一种来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰氨基葡萄糖苷酶

N-乙酰氨基葡萄糖苷酶作用于肽聚糖或几丁质,从其非还原末端水解产生β-D-N-乙酰氨基葡萄糖单体,该酶在细胞壁代谢过程中起重要作用,在医药和生物技术领域也有广泛的应用。【目的】克隆表达来源于兼性嗜碱菌Bacillus pseudofirmus 703的β-N-乙酰葡糖胺糖苷酶NagZ703,为获得乙酰氨基葡萄糖单体奠定基础。【方法】以B.pseudofirmus703基因组DNA为模板,克隆得到了β-N-乙酰氨基葡萄糖苷酶基因NagZ703,通过构建pET28a-nagZ703表达载体,在大肠杆菌BL21(DE3)中诱导表达NagZ703,利用镍柱纯化得到NagZ703纯蛋白,并对其酶学和生化性质进行分析。【结果】NagZ703与其同源蛋白多序列比对分析结果表明,NagZ703属于糖苷水解酶3家族(GH3),由2个结构域构成,催化活性中心由位于N端结构域的Arg232-His234-Arg318组成,和研究最多的Bacillussubtilis168来源的BsNagZ氨基酸的序列相似性为37%。酶学性质分析表明,以对硝基酚-β-乙酰氨基葡萄糖苷(pNP-β-GlcNAc)为底物,NagZ703的最适反应温度和pH分别为60°C和pH 6.5,比酶活为10.79 U/mg,其Km和Vmax分别为0.276 mmol/L和0.612 mmol/(mg·min)。该酶具有较好的稳定性,在50°C处理30 min,或在pH 6.0–10.5条件下,4°C保存12 h后,仍保留80%以上的酶活力。EDTA不影响该酶的活性,推测其为非金属依赖酶,且Hg2+可完全抑制酶活性。【结论】本研究将兼性嗜碱菌Bacillus pseudofirmus 703来源的β-N-乙酰葡糖胺糖苷酶NagZ703在大肠杆菌中成功表达和纯化,并分析了其酶学性质;NagZ703的最适pH为6.5,没有表现出耐盐嗜碱的特征;NagZ703能水解胶体几丁质产生GlcNAc,为酶解生产GlcNAc提供了一条可行的思路。

A new N-acetylglucosaminidase from facultative alkaliphilic Bacillus pseudofirmus 703

β-N-acetylglucosaminidases(NAGases)participate in the removal of N-acetylglucosamine(GlcNAc)from the non-reducing end of peptidoglycan or chitin,and are important for many biological functions and industrial applications.Objective]A new NAGase encoding gene Nag Z703 was cloned from a facultative alkaliphilic Bacillus pseudofirmus and expressed in Escherichia coli,for the enzymatic production of GlcNAc.Methods]The gene Nag Z703 was cloned into the expression vector pET28 a to get the recombinant plasmid pET28 a-NagZ703.The recombinant NagZ703 was induced for expression in E.coli BL21(DE3)and purified through His-Trap column.Then the purified NagZ703 was characterized.Results]The multiple sequence alignments showed that NagZ703 belonged to GH 3 NAGase with 2 domains,and the catalytic active sites were composed of 3 conserved residues(Arg232,His234 and Arg318)at the N-terminal domain.NagZ703 shared only 37%identity with the most studied BsNagZ from B.subtilis.The purified NagZ703 exhibited optimal activity at 60°C and pH 6.5,the specific activity was 10.79 U/mg,and the Km and Vmax of NagZ703 were 0.276 mmol/L and 0.612 mmol/(mg·min)toward p-nitrophenylβ-N-acetylglucosaminide,respectively.NagZ703 retained more than 80%residual activity after pre-incubation at 50°C for 30 min,or at pH 6.0–10.5 for 12 h.NagZ703 was a non-metalloenzyme because EDTA could not affect its activity,whereas Hg2+completely inhibited its activity.NagZ703 hydrolyzed colloidal chitin to produce GlcNAc in vitro.Conclusion]A NAGaseNagZ703 from facultative alkaliphilic Bacillus pseudofirmus was characterized carefully.The ability of NagZ703 to produce GlcNAc from colloidal chitin provided a promising approach for the production of GlcNAc by enzymatic hydrolysis.