Directed mutation of β-glucanases from probiotics to enhance enzymatic activity, thermal and pH stability

beta-glucanases are widely applied in biological control, brewing and feed industries; however, there are seldom studies of beta-glucanases in probiotics. Here, beta-glucanase genes were cloned from Bacillus licheniformis, Lactobacillus fermentum and L. johnsonii. beta-glucanase genes, as blg, lfg and ljg isolated from B. licheniformis, L. fermentum and L. johnsonii were prokaryotic expressed to obtain recombinant strains BL, LF and LJ, respectively. Directed mutations in these genes were introduced by sequential error-prone PCR. Results showed that beta-glucanase activities in three mutants mblg, mlfg and mljg were 1.94-, 2.72- and 1.29-fold higher than the BL, LF and LJ, respectively. Mutation sites analysis showed substitutions at Ser370Gly and Leu395Phe in mblg; Arg169His and Asn302Ser in mlfg; Val132Met, Ser226Asn, and Asp355Gly in mljg. Spatial structural predictions revealed the numbers and positions of alpha-helices and beta-strands in the three mutants were altered, which might result in beta-glucanase activity increasement. Analysis of beta-glucanase properties revealed no significant differences in the optimal temperatures and pH between mutant and wild-type strains. However, mlfg and mljg exhibited greater thermal stability at 30-50 celcius than the wild-type strains, and mblg improved pH stability compared with wild-type strain. This is the first report about beta-glucanase-encoding genes in L. fermentum and L. johnsonii. These findings provide an efficient way to improve the activity of beta-glucanase.