Dataset Information

Glucose-tolerant ?-glucosidase retrieved from a Kusaya gravy metagenome.

ABSTRACT: ?-glucosidases (BGLs) hydrolyze cello-oligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (?mM) concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in Escherichia coli (?10,000 colonies) and grown on LB agar plates containing 5-bromo-4-chloro-3-indolyl-?-D-glucoside, yielding 828 positive (blue) colonies. These were then arrayed in 96-well plates, grown in LB, and secondarily screened for activity in the presence of 10% (w/v) glucose. Seven glucose-tolerant clones were identified, each of which contained a single bgl gene. The genes were classified into two groups, differing by two nucleotides. The deduced amino acid sequences of these genes were identical (452 aa) and found to belong to the glycosyl hydrolase family 1. The recombinant protein (Ks5A7) was overproduced in E. coli as a C-terminal 6 × His-tagged protein and purified to apparent homogeneity. The molecular mass of the purified Ks5A7 was determined to be 54 kDa by SDS-PAGE, and 160 kDa by gel filtration analysis. The enzyme was optimally active at 45°C and pH 5.0-6.5 and retained full or 1.5-2-fold enhanced activity in the presence of 0.1-0.5 M glucose. It had a low KM (78 ?M with p-nitrophenyl ?-D-glucoside; 0.36 mM with cellobiose) and high V max (91 ?mol min(-1) mg(-1) with p-nitrophenyl ?-D-glucoside; 155 ?mol min(-1) mg(-1) with cellobiose) among known glucose-tolerant BGLs and was free from substrate (0.1 M cellobiose) inhibition. The efficient use of Ks5A7 in conjunction with Trichoderma reesei cellulases in enzymatic saccharification of alkaline-treated rice straw was demonstrated by increased production of glucose.

SUBMITTER: Uchiyama T

PROVIDER: S-EPMC4468940 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Glucose-tolerant β-glucosidase retrieved from a Kusaya gravy metagenome.

Uchiyama Taku T Yaoi Katusro K Miyazaki Kentaro K

Frontiers in microbiology 20150616

β-glucosidases (BGLs) hydrolyze cello-oligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (∼mM) concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great biotechnological merit. We applied a metagenomic approach to screen for such rare glucose-tolerant BGLs. A metagenomic library was created in ...[more]

PMID: 26136726

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Project description:Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel β-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel β-glucosidase - named Lfa2 - displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing β-glucose, β-galactose, β-xylose, β-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, β-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that β-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

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.

Dataset Information

Glucose-tolerant ?-glucosidase retrieved from a Kusaya gravy metagenome.

Publications

Glucose-tolerant β-glucosidase retrieved from a Kusaya gravy metagenome.

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