Increasing the thermostability of Flavobacterium meningosepticum glycerol kinase by changing Ser329 to Asp in the subunit interface region

The thermostability enhancement of Flavobacterium meningosepticum glycerol kinase (FGK) by random mutagenesis in the subunit interface region was inve stigated. A single Escherichia coli transformant, which produced a more the rmostable glycerol kinase than the parent enzyme, was obtained. The nucleot ide sequence of the gene of the mutant enzyme (FGK2615) was determined, and the four amino acid replacements were identified as Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys. Although the properties of FGK2615 were fundamentally similar to those of the parent enzyme, the thermostabil ity and Km for ATP had changed. The thermostability of FGK2615 was apparent ly increased; the temperature at which the enzyme activity is inactivated b y 50% for a 30-min incubation of FGK2615 was determined to be 72.1 degreesC which was 3.1 degreesC higher than that of the parent FGK. Four additional mutants each having a single amino acid replacement (Glu327 to Asp, Ser329 to Asp, Thr330 to Ala and Ser334 to Lys) were prepared and their thermosta bility and Km for substrates were evaluated. The effect of the substitution of Ser329 to Asp is discussed.