Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity

doi:10.1007/s10295-011-0979-7

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	Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity
	Xue, Zhiquan 1,2; Chao, Yapeng 1; Wang, Dexian 3; Wang, Meixiang 3,4; Qian, Shijun 1
刊名	JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
	2011-12-01
卷号	38 期号:12 页码:1931-1938
关键词	Amidase Rhodococcus Erythropolis Aj270 Stereoselectivity Ionic Liquid Complex Auto-inducing Medium
ISSN号	1367-5435
DOI	10.1007/s10295-011-0979-7
英文摘要	Rhodococcus erythropolis AJ270 metabolizes a wide range of nitriles via the two-step nitrile hydratase/amidase pathway. In this study, an amidase gene from R. erythropolis AJ270 was cloned and expressed in Escherichia coli BL21 (DE3). The activity reached the highest level of 22.04 U/ml in a complex auto-inducing medium using a simplified process of fermentation operation. The recombinant amidase was purified to more than 95% from the crude lysate using Ni-NTA affinity chromatography and Superose S10-300 gel filtration. The V-max and K-m values of the purified enzyme with acetamide (50 mM) were 6.89 mu mol/min/mg protein and 4.12 mM, respectively, which are similar to those of the enzyme from the wild-type cell. The enzyme converted racemic alpha-substituted amides, O-benzylated beta-hydroxy amides, and N-benzylated beta-amino amides to the corresponding (S)-acids with remarkably high enantioselectivity. The ionic liquid [BMIm][PF6] (1-butyl-3-methylimidazolium hexafluorophosphate) enhanced the activity by 1.5-fold compared with water. The adequate expression of the enzyme and excellent enantioselectivity of the recombinant amidase to a broad spectrum of amides suggest that the enzyme has prospective industrial-scale practical applications in pharmaceutical chemistry.
语种	英语
出版者	SPRINGER HEIDELBERG
WOS记录号	WOS:000300159300005
内容类型	期刊论文
源URL	[http://ir.iccas.ac.cn/handle/121111/71857]
专题	中国科学院化学研究所
通讯作者	Chao, Yapeng
作者单位	1.Chinese Acad Sci, Inst Microbiol, State Key Labs Transducer Technol, Beijing 100101, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China 3.Chinese Acad Sci, Inst Chem, Beijing Natl Lab Mol Sci, CAS Key Lab Mol Recognit & Funct, Beijing 100190, Peoples R China 4.Tsinghua Univ, Dept Chem, Minist Educ, Key Lab Bioorgan Phosphorus Chem & Chem Biol, Beijing 100084, Peoples R China
推荐引用方式 GB/T 7714	Xue, Zhiquan,Chao, Yapeng,Wang, Dexian,et al. Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity[J]. JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY,2011,38(12):1931-1938.
APA	Xue, Zhiquan,Chao, Yapeng,Wang, Dexian,Wang, Meixiang,&Qian, Shijun.(2011).Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity.JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY,38(12),1931-1938.
MLA	Xue, Zhiquan,et al."Overexpression of a recombinant amidase in a complex auto-inducing culture: purification, biochemical characterization, and regio- and stereoselectivity".JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY 38.12(2011):1931-1938.