维氏气单胞菌B565β-N-乙酰氨基葡萄糖苷酶的表达、纯化及酶学性质

Expression,Purification and Characterization of β-N-acetylglucosaminidase from Aeromonas veronii B565

以维氏气单胞菌B565基因组DNA为模板,扩增得到β-N-乙酰氨基葡萄糖苷酶nag565基因,构建nag565-p ET28a表达载体,在大肠杆菌BL21（DE3）中进行诱导表达。经过分析与预测,nag565氨基酸序列与其他气单胞菌属相似,NAG565蛋白序列分析预测其含有一个信号肽、一个碳水化合物结合结构域、两个糖苷水解酶20家族催化结构域和一个β-氨基葡萄糖苷酶C端结构域。通过NiNTA纯化获得的NAG565纯蛋白比活为7328U/mg,并对其酶学性质进行了初步研究,NAG565在p H7.0保持最高活性,并且在p H5.0-9.0范围内稳定,最适温度为37℃,0-30℃对其酶活无明显影响,并对多种金属离子和蛋白酶具有良好抗性,符合水产动物的养殖环境条件（养殖温度20-30℃,多数养殖鱼胃肠道p H值呈中性并含有蛋白酶）,为其在水产养殖等领域的应用提供了理论基础。

β-N-acetylglucosaminidase （chitobiase） gene nag565 from Aeromonas veronii B565 was cloned and expressed in E. coli. The deduced amino acid sequence of nag565 is most similar to those of other Aeromonas sp. The deduced sequence of NAG565 contained one putative signal peptide, one carbohydrate binding domain, two catalytic domain of glycosyl hydrolase family 20, and one chitobiase/beta-hexosaminidase C-terminal domain. Purified mature NAG565 has a specific activity of 7328 U/mg. NAG565 had an optimal activity at pH 7.0 and 37℃, showed good resistance to various metal ions and digestion by proteases. NAG565 retained almost all its optimal activity under aquaculture conditions （20 -30℃ and pH 7.0）. Taken together, the A. veronii B565 NAG565 may adapt to the environment of cultured fish gastrointestinal tract and may be potential aquacuhural feed additive, facilitating the effective utilization of chitin.