非对称二甲基精氨酸水解酶的表达纯化及其酶学性质的研究

中国生物工程杂志

2013, Vol. 33

Issue (4): 46-53

研究报告

非对称二甲基精氨酸水解酶的表达纯化及其酶学性质的研究

赵志敬¹, 王学忠², 沈文婷¹, 胡广¹

1. 北大工学院绍兴技术研究院生物工程中心绍兴 312000;
2. 绍兴科锐捷生物科技有限公司绍兴 312030

Expression, Purification and Characterization of Dimethylarginine Dimethylaminohydrolase

ZHAO Zhi-jing¹, WANG Xue-zhong², SHEN Wen-ting¹, HU Guang¹

1. Bioengineering Center, Shao Xing Institute of Technology, College of Engineering, Peking University, Shaoxing 312000, China;
2. Shao Xing Kreatiobio Technology Co. Ltd, Shaoxing 312030, China

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摘要： 非对称二甲基精氨酸水解酶(dimethylarginine Dimethylaminohydrolase,DDAH) 是酶学循环法检测早期急性心肌梗塞(acute myocardial infarction,AMI)的生物标记物——非对称二甲基精氨酸(asymmetric dimethylarginine, ADMA)的重要工具酶。为获得高活性DDAH,从绿脓杆菌的基因组中扩增目的基因,构建高效表达DDAH的重组菌E. coli BL 21(pET-28a-DDAH)。该菌的可溶性DDAH表达量达100mg/L,经超声破碎后的上清液通过镍柱纯化,可获得纯度达到95%以上的重组DDAH。Q柱的精制可提高它的稳定性,使其能够在常温下长期保持酶活。该重组酶的单位酶活,Km值和Vmax分别为0.5U/mg,3.04 mM和8.07mM/h。最适反应温度和pH分别为35℃和pH=6,在10℃~40℃和pH5.0~pH9.0的范围内稳定。该重组酶可形成多聚体,但并不影响酶活。最后,利用发酵罐扩大生产规模,有望进行克级生产,为新型ADMA检测试剂盒的开发和药物筛选模型提供材料基础。

关键词： DDAH; ADMA; 蛋白纯化; 酶学性质

Abstract: Dimethylarginine Dimethylaminohydrolase(DDAH) is one of the essential enzyme in enzymatic cycling detection of ADMA, a well recognized biomarker for early (acute myocardial infarction) AMI prognosis and diagnosis. To obtain DDAH with high enzymatic activity, the DDAH was cloned from the genome of Pseudomonas aeruginosa(Schroeter), and expressed in E. coli BL21 at high level of 100mg/L. Recombinant DDAH was purified to more than 95% purity from supernatant of disrupted cells with HisTrap Fast Flow and further polished by cation-exchange chromatography with Q column. The optimum reaction temperature, pH, specific activity, Km and Vmax of the purified DDAH was 35℃, 6.0, 0.5 U/mg, 3.04 mM and 8.07mM/h, respectively. No obvious activity losing was observed when the DDAH stored at termperature from 10℃ to 40℃ and pH from 5.0 to 9.0. Furthermore, more than 95% enzymatic activity was preserved after the purified DDAH was exposed to temperature of 37℃ for one week. Dimerization of the recombinant enzyme had no obvious effect on the enzymatic activity. The yield of recombinant DDAH could reach to 0.4 gram/L with high density fermentation, thus are well suitable for application in large-scale manufacturing of clinical test kit of ADMA and drug screening for DDAH.

Key words: DDAH ADMA Expression and purification Enzymatic characteristic

收稿日期: 2012-10-11 出版日期: 2013-04-25

ZTFLH:

Q786

基金资助: 浙江省绍兴市绍兴县科技项目资助项目(20111402)

通讯作者: 胡广 E-mail: hug@ie.pku.edu.cn

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引用本文:

赵志敬, 王学忠, 沈文婷, 胡广. 非对称二甲基精氨酸水解酶的表达纯化及其酶学性质的研究[J]. 中国生物工程杂志, 2013, 33(4): 46-53.

ZHAO Zhi-jing, WANG Xue-zhong, SHEN Wen-ting, HU Guang. Expression, Purification and Characterization of Dimethylarginine Dimethylaminohydrolase. China Biotechnology, 2013, 33(4): 46-53.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I4/46

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