Abstract
BackgroundN-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 β-N-acetyl glucosaminidase (NAGase) that can not only produce GlcNAc, but also synthesize N-Acetyl COSs.ResultsThe 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 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 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.ConclusionsThe 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.
Highlights
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
The protein strip in the stained gel was excised for peptide mass fingerprinting (PMF) analysis using matrixassisted laser desorption ionization-time-of-flight (MALDI-TOF MS/MS), and the results of PMF were interpreted by referencing the Mascot database [38]
Peptide fragments of purified protein were mainly detected with the amino acid sequences of YDGDTFLARLTLTNH, AMNVRYERLVKAGK, and WNQFANRLGQRELARLDGFLGGYGYRVPV, which showed 100% identity to the peptides from an annotated N-acetyl glucosaminidase (NAGase) in the complete genome of C. meiyuanensis SYBC-H1
Summary
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. Besides the direct utilization of chitin mentioned above, chitin can be used as a substrate for producing nitrogen-containing chemicals like GlcNAc, N-Acetyl chitooligosaccharides (N-Acetyl COSs), ethanolamine, N-containing furan derivatives and so on [2]. Among these chemicals, GlcNAc and N-Acetyl COSs are considered promising platform molecules, and have been widely used in the pharmaceutical, agriculture, food, and chemical industries [7, 8]. (GlcNAc) was found to have strong stimulating activity toward natural killer cells [9]. (GlcNAc) is an important building block for NOD factor synthesis [10]. (GlcNAc) and (GlcNAc) show antitumor activity against mice sarcoma 180 [11] and antimicrobial activity against fungal pathogens [12]
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