Directed Evolution of Clostridium thermocellum β-Glucosidase A Towards Enhanced Thermostability

Abstract

β-Glucosidases are key enzymes in the process of cellulose utilization. It is the last enzyme in the cellulose hydrolysis chain, which converts cellobiose to glucose. Since cellobiose is known to have a feedback inhibitory effect on a variety of cellulases, β-glucosidase can prevent this inhibition by hydrolyzing cellobiose to non-inhibitory glucose. While the optimal temperature of the Clostridium thermocellum cellulosome is 70 °C, C. thermocellum β-glucosidase A is almost inactive at such high temperatures. Thus, in the current study, a random mutagenesis directed evolutionary approach was conducted to produce a thermostable mutant with Kcat and Km, similar to those of the wild-type enzyme. The resultant mutant contained two mutations, A17S and K268N, but only the former was found to affect thermostability, whereby the inflection temperature (Ti) was increased by 6.4 °C. A17 is located near the central cavity of the native enzyme. Interestingly, multiple alignments revealed that position 17 is relatively conserved, whereby alanine is replaced only by serine. Upon the addition of the thermostable mutant to the C. thermocellum secretome for subsequent hydrolysis of microcrystalline cellulose at 70 °C, a higher soluble glucose yield (243%) was obtained compared to the activity of the secretome supplemented with the wild-type enzyme.

Isolation and characterization of thermophilic cellulose and hemicellulose degrading bacterium, Thermoanaerobacterium sp. R63 from tropical dry deciduous forest soil

“…Effect of temperature on hydrolytic activity of Thermoanaerobacterium sp. R63 was consistent with other studies of thermophilic bacteria; C. thermocellum, Bacillus licheniformis, and T. calidifontis RX-1 [32,46,47]. With their intrinsic stability and activity at high temperatures, thermophilic enzymes could be inferred to a possibility of prolonged storage and low activity loss during long-time processing under an elevated temperature condition.…”

“…thermocellum , Bacillus licheniformis , and T . calidifontis RX-1 [ 32 , 46 , 47 ]. With their intrinsic stability and activity at high temperatures, thermophilic enzymes could be inferred to a possibility of prolonged storage and low activity loss during long-time processing under an elevated temperature condition.…”

Progress in Ameliorating Beneficial Characteristics of Microbial Cellulases by Genetic Engineering Approaches for Cellulose Saccharification

“…Considering the various economic and desirable qualities of cellulases in cellulose saccharification efficiency such as high thermostability, resistance to end product inhibition, high specific activity and catalytic efficiency a lot of investigations had been carried out over the years to develop efficient enzymes utilizing various genetic/metabolic engineering techniques (Cao et al, 2018;Han et al, 2018;Saavedra et al, 2018;Zheng et al, 2018;Bashirova et al, 2019;Yoav et al, 2019;Adebami and Adebayo-Tayo, 2020;Li et al, 2020b;Wu et al, 2020). Significant efforts have also been made in discovery of novel cellulases using metagenomics (Garg et al, 2016;Song et al, 2016;Zhao et al, 2017).…”

Carbohydrate-Active Enzymes: Structure, Activity, and Reaction Products

“…A directed evolution approach was used by Yoav et al to enhance the thermostability of Clostridium thermocellum β-Glucosidase [13]. In the process of cellulose utilization, β-glucosidases are key enzymes that convert cellobiose, produced by cellulose hydrolysis, to glucose.…”

“…A thermostable mutant with kcat and Km, similar to those of the wild-type enzyme, was produced. The addition of the thermostable double mutant (A17S and K268N) to C. thermocellum secretome in order to carry out the hydrolysis of microcrystalline cellulose at 70 • C drastically increased the soluble glucose yield compared to the activity of the secretome supplemented with the wild-type enzyme [13].…”