Simultaneously optimizing multiple properties of β-glucosidase Bgl6 using combined (semi-)rational design strategies and investigation of the underlying mechanisms.

The performance of β-glucosidase during cellulose saccharification is determined by thermostability, activity and glucose tolerance. However, conflicts between them make it challenging to simultaneously optimize three properties. In this work, such a case was reported using Bgl6-M3 as a starting point. Firstly, four thermostability-enhancing mutations were obtained using computer-aided engineering strategies (mutant M7). Secondly, substrate binding pocket of M7 was reshaped, generating two mutations that increased activity but decreased glucose tolerance (mutant M9). Then a key region lining active site cavity was redesigned, resulting in three mutations that boosted glucose tolerance and activity. Finally, mutant M12 with simultaneously improved thermostability (half-life of 20-fold), activity (k/K of 5.6-fold) and glucose tolerance (ΔIC of 200 mM) was obtained. Mechanisms for property improvement were elucidated by structural analysis and molecular dynamics simulations. Overall, the strategies used here and new insights into the underlying mechanisms may provide guidance for multi-property engineering of other enzymes.