Cloning, purification and characterization of a thermostable β-galactosidase from Bacillus licheniformis strain KG9

A thermo— and alkalitolerant Bacillus licheniformis KG9 isolated from Taşlıdere hot water spring in Batman/Turkey was found to produce a thermostable β—galactosidase. Phylogenetic analysis showed that the 16S rRNA gene from B. licheniformis strain KG9 was 99.9% identical to that of the genome sequenced B. licheniformis strain DSM 13. Analysis of the B. licheniformis DSM 13 genomic sequence revealed four putative β—galactosidase genes. PCR primers based on the genome sequence of strain DSM 13 were used to isolate the corresponding β—galactosidase genes from B. licheniformis strain KG9. The calculated molecular weights of the β—galactosidases I, II, III, and IV using sequencing data were 30, 79, 74, and 79 kDa, respectively. The genes were inserted into an expression vector and recombinant β—galactosidase was produced in Escherichia coli. Of the four β—galactosidase genes identified in strain KG9, three of them were expressed as active, intracellular enzymes in E. coli. One of the recombinant enzymes, β—galactosidase III, was purified and characterized. Optimal temperature and pH was determined to be at 60 ºC and pH 6.0, respectively. Km was determined to be 1.3 mM and 13.3 mM with oNPG (ortho—nitrophenyl—β—D—galactopyranoside) and lactose as substrates, respectively, and Vmax was measured to 1.96 μmol/min and 1.55 μmol/min with oNPG and lactose, respectively.