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

A new N-acetylglucosaminidase from facultative alkaliphilic Bacillus pseudofirmus 703

摘要:

β-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.pseudofirmus 703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Bacillus subtilis 168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℃和pH 6.5，比酶活为10.79 U/mg，其K_m和V_max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℃处理30 min，或在pH 6.0-10.5条件下，4℃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。

Abstract:

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 NagZ703 was cloned from a facultative alkaliphilic Bacillus pseudofirmus and expressed in Escherichia coli, for the enzymatic production of GlcNAc. [Methods] The gene NagZ703 was cloned into the expression vector pET28a to get the recombinant plasmid pET28a-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℃ and pH 6.5, the specific activity was 10.79 U/mg, and the K_m and V_max of NagZ703 were 0.276 mmol/L and 0.612 mmol/(mg·min) toward p-nitrophenyl β-N-acetylglucosaminide, respectively. NagZ703 retained more than 80% residual activity after pre-incubation at 50℃ 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.