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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2014, Vol. 34 Issue (2): 52-58    DOI: 10.13523/j.cb.20140209
研究报告     
Thermus thermophilus HB27锰过氧化氢酶在大肠杆菌中的表达及酶学性质分析
何晓娟1,2, 薛正莲1,2, 赵志军1,2, 孙俊松1,2, 史吉平1,2
1. 安徽工程大学生物与化学工程学院 芜湖 241000;
2. 中国科学院上海高等研究院 上海 201210
Cloning of an Mn-catalase Gene from Thermus thermophilus and Its Expression in Escherichia coli
HE Xiao-juan1,2, XUE Zheng-lian1,2, ZHAO Zhi-jun1,2, SUN Jun-song1,2, SHI Ji-ping1,2
1. Biological and Chemical Engineering Department, Anhui Polytechnic University, Wuhu, 241000, China;
2. Shanghai Advanced Research Institute of Chinese Academy of Sciences, Shanghai 201210, China
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摘要： 碱性嗜热过氧化氢酶是一种重要的纺织用酶。根据大肠杆菌密码子使用偏爱性，对Thermus thermophilus HB27来源的含锰过氧化氢酶基因进行密码子优化，将优化后的基因连接至表达载体pET28a（+）上，转化到E. coli BL21（DE3）中进行诱导表达。结果表明在含有14mmol/L Mn2+浓度的培养液中以0.2 mmol/L的IPTG 42℃条件下诱导2 h的情况下，菌体破碎上清液中的酶活力可达25 U/ml。利用Ni 亲和层析柱对该Mn-CAT进行纯化，酶学性质研究表明：此酶的最适温度为70℃，最适pH为pH 10.0，在80℃保温2 h，酶活力不损失；pH9.0~11.0的环境中放置2 h后，酶活仅损失约10%，此酶具有良好的工业开发潜力。
关键词： 锰过氧化氢酶;  嗜热;  嗜碱;  表达;  酶学性质    
Abstract: Thermophilic alkaline catalase is an important textile enzyme. According to the codon bias of Escherichia coli, the gene encoding manganese catalase of Thermus thermophilus HB27 were optimized. The modified gene was inserted into the expression vector pET28a(+) and transformed into Escherichia coli BL21 (DE3). After cultured with 14 mmol/L Mn2+ and induced with 0.2 mmol/L IPTG for 2 hours at 42℃, the activity of catalase reached 25 U/ml in supernatant of cell disruption. The enzyme was purified by Ni affinity chromatography and its enzyme characterization was analyzed. The optimal temperature and pH of the catalase was 70℃ and pH10.0, respectively. when the enzyme was incubated at 80℃ for 2 hours, the catalase activity was not lost. After incubated at pH9.0~11.0 for 2 hours, its activity retained 90%. The results showed that the manganese catalase has a good potential for industrial development.
Key words: Mn-catalase    Thermophilic    Alkalophilic    Expression    Enzymatic property
收稿日期: 2013-09-25 出版日期: 2014-02-25
ZTFLH:  Q784  
通讯作者: 薛正莲     E-mail: xuezhen 0851@sina.com
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何晓娟
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史吉平

引用本文:

何晓娟, 薛正莲, 赵志军, 孙俊松, 史吉平. Thermus thermophilus HB27锰过氧化氢酶在大肠杆菌中的表达及酶学性质分析[J]. 中国生物工程杂志, 2014, 34(2): 52-58.

HE Xiao-juan, XUE Zheng-lian, ZHAO Zhi-jun, SUN Jun-song, SHI Ji-ping. Cloning of an Mn-catalase Gene from Thermus thermophilus and Its Expression in Escherichia coli. China Biotechnology, 2014, 34(2): 52-58.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140209    或    https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I2/52

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