Expression Of The Hydantoinase Genes And Screening Of Hydantoinase-producing Strains

Hydantoinases which consist of hydantoin hydrolase, N-carbamoylase and sometimes hydantoin racemase can hydrolyze D,L-5-substituted hydantoin into corresponding optically active amino acids. Therefore, the hydantoinases from micro-organisms are widely applied to the industrial production of natural and unnatural amino acids. Microbial hydantoinases are considered as industrial enzymes from the origin of life.pUC18-169 was a subcloned plasmid containing the whole operon sequence of L-hydantoinase from Arthrobacter BT801 which can convert 5-benzylhydantoin to L-phenylalanine. Hydantoin hydrolase which is responsible for the ring opening of hydantoin is one of the components of hydantoin utility enzymes of Arthrobacter BT801. N-carbamoylase is a part of hydantoinase operon which can transform N-carbamoylamino acids into the corresponding amino acids. The L-N-carbamoylase of Arthrobacter BT801, coded by the hyuC gene, is the rate-limiting and the only stereoselective enzyme. Hydantoin hydrolase gene (hyuH) and N-carbamoylase gene (hyu C) were amplified from the plasmid of pUC18-169 by PCR and, at the same time, activity expression was obtained in E.coli and Pichia pastor is GS115 respectively.To improve the production and activity of the enzyme, the hydantoin hydrolase gene (hyuH) was cloned into E.coli M15 and BL21(DE3) by using vector pQE60 and pT221 respectively. Two expression plasmids were thus constructed. In the pQE60-hyuH, the hyuH is singly controlled by the T5 promotor, while in pT221-hyuH the hyuH is co-expressed with a molecular chaperon. The expression products of recombinant plasmid were further analysis in solubility and activity. A protein band about 50kD was detected by SDS-PAGE in the recombinant cell lysate. Though the expression level of BL21(DE3)/pT221-hyuH was lower than that of M15/pQE60-hyuH, the target protein of BL21(DE3)/pT221-hyuH was principally in soluble form while the objective protein of M15/pQE60-hyuH was principally in insoluble form. Both of the products in the twostrains showed biological activity, but the former is 2 times higher than the latter.The hyuC DNA fragment was inserted into pPIC3.5K plasmid to construct the pPIC3.5K-hyuC recombinant plasmid which was then transduced into Pichia pastoris GS115 cells after being linearized by Bgl II digestion. Multi-copies insertion transformants were screened on G418 plates. The recombinant protein was proved to have biological activity of hydrolyzing N-carbamoylphenylalanine into phenylalanine through enzyme activity assay. The N-carbamoylase activity of recombinant was 2.26 and 2.15 times higher than that of Arthrobacter BT801 and DH5a/pUC18-169.A screening method that allows available, efficient and reliable selection of hydantoinase-positive micro-organisms was devised. The soil samples were collected from different region, which were enriched by using 5-phenylhydantoin as the unique nitrogen. The first detection was through screening plates, the 23 candidate strains were further selected by detection of the medial product and final product. One strain possessing hydantoinase activity were isolated and named BT811. It was proved that the BT811 was a new D-hydantoinase-producing strain by gene separation and sequence analysis.