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Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration.

Cao LC, Wang ZJ, Ren GH, Kong W, Li L, Xie W, Liu YH - Biotechnol Biofuels (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus

Thermostability of Bgl6 and the mutants. a Half-lives of Bgl6 and the mutants at 50 °C are 1 h (Bgl6), 8 h (V174A), 21 h (V174C), 5 h (A404 V), 2 h (L441F), and 48 h (M3). b Thermal inactivation curves of Bgl6 and the mutants. The T50 values are 53.1 °C (Bgl6), 57.3 °C (V174A), 57.6 °C (V174C), 55.3 °C (A404 V), 54.2 °C (L441F), and 60.7 °C (M3). Data points are the average of triplicate measurements, and error bars represent standard deviation
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Fig2: Thermostability of Bgl6 and the mutants. a Half-lives of Bgl6 and the mutants at 50 °C are 1 h (Bgl6), 8 h (V174A), 21 h (V174C), 5 h (A404 V), 2 h (L441F), and 48 h (M3). b Thermal inactivation curves of Bgl6 and the mutants. The T50 values are 53.1 °C (Bgl6), 57.3 °C (V174A), 57.6 °C (V174C), 55.3 °C (A404 V), 54.2 °C (L441F), and 60.7 °C (M3). Data points are the average of triplicate measurements, and error bars represent standard deviation

Mentions: The optimal temperature (Topt) of Bgl6 is 50 °C (Fig. 1a). The mutations increase the Topt to 60 °C (M3), which is 10 °C higher than that of the WT (Fig. 1a). At 50 °C, M3 remained about 85 % activity of that at 60 °C. Meanwhile, the optimal pH of the recombinant protein was shifted from 6.0 (Bgl6) to 5.5 (M3) by the mutations (Fig. 1b). This may benefit the utilization of this protein in the practical hydrolysis of cellulose because the optimal pHs of the cellulases employed now are about 5.0 [8, 20, 21]. The half-life of Bgl6 at 50 °C is only 1 h (Fig. 2a). The mutations result in 2–20 folds improvement on this property (Fig. 2a). Combination of three beneficial mutations further extends the half-life to 48 h (M3).Fig. 1


Engineering a novel glucose-tolerant β-glucosidase as supplementation to enhance the hydrolysis of sugarcane bagasse at high glucose concentration.

Cao LC, Wang ZJ, Ren GH, Kong W, Li L, Xie W, Liu YH - Biotechnol Biofuels (2015)

Thermostability of Bgl6 and the mutants. a Half-lives of Bgl6 and the mutants at 50 °C are 1 h (Bgl6), 8 h (V174A), 21 h (V174C), 5 h (A404 V), 2 h (L441F), and 48 h (M3). b Thermal inactivation curves of Bgl6 and the mutants. The T50 values are 53.1 °C (Bgl6), 57.3 °C (V174A), 57.6 °C (V174C), 55.3 °C (A404 V), 54.2 °C (L441F), and 60.7 °C (M3). Data points are the average of triplicate measurements, and error bars represent standard deviation
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666061&req=5

Fig2: Thermostability of Bgl6 and the mutants. a Half-lives of Bgl6 and the mutants at 50 °C are 1 h (Bgl6), 8 h (V174A), 21 h (V174C), 5 h (A404 V), 2 h (L441F), and 48 h (M3). b Thermal inactivation curves of Bgl6 and the mutants. The T50 values are 53.1 °C (Bgl6), 57.3 °C (V174A), 57.6 °C (V174C), 55.3 °C (A404 V), 54.2 °C (L441F), and 60.7 °C (M3). Data points are the average of triplicate measurements, and error bars represent standard deviation
Mentions: The optimal temperature (Topt) of Bgl6 is 50 °C (Fig. 1a). The mutations increase the Topt to 60 °C (M3), which is 10 °C higher than that of the WT (Fig. 1a). At 50 °C, M3 remained about 85 % activity of that at 60 °C. Meanwhile, the optimal pH of the recombinant protein was shifted from 6.0 (Bgl6) to 5.5 (M3) by the mutations (Fig. 1b). This may benefit the utilization of this protein in the practical hydrolysis of cellulose because the optimal pHs of the cellulases employed now are about 5.0 [8, 20, 21]. The half-life of Bgl6 at 50 °C is only 1 h (Fig. 2a). The mutations result in 2–20 folds improvement on this property (Fig. 2a). Combination of three beneficial mutations further extends the half-life to 48 h (M3).Fig. 1

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

No MeSH data available.


Related in: MedlinePlus