Enhancement of the thermostability of β-1,3-1,4-glucanase by directed evolution

摘要

In order to improve the thermostability of β-1,3-1,4-glucanase, evolutionary molecular engineering was used to evolve the β-1,3-1,4-glucanase from Bacillus subtilis ZJF-1A5. The process involves random mutation by error-prone PCR and DNA shuffling followed by screening on the filter-based assay. Two mutants, EGs1 and EGs2, were found to have four and five amino acid substitutions, respectively. These substitutions resulted in an increase in melting temperature from T_m = 62.5℃ for the wild-type enzyme to T_m = 65.5℃ for the mutant EGs1 and 67.5℃ for the mutant EGs2. However, the two mutated enzymes had opposite approaches to produce reducing sugar from lichenin with either much higher (28%) for the former or much lower (21.6%) for the latter in comparison with their parental enzymes. The results demonstrate that directed evolution is an effective approach to improve the thermostability of a mesophilic enzyme.