Abstract
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 Vmax of 3097 ± 124 μmol min−1 mg−1 towards p-nitrophenyl β-N-acetylglucosaminide and a Ki 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.
Highlights
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 Nacetylglucosamine in the bioethanol, food and pharmaceutical industries
We found that all the GlcNAcases from Microbacterium strains were annotated based on in silico analysis without biochemical characterization
A Glycoside hydrolase (GH) 20 GlcNAcase was isolated from Microbacterium sp
Summary
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 Nacetylglucosamine in the bioethanol, food and pharmaceutical industries. N-acetylglucosaminidases (GlcNAcases, EC 3.2.1.52) are glycoside hydrolases (GHs) that cut the O-glycosidic bonds formed by N-acetylglucosamine (GlcNAc) residues from the non-reducing terminals of oligosaccharides, such as chitooligosaccharides and muropeptides [1, 2]. GlcNAcases hydrolyse chitooligosaccharides to produce GlcNAc, which can be further used for the production of bioethanol [4] and single cell protein [5], the treatment of ulcerative colitis and other gastrointestinal inflammation disorders [6] and pharmaceutical therapy for osteoarthritis [7] and tumours [8]. The hydrolysis of muropeptides by GlcNAcases indicates that the enzymes function in cell wall recycling [1] and flagellum assembly of bacteria [9]. GlcNAcases exhibit many additional important biological functions and wide range of industrial applications, such as catabolism of ganglioside storage in Tay-Sachs disease [10], induction of chitinolytic enzymes [11] and synthesis of biologically important oligosaccharides [12, 13]
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