嗜碱芽孢杆菌cotA基因克隆表达及部分性质研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.08.024

摘要/Abstract

摘要： 为了获得具有高性能的多铜离子氧化酶,从极端微生物嗜碱芽孢杆菌（Bacillus clausii KSM-K16）中克隆表达了其芽孢外衣蛋白CotA。根据B. clausii KSM-K16的CotA序列及大肠杆菌密码子的偏爱性,设计和合成出该基因全长序列,构建pET28a-cotA重组表达载体,将其转化到Escherichia coli BL21（DE3）并诱导表达出重组蛋白,纯化并研究了CotA部分生化特性。重组CotA约62 kD,表现出蓝绿色并在波长609 nm有铜离子特征吸收峰,能氧化ABTS、SGZ和胆红素等底物;以SGZ为底物,最适pH和温度分别为7.5和90℃;在80℃孵育2 h,能保持70%活性;在pH4-11范围分别孵育1 h,能保持90%活性。结果显示,嗜碱芽孢杆菌CotA是一种具有漆酶和胆红素氧化酶活性的典型多铜离子氧化酶,表现出热和酸碱稳定性。

关键词: 嗜碱芽孢杆菌, 芽孢外衣蛋白, 漆酶, 多铜离子氧化酶

Abstract: In order to obtain a multi-copper oxidase with unusual features,a cotA gene from alkalophilic Bacillus clausii KSM-K16 was cloned and expressed in Escherichia coli. According to the CotA sequence from B. clausii KSM-K16 and the codon preference in E. coli,the gene of full-length sequence was designed,synthesized,and cloned into the expression vector of pET28a. Then the pET28a-cotAwas transformed to E. coli BL21（DE3）that was induced to express recombinant protein,which was subsequently purified and biochemically characterized. The purified recombinant CotA was about 62 kD,showed the blue green and had the characteristic absorption peak of Cu²⁺ at 609 nm. Also the recombinant CotA oxidized the substrates such as ABTS,SGZ and bilirubin. When SGZ as substrate,the optimal temperature and pH was 90℃ and 7.5,respectively. CotA maintained more than 70% of its original activity after incubating at 80℃ for 2 h. Furthermore,it was quite stable over pH ranging 4.0-11.0,more than 90% of its original activity after incubating at 37℃ for 1 h still remained. The results showed that the CotA from alkalophilic B. clausii was a typical multi-copper oxidase with the activities of laccase and bilirubin oxidase,and presented strong acidic-,alkali-,and thermo-stability.

Key words: Bacillus clausii, spore coat protein, laccase, multi-copper oxidase

刘友勋,闫明阳,耿园园,黄娟. 嗜碱芽孢杆菌cotA基因克隆表达及部分性质研究[J]. 生物技术通报, 2016, 32(8): 161-168.

LIU You-xun, YAN Ming-yang, GENG Yuan-yuan, HUANG Juan. Cloning,Expression,and Characterization of cotA from Alkalophilic Bacillus clausii[J]. Biotechnology Bulletin, 2016, 32(8): 161-168.

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