.4 ± 2.3 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and N,N'-diacetyl chitobiose, respectively, a V max of 3097 ± 124 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and a K i of 14.59 mM for N-acetylglucosamine inhibition. Most metal ions and chemical reagents at final concentrations of 1.0 and 10.0 mM or 0.5 and 1.0% (v/v) had little or no effect (retaining 84.5 − 131.5% activity) on the enzyme activity. The enzyme can retain more than 53.6% activity and good stability in 3.0–20.0% (w/v) NaCl. Compared with most GlcNAcases, the activity of the enzyme is considerably higher and the tolerance to salts and N-acetylglucosamine is much better. Furthermore, the enzyme had higher proportions of aspartic acid, glutamic acid, alanine, glycine, random coils and negatively charged surfaces but lower proportions of cysteine, lysine, α-helices and positively charged surfaces than its homologs. These molecular characteristics were hypothesised as potential factors in the adaptation for salt tolerance and high activity of the GH 20 GlcNAcase.ConclusionsBiochemical characterization revealed that the GlcNAcase had novel salt–GlcNAc tolerance and high activity. These characteristics suggest that the enzyme has versatile potential in biotechnological applications, such as bioconversion of chitin waste and the processing of marine materials and saline foods. Molecular characterization provided an understanding of the molecular–function relationships for the salt tolerance and high activity of the GH 20 GlcNAcase.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0358-1) contains supplementary material, which is available to authorized users.

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A β-N-acetylhexosaminidase from Symbiobacterium thermophilum; gene cloning, overexpression, purification and characterization

Cited by 21 publications

References 19 publications

A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

A novel β-N-acetyl glucosaminidase from Chitinolyticbacter meiyuanensis possessing transglycosylation activity and its use in generating long-chain N-acetyl chitooligosaccharides

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

Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance

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