Cloning of D-hydantoinase Gene from Pseudomonas and Its Expression in E.coli
- VernacularTitle:假单胞菌海因酶基因的克隆及其在大肠杆菌中的表达
- Author:
Zhiqiang LI
;
Jingjing LIU
;
Zhuoyi HU
;
Zhenghua WANG
;
Xin MING
- Publication Type:Journal Article
- From:
Journal of China Pharmaceutical University
2001;(3):227-230
- CountryChina
- Language:Chinese
-
Abstract:
AIM The purpose is to construct D-hydantoinase genetic engineering strain for the purpose of the industrial production of D-p-hydroxyphenylglycine. METHODS D-hydantoinase gene was created from Pseudomonas putida 9801 by PCR technique and inserted into pMD18-T vector. The recombinant plasmid was transformed into several Escherichia coli strains. The positive transformants with D-hydantoinase activity were obtained by the two step screening, digoxigenin DNA labeling in situ hybridization and D-hydantoinase activity assay. RESULTS The D-hydantoinase activity of the genetic engineering strain E.coli BL21/pMD-dht was 1700 U*L-1 and increased as high as 8 times compared with those of wild-type strain Pseudomonas putida 9801. The subunit molecular weight of recombinant D-hydantoinase was about 53 kDa measured by SDS-PAGE. The amount of the recombinant D-hydantoinase was about 20 percent of total bacterial soluble proteins. CONCLUSION The genetic engineering strain E.coli BL21/pMD-dht possesses the initial industrial production prospects.
