Improvement of the catalytic performance of glycerol kinase from Bacillus subtilis by chromosomal site-directed mutagenesis

Glycerol kinase is the key enzyme in glycerol metabolism, and its catalytic efficiency has an important effect on glycerol utilization. Based on an analysis of the glycerol utilization pathway and regulation mechanism in B . subtilis , we conducted site-directed mutagenesis of the key glycerol kinase gene ( glpK ) on the chromosome to improve the glycerol utilization efficiency of Bacillus subtilis . Recombinant wild-type Bacillus subtilis glycerol kinase ( Bsu GlpK WT ) and two mutants ( Bsu GlpK M270I and Bsu GlpK S71V ) were successfully overexpressed in Escherichia coli BL21(DE3) and purified by Ni-IDA metal chelate chromatography. The specific activity of the Bsu GlpK M270I mutant (62.6 U/mg) was significantly higher (296.2%) than that of wild-type Bsu GlpK WT (15.8 U/mg). By contrast, the mutant Bsu GlpK S71V (4.89 U/mg) exhibited lower (69.1%) activity than Bsu GlpK WT , which suggested that variant S71V exhibited reduced catalytic efficiency for the substrate. Furthermore, the mutant strain B . subtilis M270I was constructed using a markerless delivery system, and exhibited a higher specific growth rate (improved by 11.3%, from 0.453 ± 0.012 to 0.511 ± 0.017 h −1 ) and higher maximal biomass (cell dry weight increased by 16%, from 0.577 ± 0.033 to 0.721 ± 0.015 g/L) than the parental strain with a shortened lag phase (2 ~ 4 h shorter) in M9 minimal medium with glycerol. These results indicate that the mutated glpK resulted in improved glycerol utilization, which has broad application prospects.