Hitoshi Sagai

Cloning, sequencing, and overexpression in Escherichia coli of a sarcosine oxidase-encoding gene linked to the Bacillus creatinase gene

Abstract Bacillus sp. B-0618 produces both creatinase (Cre; creatine amidinohydrolase; EC 3.5.3.3) and sarcosine oxidase (Sox; EC 1.5.3.1) enzymes when grown in the presence of an inducer, choline chloride. A genomic library of Bacillus sp. B-0618, prepared in the plasmid vector pACYC184, was screened to obtain a gene (sox) encoding Sox by a convenient colorimetric assay. A plasmid, pOXI101, isolated from a sox-positive clone, contained a 14.2-kb insert of Bacillus DNA. The nucleotide sequence of a 1.7-kb segment containing the sox gene was determined, and it was found that an open reading frame encoding a protein consisting of 390 amino acids was located upstream from the cre structural gene cloned previously. When a 1.6-kb XhoI-BglII fragment of pOXI101 was inserted into the pUC118 vector and introduced into Escherichia coli, transformants cultured in the absence of the inducer produced Sox about 50-fold more than Bacillus sp. B-0618 cultured in the presence of the inducer. The Bacillus Sox had the -11Gly-X-13Gly-X-X-16Gly- sequence motif that is highly conserved in flavoproteins. We created an FAD-free Sox by changing 13Gly to Asp in the motif of the parental Sox by oligodeoxynucleotide-directed mutagenesis. The mutant protein no longer expressed the Sox activity, even on the addition of FAD.

Molecular cloning and high expression of the Bacillus creatinase gene in Escherichia coli

Abstract 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.