食品科学 ›› 2020, Vol. 41 ›› Issue (8): 123-129.doi: 10.7506/spkx1002-6630-20190221-132

• 生物工程 • 上一篇    下一篇

解淀粉芽孢杆菌β-N-乙酰氨基葡萄糖苷酶基因的克隆表达及其酶学表征

陈宝莉,秦臻,赵黎明   

  1. (华东理工大学生物工程学院,发酵工业分离提取技术研发中心,生物反应器工程国家重点实验室,上海 200237)
  • 出版日期:2020-04-25 发布日期:2020-04-20
  • 基金资助:
    上海市教育委员会和上海市教育发展基金会“曙光计划”项目(15SG28);高等学校学科创新引智计划项目(B18022)

Gene Cloning, Expression and Characterization of β-N-Acetylglucosaminidase from Bacillus amyloliquefaciens

CHEN Baoli, QIN Zhen, ZHAO Liming   

  1. (State Key Laboratory of Bioreactor Engineering, R&D Center of Separation and Extraction Technology in Fermentation Industry, School of Biotechnology, East China University of Science and Technology, Shanghai 200237, China)
  • Online:2020-04-25 Published:2020-04-20

摘要: 从解淀粉芽孢杆菌(Bacillus amyloliquefaciens YX-01)中克隆得到一个新型β-N-乙酰氨基葡萄糖苷酶基因(BaNagase),并成功在大肠杆菌中异源表达。BaNagase编码616 个氨基酸,与B. subtilis来源的β-N-乙酰氨基葡萄糖苷酶(AIY91451.1)氨基酸序列相似性最高,为76.97%。通过对比分析,BaNagase属于糖苷水解酶3家族。纯化获得的BaNagase比活力为16.42 U/mg,最适温度65 ℃,最适pH 6.0,且在低于55 ℃条件下能保持高于70%的酶活力。BaNagase展现出高的热稳定性和严格的底物特异性,为其高效制备N-乙酰氨基葡萄糖提供了理论支持。

关键词: β-N-乙酰氨基葡萄糖苷酶, 解淀粉芽孢杆菌, 克隆, 酶学性质, 糖苷水解酶3家族

Abstract: In this study, a novel β-N-acetylglucosaminidase gene was cloned from Bacillus amyloliquefaciens YX-01 and successfully expressed in Escherichia coli BL21. The recombinant protein (defined as BaNagase) was purified by Ni-IDA affinity chromatography and analyzed. The results showed that it encoded 616 amino acids, with the highest similarity (76.97%) to the sequence of β-N-acetylglucosidase (AIY91451.1) from B. subtilis. According to the phylogenetic analysis and multiple sequence alignments, BaNagase belonged to glycoside hydrolase (GH) family 3. The recombinant BaNagase showed specific activity of 16.42 U/mg toward the substrate pNP-GlcNAc. Its optimal pH value was 6.0 and the optimal reaction temperature was 65 ℃. BaNagase showed good stability below 55 ℃ at which more than 70% of initial activity was still retained. BaNagase exhibited strong thermostability and strict substrate specificity. This study provides a theoretical basis for efficient preparation of N-acetylglucosamine.

Key words: β-N-acetylglucosaminidase, Bacillus amyloliquefaciens, cloning, characterization, glycoside hydrolase family 3

中图分类号: