A novel bacterial β-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 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 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. 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.

Characterization of a New Multifunctional GH20 β-N-Acetylglucosaminidase From Chitinibacter sp. GC72 and Its Application in Converting Chitin Into N-Acetyl Glucosamine

“…(57.5 kDa), V. harveyi 650 (73 kDa; Suginta et al, 2010 ), and Streptomyces thermoviolaceus (60 kDa; Kubota et al, 2004 ). However, the M w of NAGaseA is similar to the previously reported GH20 NAGase from C. meiyuanensis with a molecular mass of 92,571 Da ( Zhang et al, 2020b ). The specific activity of recombinant NAGaseA exhibited a 1.39-fold increase from 270.17 to 373.29 U/mg with a protein recovery of 78.6% yield after purification ( Supplementary Table 1 ).…”

“…F-3 (60°C; Sun et al, 2019 ), and Paraglaciecola hydrolytica S66 (50°C; Visnapuu et al, 2020 ). As for the thermostability profile ( Figure 3B ), the activity dropped rapidly after incubation at temperatures above 40°C, suggesting the poor thermostability of NAGaseA, which was similar to that of GH20 GlcNAGases from C. meiyuanensis ( Zhang et al, 2020b ), Microbacterium sp. HJ5 ( Zhou et al, 2017b ) and Aeromonas sp.…”

“…The temperature and pH profiles of recombinant NAGaseA were investigated in Figure 3 . As shown in Figure 3A , the recombinant NAGaseA displayed the optimal temperature at 40°C, which was consisted of NAGase from C. Meiyuanensis SYBC-H1 (40°C; Zhang et al, 2020b ), but different from NAGases from S. marcescens (52°C; Tews et al, 1996b ), Microbacterium sp. HJ5 (45°C; Zhou et al, 2017b ), Streptomyces sp.…”

“…The NAGaseA protein was eluted with buffer B (50 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, and pH 7.4) under a flow rate of 3 ml/min. The eluted protein was collected, concentrated, and exchanged with 20 mM phosphate buffer (pH 7.0) via ultrafiltration and stored at 4°C before use ( Zhang et al, 2020b ).…”

Property and Function of a Novel Chitinase Containing Dual Catalytic Domains Capable of Converting Chitin Into N-Acetyl-D-Glucosamine

“…GC72 (pH 6.8) (Gao et al, 2015), and Paenibacillus pasadenensis NCIM 5434 (pH 10.0) (Loni et al, 2014) was different from CmChi3. With regard to pH stability, CmChi3 retained more than 60% of its activity after storage at pH 5.0-8.5 for 2 h. This result is comparable to those of other chitinases from C. meiyuanensis, including CmChi1 (pH 5.2-8.2) (Zhang et al, 2018) and CmNAGase (pH 4.0-8.5) (Zhang et al, 2020a).…”