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An engineered GH1 ?-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

ABSTRACT: ?-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of ?-1, 4-glycosidic linkages, ?-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal ?-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 ?-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 ?-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

SUBMITTER: Santos CA

PROVIDER: S-EPMC6426972 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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An engineered GH1 β-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

Santos Clelton A CA Morais Mariana A B MAB Terrett Oliver M OM Lyczakowski Jan J JJ Zanphorlin Letícia M LM Ferreira-Filho Jaire A JA Tonoli Celisa C C CCC Murakami Mario T MT Dupree Paul P Souza Anete P AP

Scientific reports 20190320 1

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. ...[more]

PMID: 30894609

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Project description:Introductionβ-Glucosidase serves as the pivotal rate-limiting enzyme in the cellulose degradation process, facilitating the hydrolysis of cellobiose and cellooligosaccharides into glucose. However, the widespread application of numerous β-glucosidases is hindered by their limited thermostability and low glucose tolerance, particularly in elevated-temperature and high-glucose environments.MethodsThis study presents an analysis of a β-glucosidase gene belonging to the GH1 family, denoted lqbg8, which was isolated from the metagenomic repository of Hehua hot spring located in Tengchong, China. Subsequently, the gene was cloned and heterologously expressed in Escherichia coli BL21(DE3). Post expression, the recombinant β-glucosidase (LQBG8) underwent purification through a Ni affinity chromatography column, thereby enabling the in-depth exploration of its enzymatic properties.ResultsLQBG8 had an optimal temperature of 70°C and an optimum pH of 5.6. LQBG8 retained 100 and 70% of its maximum activity after 2-h incubation periods at 65°C and 70°C, respectively. Moreover, even following exposure to pH ranges of 3.0-10.0 for 24 h, LQBG8 retained approximately 80% of its initial activity. Notably, the enzymatic prowess of LQBG8 remained substantial at glucose concentrations of up to 3 M, with a retention of over 60% relative activity. The kinetic parameters of LQBG8 were characterized using cellobiose as substrate, with Km and Vmax values of 28 ± 1.9 mg/mL and 55 ± 3.2 μmol/min/mg, respectively. Furthermore, the introduction of LQBG8 (at a concentration of 0.03 mg/mL) into a conventional cellulase reaction system led to an impressive 43.7% augmentation in glucose yield from corn stover over a 24-h period. Molecular dynamics simulations offered valuable insights into LQBG8's thermophilic nature, attributing its robust stability to reduced fluctuations, conformational changes, and heightened structural rigidity in comparison to mesophilic β-glucosidases.DiscussionIn summation, its thermophilic, thermostable, and glucose-tolerant attributes, render LQBG8 ripe for potential applications across diverse domains encompassing food, feed, and the production of lignocellulosic ethanol.

Dataset Information

An engineered GH1 ?-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

Publications

An engineered GH1 β-glucosidase displays enhanced glucose tolerance and increased sugar release from lignocellulosic materials.

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