Isolation of a hich-activity promoter for tyrosinase gene

A promoter-probe plasmid pKK232-8 was used as a vector which functioned in E. coli. The chromosome DNA of Pseudomonas maltophilia was partially digested by BamH I -Hind III , recombinated in vitro and transformed into E. coli. The transformants were selected on Ampicillin(Ap) and chloramphenicol (Cm) containing resistance plates. From random-seleted 54 strains of transformants we obtained transformant E. coli PASI which level of Cm resistance got to 1 000 mg/L. The recombinant plasmid from E. coli-PASI was named pASI. The results of analysis by restriction endonucleases digestion and molecular hybridization showed that recombinant plasmid pASI carried an inserted fragment with size of 1. 4 kb from chromosome DNA of P. maltophilia. The result of analysis by SDS-PAGE indicated that transformant E. coliPASI expressed an additionaly band of CAT enzyme (22 × 10³). The mel gene on recombinant plasmid pWSY, which is from P. maltophilia AT18, was subcloned into downstream of the promoter fragment of pASI to construct engineering strain E. coliAWS. The experiment result showed that melanin productivity of E. coliAWS increased 70. 6% than that of E. coli WSY strain.