Enhanced Thermostability and Enzymatic Activity of Cel6A Variants from Thermobifida fusca by Empirical Domain Engineering (Short Title: Domain Engineering of Cel6A).

Imran Ali,Matheus Froeyen,Rashid Ahmed,Muhammad Tariq,Rehana Asghar,Muhammad Waheed Akhtar,Hina Khalid,Huda Ahmed Alghamdi,Fatima Tanveer,Muhammad Usman Mirza,Hafiz Muzzammel Rehman

doi:10.3390/biology9080214

Abstract

Cellulases are a set of lignocellulolytic enzymes, capable of producing eco-friendly low-cost renewable bioethanol. However, low stability and hydrolytic activity limit their wide-scale applicability at the industrial scale. In this work, we report the domain engineering of endoglucanase (cel6A) of Thermobifida fusca to improve their catalytic activity and thermal stability. Later, enzymatic activity and thermostability of the most efficient variant named as cel6A.CBC was analyzed by molecular dynamics simulations. This variant demonstrated profound activity against soluble and insoluble cellulosic substrates like filter paper, alkali-treated bagasse, regenerated amorphous cellulose (RAC), and bacterial microcrystalline cellulose. The variant cel6A.CBC showed the highest catalysis of carboxymethyl cellulose (CMC) and other related insoluble substrates at a pH of 6.0 and a temperature of 60 °C. Furthermore, a sound rationale was observed between experimental findings and molecular modeling of cel6A.CBC which revealed thermostability of cel6A.CBC at 26.85, 60.85, and 74.85 °C as well as structural flexibility at 126.85 °C. Therefore, a thermostable derivative of cel6A engineered in the present work has enhanced biological performance and can be a useful construct for the mass production of bioethanol from plant biomass.

Highlights

Cellulose is the most abundant plant biomass found on earth and is extremely important for mankind due to its diverse applications
The cel6A construct with higher thermostability was investigated structurally through long-run molecular dynamics simulations to get an insight of the thermostable behavior of enzyme at different comparable temperatures
Enzymatic activities of native cel6A were almost similar to that of cel6A.BC construct on soluble and insoluble substrates, which we reported previously [49,50], Similar results were reported in a study in which an extra catalytic domain was added to the CelA of C. thermocellum [65]

Summary

Introduction

Cellulose is the most abundant plant biomass found on earth and is extremely important for mankind due to its diverse applications. The evolving use of renewable non-food cellulosic plant biomass is a very attractive option for biofuel production. Biological depolymerization of cellulose biomass is accomplished by various types of cellulases, endoglucanases, exoglucanases, and β-glucosidases. Endoglucanase cleaves cellulose polymer into oligopolymers of varying lengths, while exoglucanases attack reducing and/or non-reducing ends of cellulose yielding glucose or cellobioses, β-glucosidases convert cellobioses into fermentable sugar monomers. Despite the tremendous potential of cellulases in the conversion of plant biomass into useable bioethanol, their widespread application is restricted by their high cost and low efficacy, especially under harsh industrial conditions [1,2,3,4,5]. Enzymes with high thermostability are advantageous in saccharification processes owing to their better penetrating ability into lignocellulosic biomass for disorganization [6]

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Conclusion