Evolution of a Thermophilic Bacterial β Glucosidase towards Increased Activity at Reduced
The enzymatic degradation of cellulose derived from lignocellulosic biomass is commonly conducted at temperatures of 55 °C to 65 °C with a mixture of different classes of cellulases. Thereby insufficient β-glucosidase activity often represents the bottleneck in the saccharification process. β-Glucosidases with appropriate abilities, such as high activity, high stability and low product inhibition, are rarely described within this temperature range. To overcome this bottleneck, two strategies of protein engineering – a semi-rational strategy based on site-saturation mutagenesis of bioinformatically predicted hot-spot positions and a random mutagenesis strategy – were applied to the thermophilic β-glucosidase BglA from Caldicellulosiruptor saccharolyticus (CsBglA). The combination of beneficial mutations identified from both strategies resulted in the variant CsBglA L64R Y73F T221N H324L (CsBglA LYTH). CsBglA LYTH offered an increased activity of 552 U/mg (CsBglA 359 U/mg) and a 2.5-fold increase in kcat/KM at 55 °C while retaining its initially stability at this temperature. Taking into account its low product inhibition (IC50 of 0.29 M glucose), CsBglA LYTH is predestined by a unique combination of desired abilities for current state-of-the-art processes aimed at the enzymatic degradation of cellulose.