Dataset Information

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

ABSTRACT: ?-glucosidases catalyze the hydrolysis ?-1,4, ?-1,3 and ?-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl ?-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize a family 3 glycosyl hydrolase (GH3) ?-glucosidase (Bgl) produced by Malbranchea pulchella (MpBgl3) grown on cellobiose as the sole carbon source. Kinetic characterization revealed that the MpBgl3 was highly tolerant to glucose, which is in contrast to many Bgls that are completely inhibited by glucose. A 3D model of MpBgl3 was generated by molecular modeling and used for the evaluation of structural differences with a Bgl3 that is inhibited by glucose. Taken together, our results provide new clues to understand the glucose tolerance in GH3 ?-glucosidases.

SUBMITTER: Monteiro LMO

PROVIDER: S-EPMC7181827 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

Monteiro Lummy Maria Oliveira LMO Vici Ana Claudia AC Pinheiro Matheus Pinto MP Heinen Paulo Ricardo PR de Oliveira Arthur Henrique Cavalcante AHC Ward Richard John RJ Prade Rolf Alexander RA Buckeridge Marcos S MS Polizeli Maria de Lourdes Teixeira de Moraes MLTM

Scientific reports 20200424 1

β-glucosidases catalyze the hydrolysis β-1,4, β-1,3 and β-1,6 glucosidic linkages from non-reducing end of short chain oligosaccharides, alkyl and aryl β-D-glucosides and disaccharides. They catalyze the rate-limiting reaction in the conversion of cellobiose to glucose in the saccharification of cellulose for second-generation ethanol production, and due to this important role the search for glucose tolerant enzymes is of biochemical and biotechnological importance. In this study we characterize ...[more]

PMID: 32332833

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Project description:BACKGROUND:?-Glucosidase is an important member of the biomass-degrading enzyme system, and plays vital roles in enzymatic saccharification for biofuels production. Candidates with high activity and great stability over high temperature and varied pHs are always preferred in industrial practice. To achieve cost-effective biomass conversion, exploring natural enzymes, developing high level expression systems and engineering superior mutants are effective approaches commonly used. RESULTS:A newly identified ?-glucosidase of GH3, Bgl3A, from Talaromyces leycettanus JCM12802, was overexpressed in yeast strain Pichia pastoris GS115, yielding a crude enzyme activity of 6000 U/ml in a 3 L fermentation tank. The purified enzyme exhibited outstanding enzymatic properties, including favorable temperature and pH optima (75 °C and pH 4.5), good thermostability (maintaining stable at 60 °C), and high catalytic performance (with a specific activity and catalytic efficiency of 905 U/mg and 9096/s/mM on pNPG, respectively). However, the narrow stability of Bgl3A at pH 4.0-5.0 would limit its industrial applications. Further site-directed mutagenesis indicated the role of excessive O-glycosylation in pH liability. By removing the potential O-glycosylation sites, two mutants showed improved pH stability over a broader pH range (3.0-10.0). Besides, with better stability under pH 5.0 and 50 °C compared with wild type Bgl3A, saccharification efficiency of mutant M1 was improved substantially cooperating with cellulase Celluclast 1.5L. And mutant M1 reached approximately equivalent saccharification performance to commercial ?-glucosidase Novozyme 188 with identical ?-glucosidase activity, suggesting its great prospect in biofuels production. CONCLUSIONS:In this study, we overexpressed a novel ?-glucosidase Bgl3A with high specific activity and high catalytic efficiency in P. pastoris. We further proved the negative effect of excessive O-glycosylation on the pH stability of Bgl3A, and enhanced the pH stability by reducing the O-glycosylation. And the enhanced mutants showed much better application prospect with substantially improved saccharification efficiency on cellulosic materials.

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

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

Publications

A Highly Glucose Tolerant ß-Glucosidase from Malbranchea pulchella (MpBg3) Enables Cellulose Saccharification.

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