A Novel High Glucose-Tolerant β-Glucosidase: Targeted Computational Approach for Metagenomic Screening.

Shohreh Ariaeenejad,Mahsa Mohammadi,Atefeh Ghasemi Pirbalooti,Mehdi Mirzaei,Kaveh Kavousi,Safura Nooshi-Nedamani,Ghasem Hosseini Salekdeh,SeyedSoheil Mirghaderi,Mahdie Rahban

doi:10.3389/fbioe.2020.00813

Abstract

The rate-limiting component of cellulase for efficient degradation of lignocellulosic biomass through the enzymatic route depends on glucosidase’s sensitivity to the end product (glucose). Therefore, there is still a keen interest in finding glucose-tolerant β-glucosidase (BGL) that is active at high glucose concentrations. The main objective of this study was to identify, isolate, and characterize novel highly glucose-tolerant and halotolerant β-glucosidase gene (PersiBGL1) from the mixed genome DNA of sheep rumen metagenome as a suitable environment for efficient cellulase by computationally guided experiments instead of costly functional screening. At first, an in silico screening approach was utilized to find primary candidate enzymes with superior properties. The structure-dependent mechanism of glucose tolerance was investigated for candidate enzymes. Among the computationally selected candidates, PersiBGL1 was cloned, isolated, and structurally characterized, which achieved very high activity in relatively high temperatures and alkaline pH and was successfully used for the hydrolysis of cellobiose. This enzyme exhibits a very high glucose tolerance, with the highest inhibition constant Ki (8.8 M) among BGLs reported so far and retained 75% of its initial activity in the presence of 10 M glucose. Furthermore, a group of multivalent metal, including Mg2+, Mn2+, and Ca2+, as a cofactor, could improve the catalytic efficiency of PersiBGL1. Our results demonstrated the power of computational selected candidates to discover novel glucose tolerance BGL, effective for the bioconversion of lignocellulosic biomass.

Highlights

The term cellulosic biomass, known as lignocellulose, refers to any constituent of plants or plant-derived biodegradable matter, namely, wood, grasses, and other feedstock across the world
Assembling and screening raw metagenome data, predicting potential BGLs, identifying the BGL enzymes having all the desired features, selecting the most appropriate candidates, 3D modeling of the selected enzymes, and investigating the molecular mechanisms behind these important features, all done before starting the laboratory phase, result in highly targeted and highly successful laboratory tasks (Figure 1)
It was notably insensitive to glucose as a competitive inhibitor, and even its catalytic activity soared by the addition of some ionic salt

Summary

Introduction

The term cellulosic biomass, known as lignocellulose, refers to any constituent of plants or plant-derived biodegradable matter, namely, wood, grasses, and other feedstock across the world. Lignocellulose is mainly composed of three structural polymers (cellulose, hemicellulose, and lignin). The use of fossil fuel and the problems associated with it, such as contributing to environmental pollution and the fact that nature cannot replenish fossil energy sources, have forced us to forage for an alternative and renewable source of energy (Singhania et al, 2017). In this respect, bioethanol production is receiving abundant consideration as a promising candidate for biofuel, and substances that participate in biofuel production are of great economic importance (Zhang et al, 2012)

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