Limits...
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

Effects of cellobiose concentration on the initial reaction rates of Bgl6 (filled square) and M3 (filled circle). The reactions were performed at 50 °C and pH 6.0 with different concentrations of celobiose (0.5–15 %, w/v) as substrate. 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
getmorefigures.php?uid=PMC4666061&req=5

Fig3: Effects of cellobiose concentration on the initial reaction rates of Bgl6 (filled square) and M3 (filled circle). The reactions were performed at 50 °C and pH 6.0 with different concentrations of celobiose (0.5–15 %, w/v) as substrate. Data points are the average of triplicate measurements, and error bars represent standard deviation

Mentions: Bgl6 is active towards cellobiose, cellotriose, cellotetrose, and cellopentose (Table 1, Additional file 1: Figure S4). This property is favorable for the usage of this enzyme in cellulose refining as it helps relieve the inhibitory effect of the saccharides to EGs and CBHs during the saccharification process of cellulose [18]. Meanwhile, the recombinant Bgl6 remained highly active towards cellobiose at the concentration of 15 % (w/v, about 440 mM, Fig. 3), and this feature is different from many other β-glucosidases, which are often inhibited by cellobiose at millimolar concentration range. For instance, the β-glucosidase from Orpinomyces sp. Strain PC-2 was inhibited when the cellobiose concentration was higher than 1.5 mM [24]; the β-glucosidase from Trichoderma viride was inhibited when the cellobiose concentration was higher than 8 mM [25], and the β-glucosidases from Acremonium thermophilum (AtBG3), Thermoascus aurantiacus (TaBG3), and Aspergillus sp. (N188BG) were inhibited when the cellobiose concentration was higher than 5 mM [7]. The substrate inhibition is due to the occurrence of transglycosylation reaction, which is under kinetic control [26, 27]. Although all cellobiose and transglycosylation products will eventually be hydrolyzed to glucose, transglycosylation competes with hydrolysis and thus will hinder the efficient degradation of cellulose [8, 27, 28]. Therefore, the cellobiose-tolerant β-glucosidases, such as Bgl6 and mutant M3 (Fig. 3), may be more suitable under industrial condition where the typical cellobiose concentrations are tens of millimolar [29].Table 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)

Effects of cellobiose concentration on the initial reaction rates of Bgl6 (filled square) and M3 (filled circle). The reactions were performed at 50 °C and pH 6.0 with different concentrations of celobiose (0.5–15 %, w/v) as substrate. 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

Fig3: Effects of cellobiose concentration on the initial reaction rates of Bgl6 (filled square) and M3 (filled circle). The reactions were performed at 50 °C and pH 6.0 with different concentrations of celobiose (0.5–15 %, w/v) as substrate. Data points are the average of triplicate measurements, and error bars represent standard deviation
Mentions: Bgl6 is active towards cellobiose, cellotriose, cellotetrose, and cellopentose (Table 1, Additional file 1: Figure S4). This property is favorable for the usage of this enzyme in cellulose refining as it helps relieve the inhibitory effect of the saccharides to EGs and CBHs during the saccharification process of cellulose [18]. Meanwhile, the recombinant Bgl6 remained highly active towards cellobiose at the concentration of 15 % (w/v, about 440 mM, Fig. 3), and this feature is different from many other β-glucosidases, which are often inhibited by cellobiose at millimolar concentration range. For instance, the β-glucosidase from Orpinomyces sp. Strain PC-2 was inhibited when the cellobiose concentration was higher than 1.5 mM [24]; the β-glucosidase from Trichoderma viride was inhibited when the cellobiose concentration was higher than 8 mM [25], and the β-glucosidases from Acremonium thermophilum (AtBG3), Thermoascus aurantiacus (TaBG3), and Aspergillus sp. (N188BG) were inhibited when the cellobiose concentration was higher than 5 mM [7]. The substrate inhibition is due to the occurrence of transglycosylation reaction, which is under kinetic control [26, 27]. Although all cellobiose and transglycosylation products will eventually be hydrolyzed to glucose, transglycosylation competes with hydrolysis and thus will hinder the efficient degradation of cellulose [8, 27, 28]. Therefore, the cellobiose-tolerant β-glucosidases, such as Bgl6 and mutant M3 (Fig. 3), may be more suitable under industrial condition where the typical cellobiose concentrations are tens of millimolar [29].Table 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