In-Silico Characterization of Glycosyl Hydrolase Family 1 β-Glucosidase from Trichoderma asperellum UPM1.

Mohamad Farhan Mohamad Sobri,Norhayati Ramli,Farah Diba Abu Bakar,Suraini Abd-Aziz

doi:10.3390/ijms21114035

Abstract

β-glucosidases (Bgl) are widely utilized for releasing non-reducing terminal glucosyl residues. Nevertheless, feedback inhibition by glucose end product has limited its application. A noticeable exception has been found for β-glucosidases of the glycoside hydrolase (GH) family 1, which exhibit tolerance and even stimulation by glucose. In this study, using local isolate Trichoderma asperellum UPM1, the gene encoding β-glucosidase from GH family 1, hereafter designated as TaBgl2, was isolated and characterized via in-silico analyses. A comparison of enzyme activity was subsequently made by heterologous expression in Escherichia coli BL21(DE3). The presence of N-terminal signature, cis-peptide bonds, conserved active site motifs, non-proline cis peptide bonds, substrate binding, and a lone conserved stabilizing tryptophan (W) residue confirms the identity of Trichoderma sp. GH family 1 β-glucosidase isolated. Glucose tolerance was suggested by the presence of 14 of 22 known consensus residues, along with corresponding residues L167 and P172, crucial in the retention of the active site’s narrow cavity. Retention of 40% of relative hydrolytic activity on ρ-nitrophenyl-β-D-glucopyranoside (ρNPG) in a concentration of 0.2 M glucose was comparable to that of GH family 1 β-glucosidase (Cel1A) from Trichoderma reesei. This research thus underlines the potential in the prediction of enzymatic function, and of industrial importance, glucose tolerance of family 1 β-glucosidases following relevant in-silico analyses.

Highlights

Trichoderma sp. is a genus of fungi that has been the subject of several studies, ranging from phylogeny, distribution, defense mechanism, host interaction, production and secretion of enzymes, sexual development, and responses to changes in the environment [1]
By means of nucleotide sequence analyzed using Basic Local Alignment Search Tool (BLAST) (Supplementary Table S1), isolation and sequencing of cDNA obtained was found to code for β-glucosidase of GH family 1 (GH1), hereafter designated as TaBgl2
Translation into the corresponding amino acids led to identification of an open reading frame (ORF) of 1398 bp, G-C content of 55.65%, with subsequent protein BLAST elucidating a β-glucosidase, 465 amino acids in length, weighing approximately 52 kDa

Summary

Introduction

Trichoderma sp. is a genus of fungi that has been the subject of several studies, ranging from phylogeny, distribution, defense mechanism, host interaction, production and secretion of enzymes, sexual development, and responses to changes in the environment [1]. There has been extensive interest in its capacity for cellulase production, resulting in its application in biotechnology and subsequent industrial fields, such as bioethanol production from lignocellulosic materials, textile fields, and feed production [2,3] Efforts on this front have involved Trichoderma reesei, making it a fungal model organism, of focus for both industrial and academic research teams [4]. In the process of cellulose saccharification, occurrences of substrate and production inhibition are not uncommon In substrate inhibition, this occurs on a two-domain structure found on cellobiohydrolase, of which for a given fixed enzyme load, rise in substrate concentration results in increase of time/distance necessary for cellobiohydrolases to act on the chain ends resulting from endoglucanase activity [7]. To achieve increased performance of hydrolysis, efforts have included balancing the enzyme cocktail and tailoring specific enzymes [10]

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Results

Conclusion