(166e) Enzyme Engineering of Beta-Glucosidases for Enhanced Thermal Stability and Activity

Biomass derived from various sources, such as forest and agricultural residues, can be used for production of high-value chemicals and other energy-related products. The biorefinery approach can increase the efficiency of agricultural land usage as well as help to develop more sustainable processes. Lignocellulosic biomass, an abundant biomass resource rich in carbon, is widely used for producing chemicals and biofuels. While hydrolysis of cellulose into its monomeric unit glucose is an important step toward the efficient biorefinery, an enzyme catalyzing this reaction, β-glucosidase, suffers from limitations, such as low activity, low thermal stability and product inhibition. Thus, an approach to engineer β-glucosidase to overcome these limitations would enhance the overall yield and the efficiency of the biorefinery process. Here, we report our approach to engineer β-glucosidases for enhanced thermal stability and activity. To this end, β-glucosidases from Thermoanaerobacterium aotearoense (TaBGL) and Pyrococcus furiosus (PfBGL) were chosen as parental enzymes. The wild-type TaBGL and PfBGL were recombinantly produced from Escherichia coli and purified. The enzymes were then subjected to activity assays using p-nitrophenyl-β-D-glucopyranoside (pNP-Glc) as a substrate. The steady state kinetic parameters of k_cat, K_m and K_I were 335 s^-1, 0.25 mM and 169 mM, respectively, for TaBGL at 60 °C and pH 6 and 303 s^-1, 0.34 mM and 71.2 mM, respectively, for PfBGL at 80 °C and pH 5.5. The half-lives (t_1/2) for TaBGL and PfBGL were 9 minutes at 60 °C and ~100 days s at 80 °C, respectively. Combinatorial libraries of these enzymes were created either using error prone PCR or guided by a bioinformatic analysis, HotSpot, to enhance thermal stability of TaBGL and increase enzymatic activity of PfBGL at lower temperatures. The agar plate based screening method using esculin hydrate and ferric ammonium citrate has been developed for rapid evaluation of the enzyme mutants for thermal stability and enzymatic activity and used together with 96-well based activity assays with pNP-Glc. Active mutants were identified from the screenings and are being characterized for their activity, thermal stability and glucose inhibition. We will also discuss sequence characteristics important in enzymatic activity, thermal stability and glucose inhibition of β-glucosidases determined from comparative studies based on a collection of literature and our own experimental results.