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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2015, Vol. 35 Issue (2): 38-44    DOI: 10.13523/j.cb.20150206
研究报告     
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
刘阳1, 杨雅麟2, 张宇婷2, 冉超2, 周志刚1,2
1. 华中农业大学水产学院 武汉 430070;
2. 中国农业科学院饲料研究所 北京 100081
Expression, Purification and Characterization of β-N-acetylglucosaminidase from Aeromonas veronii B565
LIU Yang1, YANG Ya-lin2, ZHANG Yu-ting2, RAN Chao2, ZHOU Zhi-gang1,2
1. Fisheries Colloge of Huazhong Agricultural University, Wuhan 430070, China;
2. Feed Research Institute of Chinese Academy of Agriculture Sciences, Beijing 100081, China
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摘要:

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 -pET28a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Ni-NTA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pH7.0保持最高活性,并且在pH5.0~9.0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pH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。

关键词: 维氏气单胞菌&beta-N-乙酰氨基葡萄糖苷酶表达酶学性质    
Abstract:

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 aquacultural feed additive, facilitating the effective utilization of chitin.

Key words: Aeromonas veronii    β-N-acetylglucosaminidase    Expression    Enzymatic properties
收稿日期: 2014-10-23 出版日期: 2015-02-25
ZTFLH:  Q789  
基金资助:

"十二五"国家科技计划农村领域项目(2013BAD10B01-2-3,2012BAD25BD203)、北京市鲟鱼、鲑鳟鱼产业技术体系项目(SCGWZJ20141104-4)、海洋公益性行业科研专项经费项目(201305022-6)资助项目

通讯作者: 周志刚     E-mail: zhou_zg@msn.com
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引用本文:

刘阳, 杨雅麟, 张宇婷, 冉超, 周志刚. 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[J]. 中国生物工程杂志, 2015, 35(2): 38-44.

LIU Yang, YANG Ya-lin, ZHANG Yu-ting, RAN Chao, ZHOU Zhi-gang. Expression, Purification and Characterization of β-N-acetylglucosaminidase from Aeromonas veronii B565. China Biotechnology, 2015, 35(2): 38-44.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150206        https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I2/38


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