A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine.
A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.
Kao
Chao-Hung
CH
Department of Biotechnology, Hungkuang University, Taichung 433, Taiwan.
Lo
Hsueh-Hsia
HH
Hsu
Shih-Kuang
SK
Hsu
Wen-Hwei
WH
D000581:Amidohydrolases / Q000235:genetics* / Q000378:metabolism*; D000613:Aminobutyrates / Q000378:metabolism*; D001407:Bacillus / Q000235:genetics; D001709:Biotechnology / Q000379:methods; D002021:Buffers; D002384:Catalysis; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D006827:Hydantoins / Q000378:metabolism; D008345:Manganese; D015202:Protein Engineering / Q000379:methods*; D013237:Stereoisomerism; D014169:Transformation, Bacterial
D000613:Aminobutyrates; D002021:Buffers; D029968:Escherichia coli Proteins; D006827:Hydantoins; D008345:Manganese; C014328:2-amino-4-phenylbutyric acid; D000581:Amidohydrolases; C104561:N-carbamoyl-L-amino-acid hydrolase; C020047:dihydropyrimidinase