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Distinctive molecular and biochemical characteristics of a glycoside hydrolase family 20 β-N-acetylglucosaminidase and salt tolerance
- Source :
- BMC Biotechnology, BMC Biotechnology, Vol 17, Iss 1, Pp 1-15 (2017)
- Publication Year :
- 2017
- Publisher :
- Springer Science and Business Media LLC, 2017.
-
Abstract
- Background Enzymatic 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. Results A 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. Conclusions Biochemical 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 material The online version of this article (doi:10.1186/s12896-017-0358-1) contains supplementary material, which is available to authorized users.
- Subjects :
- 0301 basic medicine
Glycoside Hydrolases
Protein Conformation
lcsh:Biotechnology
Microbacterium
Lysine
Chitobiose
Acetylglucosamine
Substrate Specificity
Microbiology
03 medical and health sciences
chemistry.chemical_compound
Chitin
lcsh:TP248.13-248.65
Acetylglucosaminidase
Enzyme Stability
Aspartic acid
Glycoside hydrolase
chemistry.chemical_classification
Binding Sites
biology
Salt Tolerance
biology.organism_classification
Enzyme assay
Activity
Actinobacteria
Enzyme Activation
N-Acetylglucosaminide tolerance
030104 developmental biology
Enzyme
Biochemistry
chemistry
biology.protein
Salts
β-N-Acetylglucosaminidase
Protein Binding
Research Article
Biotechnology
Subjects
Details
- ISSN :
- 14726750
- Volume :
- 17
- Database :
- OpenAIRE
- Journal :
- BMC Biotechnology
- Accession number :
- edsair.doi.dedup.....d5ae62460a327cb8e37ad07d78419cf9