RENATURATION, PURIFICATION, AND CHARACTERIZATION OF RECOMBINANT D-2-HYDROXYISOCAPROATE DEHYDROGENASE FROM ESCHERICHIA-COLI

被引：1

作者：

AGRAZ, A

PAULSEN, J

BORNER, B

HUSTEDT, H

机构：

[1] GESELL BIOTECHNOL FORSCH MBH,D-38124 BRAUNSCHWEIG,GERMANY

[2] CTR INGN GENET & BIOTECNOL,HAVANA,CUBA

来源：

ENZYME AND MICROBIAL TECHNOLOGY | 1995年 / 17卷 / 06期

关键词：

RECOMBINANT PROTEIN; D-2-HYDROXYISOCAPROATE DEHYDROGENASE; INCLUSION BODIES; RENATURATION;

D O I：

10.1016/0141-0229(94)00097-B

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Conditions for renaturation and purification of recombinant D-2-hydroxyisocaproate dehydrogenase (r-DHicDH) produced in E. coli in the form of inclusion bodies were investigated. Urea (8 M) was found to extract the enzyme from the pellet fraction efficiently. Renaturation by dialysis against 20 mM sodium phosphate buffer, pH 7.0, a protein concentration of 2 mg ml(-1), and a temperature of 4-8 degrees C was found to be optimal and even at a protein concentration of 10 mg ml(-1) 85% of enzyme activity could be recovered. Protein was further purified by ion-exchange chromatography on a Mono Q column. An enzyme preparation of greater than 95% purity was obtained with an overall yield of about 50%. r-DHicDH shows identical properties within experimental error with respect to molecular weight, temperature dependence of enzyme activity, pH optimum of the enzyme reaction, and K-M values for 2-ketoisocaproate and NADH as the native enzyme from Lactobacillus casei.

引用

页码：558 / 563

页数：6

共 20 条

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