Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Xia, Wei; Xu, Xinxin; Qian, Lichun; Shi, Pengjun; Bai, Yingguo; Luo, Huiying; Ma, Rui; Yao, Bin

doi:10.1186/s13068-016-0560-8

Cited by 60 publications

(62 citation statements)

References 61 publications

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“…Likewise, variant N342V of EG II from T. reesei exhibited an optimal activity at pH 5.8, corresponding to a basic shift of one pH unit compared with the wild type enzyme, and had improved catalytic efficiency (1.5-fold of k cat /K m ) for the main substrates at pH 6.2 [155,156]. Additionally, two variants (M1, M2) of β-glucosidase from T. leycettanus JCM12802 showed improved pH stability over a broader pH range (3.0-10.0) compared with the wild type (pH stability 4.5) [157].…”

Section: Engineering Cellulase For Ph Stabilitymentioning

confidence: 98%

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Contreras

Pramanik

Рожкова

et al. 2020

IJMS

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Lignocellulosic biomass is a most promising feedstock in the production of second-generation biofuels. Efficient degradation of lignocellulosic biomass requires a synergistic action of several cellulases and hemicellulases. Cellulases depolymerize cellulose, the main polymer of the lignocellulosic biomass, to its building blocks. The production of cellulase cocktails has been widely explored, however, there are still some main challenges that enzymes need to overcome in order to develop a sustainable production of bioethanol. The main challenges include low activity, product inhibition, and the need to perform fine-tuning of a cellulase cocktail for each type of biomass. Protein engineering and directed evolution are powerful technologies to improve enzyme properties such as increased activity, decreased product inhibition, increased thermal stability, improved performance in non-conventional media, and pH stability, which will lead to a production of more efficient cocktails. In this review, we focus on recent advances in cellulase cocktail production, its current challenges, protein engineering as an efficient strategy to engineer cellulases, and our view on future prospects in the generation of tailored cellulases for biofuel production.

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Section: Engineering Cellulase For Ph Stabilitymentioning

confidence: 98%

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Contreras

Pramanik

Рожкова

et al. 2020

IJMS

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“…According to their substrate preferences, β-glucosidases can be classified into three groups: cellobiases, with high substrate specificity towards cellooligosaccharides, aryl-β-glucosidases, very specific for synthetic substrates such as p-nitrophenyl-β-D-glucopyranoside (pNPG), and β-glucosidases with broad substrate specificity, that combine both activities [17]. For BGL-1, the k cat values calculated for hydrolysis of cellooligosaccharides were relatively good, but its K m values were poor when compared with those observed for BGLs from the GH3 family [5,14,15,23]. This result confirms the low affinity of BGL-1 for these substrates, similarly to other GH1 β-glucosidases [16,19,[29][30][31][32], which could limit its applicability in hydrolytic processes despite its enormous glucotolerance.…”

Section: Discussionmentioning

confidence: 99%

A glucotolerant b-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds

Méndez-Líter

Nieto-Domínguez

Toro

et al. 2020

Preprint

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Background: The interest for finding novel b-glucosidases that can improve the yields to produce second-generation (2G) biofuels is still very high. One of the most desired features for these enzymes is glucose tolerance, which enables their optimal activity under high-glucose concentrations. Besides, there is an additional focus of attention on finding novel enzymatic alternatives for glycoside synthesis, for which a mutated version of glycosidases, named glycosynthases, has gained much interest in recent years. Results: In this work, a glucotolerant β-glucosidase (BGL-1) from the ascomycete fungus Talaromyces amestolkiae has been heterologously expressed in Pichia pastoris , purified, and characterized. The enzyme showed good efficiency on p NPG ( K m =3.36 ± 0.7 mM, k cat =898.31 s -1 ), but its activity on cellooligosaccharides, the natural substrates of these enzymes, was much lower, which could limit its exploitation in lignocellulose degradation applications. Interestingly, when examining the substrate specificity of BGL-1, it showed to be more active on sophorose, the b-1,2 disaccharide of glucose, than on cellobiose. Besides, the transglycosylation profile of BGL-1 was examined, and, for expanding its synthetic capacities, it was converted into a glycosynthase. The mutant enzyme, named BGL-1-E521G, was able to use α-D-glucosyl-fluoride as donor in glycosylation reactions, and synthesized glucosylated derivatives of different p NP-sugars in a regioselective manner, as well as of some phenolic compounds of industrial interest, such as epigallocatechin gallate (EGCG). Conclusions: In this work, we report the characterization of a novel glucotolerant 1,2-β-glucosidase, which also has a considerable activity on 1,4-β-glucosyl bonds, that has been cloned in P. pastoris , produced, purified and characterized. In addition, the enzyme was converted into an efficient glycosynthase, able to transfer glucose molecules to a diversity of acceptors for obtaining compounds of interest. The remarkable capacities of BGL-1 and its glycosynthase mutant, both in hydrolysis and synthesis, suggest that it could be an interesting tool for biotechnological applications.

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“…The conformational stability and catalytic activity of enzymes are in direct relationship to their pH [59]. To determine the optimal pH value for the hydrolysis of PNPG by leukocyte GCase, we examined the effects of variable pH (4.0-5.5) at a constant substrate concentration (5mM).…”

Section: Determination Of Optimal Phmentioning

confidence: 99%

Enzyme Kinetics and Inhibition Parameters of Human Leukocyte Glucosylceramidase

Karataş

Dogan

Spahiu³

et al. 2020

Preprint

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Glucosylceramidase (GCase) is a lysosomal enzyme that catalyzes the cleavage of β-glucosidic linkage of glucocerebroside (GC) into glucose and ceramide; thereby, plays an essential function in the degradation of complex lipids and the turnover of cellular membranes.The growing list of 460 mutations in the gene coding for it—glucosylceramidase beta acid 1 (GBA1)—is reported to abolish its catalytic activity and decrease its enzyme stability, associating it with severe health conditions such as Gaucher disease (GD), Parkinson Disease and Dementia with Lewy bodies.Although the three-dimensional structure of wild type glucosylceramidase is elucidated, little is known about its features in human cells. Moreover, alternative sources of GCase that prove to be effective in the treatment of diseases with enzyme treatment therapies, impose the need for simple and cost-effective procedures to study the enzyme behaviour. This work, for the first time, shows a well established, yet simple, cost- and time-efficient protocol for the study of GCase enzyme in human leukocytes by the artificial substrate PNPG. Characterization of the enzyme in human leukocytes for activation parameters (optimal pH, Km, and Vmax) and enzyme inhibition, was done. The results indicate that the optimum pH of GCase enzyme with PNPG is 5.1. The Km and Vmax values were 12.6mM and 333 U/mg, respectively. Gluconolactone successfully inhibits GCase in a competitive manner, with a Ki value of 0.023 mM and IC 50 of 0.047 mM. Glucose inhibition was uncompetitive with a Ki of 1.94 mM and IC50 of 55.3 mM. This is the first report for the inhibitory effect of glucose, δ-gluconolactone on leukocyte GCase activity.

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Engineering a highly active thermophilic β-glucosidase to enhance its pH stability and saccharification performance

Cited by 60 publications

References 61 publications

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

Engineering Robust Cellulases for Tailored Lignocellulosic Degradation Cocktails

A glucotolerant b-glucosidase from the fungus Talaromyces amestolkiae and its conversion into a glycosynthase for glycosylation of phenolic compounds

Enzyme Kinetics and Inhibition Parameters of Human Leukocyte Glucosylceramidase

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