CLONING OF A LIGNOSTILLBENE-ALPHA,BETA-DIOXYGENASE ISOZYME GENE FROM PSEUDOMONAS-PAUCIMOBILIS TMY1009

A genomic DNA library of Pseudomonas paucimobilis TMY1009 was construc ted using a cosmid vector pWE15. Screening of the library for lignosti lbene-alpha,beta-dioxygenase (LSD) isozyme genes was done with a commo n probe for the alpha, beta, and gamma subunits, which composed LSD is ozymes, The positive clones obtained by colony hybridization were furt her confirmed by Southern hybridization, A 4.2-kb BamHI-HindIII fragme nt hybridized with the probe was subcloned into pUC118 to yield the pl asmid pKHE1700. Escherichia coli MV1184 carrying pKHE1700 produced one of the LSD isozymes, The cloned LSD was purified and compared with LS D isozymes from P. paucimobilis TMY1009. The behavior on column chroma tographies through all purification steps accorded with that of LSD-II I. Furthermore, the mobility on polyacrylamide gel electrophoresis, th e elution profile on reversed-phase HPLC, and the partial amino acid s equence were common to the cloned LSD and the native LSD-III. Thus the cloned LSD could be identified as LSD-III. It was found that the gene of LSD-III (IsdB) was composed of 489 codons,