Enhancement of thermostability of Bacillus subtilis endoglucanase by error-prone PCR and DNA shuffling

The Bacillus subtilis endo-β-1,4-glucanase gene (beg), which encodes the enzyme BEG that comprises 499 amino acid residues, was mutated by error-prone polymerase chain reaction and DNA shuffling to make variants with improved functionalities. The mutated DNAs were transformed into Escherichia coli DH5α, and among the 1370 transformants, a positive clone 8-20 was obtained finally based on a halo assay and thermostability and alkaline tolerance analyses. The mutated enzyme BEG8-20 of clone 8-20 was changed at seven amino acid residues compared to the wild-type enzyme BEGwt: K45E, I102Y, M112V, D226Y, D295E, L423S, and D460G. The optimum temperature and pH of BEG8-20 were nearly the same as those of BEGwt. However, the thermostability of BEG8-20 was increased 1.53-fold (21.4 min vs. 14.0 min) based on the half-life of the residual activity at 70 °C. This increase in enzyme thermostability is advantageous for the hydrolysis of lignocellulosic materials to produce fermentable sugars. In the activity staining experiment, only a truncated smaller enzyme was observed for BEG8-20, whereas two forms, a matured (52 kDa) and a truncated smaller (34.5 kDa) enzyme, were observed for BEGwt owing to proteolytic internal cleavage at the linker region between the 297th and 356th amino acid residues. These results indicate that amino acid substitutions in the mutant enzyme have rendered the protein prone to cleavage at the C-terminal region.