Expression Of Hydantoinase Genes And Screening Hydantoinase-positive Micro-organisms

This paper comprises two part research of L-hydantoinase and D-hydantoinase.A screening method that allows available, efficient, and reliable detection and selection of hydantoinase-positive micro-organisms was devised. The soil samples were collected from different region. Soil sample were enriched by using 5-p-hydroxyphenylhydantoin as the sole nitrogen. The first detection was through screening plate , the 49 doubtable strains were further screened by assay the production of medial product and end product. 1 strain possessing hydantoin-hydrolysing activity was isolated and named BT821. To synthesized thermotolerant D-hydantoin hydrolase genes by chemic method, we researched the enzyme characters. It's activity is more higher at 55°C than at 37°C.A novel expression vector of pOK12-Trx was constructed with T5 promoter of pQE60, and the thioredoxin gene(Trx) was functionally expressed with the vector. It can decrease to form inclusion bodies.BT811 is a D-hydantoinase producing strain which is able to convert 5-phenylhydantoin to D-phenylglycine. D-hydantoin hydrolase and D-carbamoylase genes generated by PCR and cloned into E. coli using vectors pT221 and pQE60. The two genes were highly expressed in E. coli DH5a and JM109 under the control of T7 and T5 promoter respectively and its expression products were analyzed. In the pT221-hyuH, the hyuH is co-expressed with a molecular chaperon in pT221, while in pQE60-hyuC the hyuC is singly expressed under control of the T5 promotor in pQE60. The expressed products, 53kD and 43kD respectively, were detected by SDS-PAGE in the recombinant cell lysate. The objective proteins are both principally in soluble form. Both the products in the two stains showed biological activity, and obtained obvious improvement.The hydantoin hydrolase activity of the recombinant DH5a/pT221-hyuH is 2.8 times higher than that of BT811.The carbamoylase activity of the recombinant JM109/pQE60-hyuC is 1.6 times higher than that of BT811.Arthrobacter BT801 is a L-hydantoinase producing strain isolated from soil. L-carbamoylase genes was expressed with pQE60 vector. Using ion-exchange chromatography, we were able to purify the L- carbamoylase 144-fold to homogeneity in two steps. In order to activate the L-hydantoin hydrolase by 100mmol/L NaHCO₃ and 100μmol/L Co²⁺, the activity of L-hydantoin...