Thermostable beta-glycosidases as processing enzymes in biorefineries

Glycoside hydrolases are enzymes that cleave the glycosidic bonds in carbohydrates and glycoconjugates. Cellulases, xylanases and other glycosidases play a vital role in producing sugars from lignocellulosic biomass, which then be converted to fuels and chemicals to replace fossil-based fuel. Xylan is the second most abundant polysaccharide next to cellulose, plays a significant role in the total hydrolysis of biomass. Xylanases can be used in the processing of biomass to monosaccharides or other products. This study aims to analyse the working capacity of GH10 xylanases obtained from T. flexuosa (TfXYN10A) and T. aurantiacus (TasXYN10A) at high temperature and in the presence of ionic liquids and to understand the various effects of active site mutations in a disaccharide hydrolysing GH1 beta-glycosidase from S. acidocaldarius. TfXYN10A expressed in E. coli produced more thermostable enzyme than the same expressed in T. reesei, probably due to the destabilizing effect of glycosylation in T. reesei. CBM mediated reduction and substrate mediated increase of thermostability showed the significance of covalent and non-covalent molecular contacts by the intramolecular or external environment in the enzyme's activity and stability. Analysis of the end-products of xylan hydrolysis exhibited high temperature apparently reduced the binding of short substrate. Ionic liquids (IL) are salts with melting point below 100°C and are used in lignocellulosic biomass hydrolysis. ILs adversely affect the enzyme stability and activity, though they can be beneficial in the pretreatment of lignocellulose before the enzymatic hydrolysis. TfXYN10A and TasXYN10A can remain significantly active in the presence of 30–40% of ILs, but restrains the activity in > 50% IL. Also, the optimum temperature for hydrolytic activity decreases with increase in the IL concentration. With both xylanases, ILs affect the activity more than the stability. Thermostability tends to cease the unfolding of enzyme caused by ILs, but the active site remains available for inhibition by ionic liquids. Elevated Km in the presence of ILs suggests the idea of competitive inhibition between the substrate and ILs, this is further confirmed by molecular docking studies. S. acidocaldarius β-glycosidase was studied to explore the effect of active site mutations on the enzyme activity, especially on pH activity. One mutant (V212T) proved its efficiency by increasing the thermostability and extending the pH activity range to more acidic pH. Despite of the increased Km in V212T, the hydrolytic activity increased significantly at higher substrate concentrations and the total activity is higher with lactose than cellobiose. Change in the end-product inhibition with glucose, turned out not to be the reason for increased activity with cellobiose, but apparently was a reason for increased activity with lactose. Further studies revealed that V212T had a remarkable lowering effect on the transglycosylation activity.