Cloning, Sequencing, and Overexpression in Escherichia coli of a Sarcosine Oxidase-Encoding Gene Linked to the Bacillus Creatinase Gene

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Bacillus sp. B-0618 produces both creatinase (Cre; creatine amidinohydrolase; EC3.5.3.3) and sarcosine oxidase(Sox; EC1.5.3.1) enzymes when grown in the presence of an inducer, choline chloride. A genomic library pf Bacillus sp. B-06918,prepared in the plasmid vector pACYC184,was screened to obtain a gene (sox) encoding Sox by a convenient colormetric 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 clone previously. When a 1.6-kb XhoI-BglII fragement of pOXI101 was inserted into the pUC118 vector and introduced into Escherichia coli, transforments 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 -^<11>Gly-X-^<13>Gly-X-X-^<16>Gly- sequence motif that is highly conserved in flavoproteins. We created an FAD-free Sox by chaning ^<13>Gly 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.
社団法人日本生物工学会の論文
1994-03-25