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| 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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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.
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Received: 11 October 2012
Published: 25 April 2013
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