MOLECULAR-CLONING AND HIGH EXPRESSION OF THE BACILLUS-CREATINASE GENE IN ESCHERICHIA-COLI

A genomic library of Bacillus sp. B-0618, prepared with the plasmid vector pACYC184, was screened to obtain a gene encoding creatinase (creatine amidinohydrolase; EC 3.5.3.3) by a direct coloration assay. A plasmid pCR1 isolated from a creatinase positive clone contained a 3.7-kb insert of the Bacillus chromosomal DNA. We determined the nucleotide sequence of a 1.55-kb segment containing the creatinase gene and found an open reading frame that coded for a protein consisting of 411 amino acids, with a calculated molecular mass of 46,946 daltons. The translated protein sequence of the creatinase gene from the Bacillus strain was 67% homologous to those of Pseudomonas and Flavobacterium. Although the creatinase of Bacillus sp. B-0618 was induced by choline chloride, Escherichia coli harboring pCR1 expressed 60-fold more creatinase activity intracellularly than did the producing strain, even in the absence of the inducer.