[Gene technology to overproduce the enzymes useful as diagnostic reagents]

We cloned the genes encoding the microbial enzymes described below and discussed the possibility as the diagnostic reagents: Creatinase(CR) and sarcosine oxidase(SOX) are useful for a diagnostic measurement of creatinine in combination with creatinase. Bacillus sp. B-0618 produces both CR and SOX enzymes when grown in the presence of an inducer, choline chloride. We cloned these two genes encoding CR and SOX by using recombinant DNA techniques. We found that the SOX-encoding gene is located upstream from the CR-encoding gene. When the CR- and SOX-encoding genes were independently inserted into the pUC-based vector and introduced into Escherichia coli, the transformants produced 15.5-fold more CR and 50-fold more SOX than Bacillus sp. B-0618, respectively, in the absence of the inducer. Although the Bacillus SOX is a flavoprotein, we created an FAD-free SOX by site-directed DNA mutagenesis. The mutant protein no longer expressed the SOX activity. Cholesterol esterase is useful for a diagnostic measurement of total cholesterol in combination with cholesterol oxidase. We cloned a gene encoding the cholesterol esterase enzyme. When the gene was introduced into the non-producing Streptomyces strain, the transformant produced the cholesterol esterase. Phospholipase A2 is a diverse family of enzymes that hydrolyze the sn-2 fatty acyl ester bond of phosphoglycerides producing free fatty acids and lysophospholipids. The enzymes are present in pancreatic juice and in the venoms of snakes and bees, where they serve digestive functions.(ABSTRACT TRUNCATED AT 250 WORDS)