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2025年7月31日 星期四
  研究论文 本期目录 | 过刊浏览 | 高级检索 |
环状芽孢杆菌乳糖酶基因在大肠杆菌中的表达及酶学性质分析
王元火1 姚 斌1** 袁铁铮1 操时树1 张 伟2 王亚茹1 范云六2
(1.中国农业科学院饲料研究所, 北京 100081;  2.中国农业科学院生物技术研究所, 北京 100081)
Expression of Bacillus circullans Lactase Gene in E. coli
and Properties of Expressed Lactase
Wang Yuanhuo1 Yao Bin1** Yuan Tiezheng1 Cao Shishu1 Zhang Wei2 Wang Yaru1 Fan Yunliu2
   (1. Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing  100081, China;  2 .Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing  100081, China)
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摘要 摘要: 将环状芽孢杆菌(Bacillus circulans )中的乳糖酶基因在大肠杆菌(Escherichia coli )中进行了高效表达, 并测定了表达的乳糖酶的基本酶学性质。结果表明, 表达的乳糖酶有生物学活性, 但与来源于环状芽孢杆菌的原始酶相比, 其酶促反应的最适pH、最适温度、Km值, 以及酶的pH稳定性和温度稳定性等酶学性质均有较大变化。表达的乳糖酶最适pH为5.0, 低于原始酶的pH 6.5; 最适温度为37 ℃, 而原始酶为55 ℃; 其耐酸性、耐热性及对金属离子的抗性等方面比原酶有所提高; 而且表达的乳糖酶Km比原始酶小285倍、Vmax比原始酶大5.4倍,表明经大肠杆菌表达的乳糖酶有较高的底物亲和力,酶促反应效率更高。从乳糖酶的单位表达量来看, 原始酶在环状芽孢杆菌的表达量为20.98 U/mL,而在大肠杆菌中的表达量提高到33.102  U/mL。
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王元火
姚斌
**袁铁铮
操时树
张伟
王亚茹
范云六
关键词 关键词:环状芽孢杆菌 乳糖酶 基因表达 酶学性质    
AbstractAbstract : The lactase gene from Bacillus circulans was overexpressed in Escherichia coli and the expressed lactase had normal bioactivity. The properties of purified lactase expressed in E. coli and produced by Bacillus circulans were determined. The results revealed, between the lactase expressed in E. coli and produced by Bacillus circulans, that there had notable difference in enzyme properties in optimal pH value and pH stability, optimal temperature and temperature stability, Km  value. The optimal pH value of the lactase expressed in E. coli was 5.0, whereas that of origin lactase produced by Bacillus circulans was 6.5. The optimal temperature of expressed lactase was 37 ℃ and 55 ℃, whereas that of origin lactase was 55 ℃. After treated for 2 h under pH 4.0, the remaining enzyme activity of expressed lactase was 85%, whereas that of origin lactase was only 5%. The thermal stability of expressed lactase was better than that of origin lactase, after treated for 10 minutes under 60 ℃, the remaining enzyme activity of expressed lactase was 30% , whereas origin lactase almost lost all of its activity. The expressed lactase had better metal ion resistance to Cu2+, Mn2+ and Fe2+, the remaining enzyme activity of expressed lactase and origin lactase in reaction system with 1mmol/L Fe2+ was 96% and 22%, respectively. The Km  and Vmax values of expressed lactase had 285-fold decrease and 5.4-fold increase, respectively, compared to those of origin lactase. The lactase was overexpressed in E. coli, the expression level of lactase was high up to 33.102 U/mL. The properties of the lactase expressed in E. coli including high activity in pH 3.5~8.5, good thermal stability and metal ion resistance demonstrate that the enzyme is a good candidate in dairy industry.
Key wordsKey words: Bacillus circulans    lactase    gene expression    enzyme properties
收稿日期: 1900-01-01     
引用本文:   
王元火1 姚 斌1** 袁铁铮1 操时树1 张 伟2 王亚茹1 范云六2. 环状芽孢杆菌乳糖酶基因在大肠杆菌中的表达及酶学性质分析[J]. , 2003, 11(1): 83-88.
Wang Yuanhuo1 Yao Bin1** Yuan Tiezheng1 Cao Shishu1 Zhang Wei2 Wang Yaru1 Fan Yunliu2. Expression of Bacillus circullans Lactase Gene in E. coli
and Properties of Expressed Lactase
. , 2003, 11(1): 83-88.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/      或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2003/V11/I1/83
 
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