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A Novel High Glucose-Tolerant β-Glucosidase: Targeted Computational Approach for Metagenomic Screening.

ABSTRACT: The rate-limiting component of cellulase for efficient degradation of lignocellulosic biomass through the enzymatic route depends on glucosidase's sensitivity to the end product (glucose). Therefore, there is still a keen interest in finding glucose-tolerant β-glucosidase (BGL) that is active at high glucose concentrations. The main objective of this study was to identify, isolate, and characterize novel highly glucose-tolerant and halotolerant β-glucosidase gene (PersiBGL1) from the mixed genome DNA of sheep rumen metagenome as a suitable environment for efficient cellulase by computationally guided experiments instead of costly functional screening. At first, an in silico screening approach was utilized to find primary candidate enzymes with superior properties. The structure-dependent mechanism of glucose tolerance was investigated for candidate enzymes. Among the computationally selected candidates, PersiBGL1 was cloned, isolated, and structurally characterized, which achieved very high activity in relatively high temperatures and alkaline pH and was successfully used for the hydrolysis of cellobiose. This enzyme exhibits a very high glucose tolerance, with the highest inhibition constant K _i (8.8 M) among BGLs reported so far and retained 75% of its initial activity in the presence of 10 M glucose. Furthermore, a group of multivalent metal, including Mg²⁺, Mn²⁺, and Ca²⁺, as a cofactor, could improve the catalytic efficiency of PersiBGL1. Our results demonstrated the power of computational selected candidates to discover novel glucose tolerance BGL, effective for the bioconversion of lignocellulosic biomass.

SUBMITTER: Ariaeenejad S

PROVIDER: S-EPMC7406677 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:?-Glucosidase is an important component of the cellulase enzyme system. It does not only participate in cellulose degradation, it also plays an important role in hydrolyzing cellulose to fermentable glucose by relieving the inhibition of exoglucanase and endoglucanase from cellobiose. Therefore, the glucose-tolerant ?-glucosidase with high specific activity for cellobiose might be a potent candidate for industrial applications. RESULTS:The ?-glucosidase gene bgl that encodes a 443-amino-acid protein was cloned and over-expressed from Thermoanaerobacterium thermosaccharolyticum DSM 571 in Escherichia coli. The phylogenetic trees of ?-glucosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the BGL was a novel ?-glucosidase. By replacing the rare codons for the N-terminal amino acids of the target protein, the expression level of bgl was increased from 6.6 to 11.2 U/mg in LB medium. Recombinant BGL was purified by heat treatment followed by Ni-NTA affinity. The optimal activity was at pH 6.4 and 70°C. The purified enzyme was stable over pH range of 5.2-7.6 and had a 1 h half life at 68°C. The activity of BGL was significantly enhanced by Fe2+ and Mn2+. The Vmax of 64 U/mg and 120 U/mg were found for p-nitrophenyl-?-D-glucopyranoside (Km value of 0.62?mM) and cellobiose (Km value of 7.9?mM), respectively. It displayed high tolerance to glucose and cellobiose. The Kcat for cellobiose was 67.7?s-1 at 60°C and pH 6.4, when the concentration of cellobiose was 290?mM. It was activated by glucose at concentrations lower that 200?mM. With glucose further increasing, the enzyme activity of BGL was gradually inhibited, but remained 50% of the original value in even as high as 600?mM glucose. CONCLUSIONS:The article provides a useful novel ?-glucosidase which displayed favorable properties: high glucose and cellobiose tolerance, independence of metal ions, and high hydrolysis activity on cellobiose.

Project description:BACKGROUND:Most ?-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 ?-glucosidases, which are still active at high glucose concentration. Although many ?-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 ?-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.

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A Novel High Glucose-Tolerant β-Glucosidase: Targeted Computational Approach for Metagenomic Screening.

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