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 °C and pH 8.0. This enzyme was completely thermostable at 50 °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-β-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 10 mM 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.