Development of a β-glucosidase improved for glucose retroinhibition for cellulosic ethanol production: an integrated bioinformatics and genetic engineering approach.

backgroundThe global energy crisis, driven by economic growth and the increasing demand for energy, highlights the urgency of searching for alternative energy sources to mitigate environmental pollution and climate change. β-Glucosidases act in the final step of the enzymatic hydrolysis of cellulose, cleaving the β-1,4-glycosidic bonds in cellobiose to produce second-generation ethanol. However, these enzymes are easily inhibited by glucose, their final product, which limits the production of this biofuel. Genetic engineering combined with bioinformatics tools can improve key enzymatic characteristics, such as catalytic activity and glucose tolerance, in a more precise, faster, and cost-effective manner compared to traditional methods. In this work, a variant of a β-glucosidase from the GH1 family, isolated from the microbial community of Amazonian soil (Brazil), with enhanced catalytic activity and improved for glucose retroinhibition, was developed.

resultsBioinformatics analyses suggested the substitution of tryptophan at position 404 with leucine. The produced variant (W404L) was expressed in Escherichia coli and showed activity 3.2 times higher in the presence of glucose than the non-mutated control. Moreover, the partially purified mutated variant of β-glucosidase exhibited a 26-fold increase in catalytic activity compared to the original form of the enzyme. The results confirmed that the mutation proposed by computational analyses had a significant impact on enzyme catalytic activity and glucose retroinhibition.

conclusionsThis new variant may become a promising alternative to reduce the costs of enzyme cocktails used in the hydrolysis of lignocellulosic biomass used as a raw material in the production of second-generation ethanol.