title: Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration.
abstract: BACKGROUND
  Most β-glucosidases reported are sensitive to the end product (glucose), making it the rate limiting component of cellulase for efficient degradation of cellulose through enzymatic route. Thus, there are ongoing interests in searching for glucose-tolerant β-glucosidases, which are still active at high glucose concentration. Although many β-glucosidases with different glucose-tolerance levels have been isolated and characterized in the past decades, the effects of glucose-tolerance on the hydrolysis of cellulose are not thoroughly studied.
  
  
  RESULTS
  In the present study, a novel β-glucosidase (Bgl6) with the half maximal inhibitory concentration (IC 50) of 3.5 M glucose was isolated from a metagenomic library and characterized. However, its poor thermostability at 50 °C hindered the employment in cellulose hydrolysis. To improve its thermostability, random mutagenesis was performed. A thermostable mutant, M3, with three amino acid substitutions was obtained. The half-life of M3 at 50 °C is 48 h, while that of Bgl6 is 1 h. The K cat/K m value of M3 is 3-fold higher than that of Bgl6. The mutations maintained its high glucose-tolerance with IC 50 of 3.0 M for M3. In a 10-h hydrolysis of cellobiose, M3 completely converted cellobiose to glucose, while Bgl6 reached a conversion of 80 %. Then their synergistic effects with the commercial cellulase (Celluclast 1.5 L) on hydrolyzing pretreated sugarcane bagasse (SCB) were investigated. The supplementation of Bgl6 or mutant M3 to Celluclast 1.5 L significantly improved the SCB conversion from 64 % (Celluclast 1.5 L alone) to 79 % (Bgl6) and 94 % (M3), respectively. To further evaluate the application potential of M3 in high-solids cellulose hydrolysis, such reactions were performed at initial glucose concentration of 20-500 mM. Results showed that the supplementation of mutant M3 enhanced the glucose production from SCB under all the conditions tested, improving the SCB conversion by 14-35 %.
  
  
  CONCLUSIONS
  These results not only clearly revealed the significant role of glucose-tolerance in cellulose hydrolysis, but also showed that mutant M3 may be a potent candidate for high-solids cellulose refining.
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China ; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Li-Chuang
authors.initials: LC
authors.lastname: Cao
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China ; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Wang
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China ; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Guang-Hui
authors.initials: GH
authors.lastname: Ren
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China ; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Kong
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China ; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Liang
authors.initials: L
authors.lastname: Li
authors.affiliation: State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Wei
authors.initials: W
authors.lastname: Xie
authors.affiliation: School of Life Sciences, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China ; South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, Sun Yat-sen University, Guangzhou, 510275 People's Republic of China.
authors.forename: Yu-Huan
authors.initials: YH
authors.lastname: Liu
keywords: Cellulase; Cellulose refining; Directed evolution; Glucose-tolerance; Metagenomic library; Thermostability; β-Glucosidase