Enhanced Thermostability and Enzymatic Activity of cel6A Variants from Thermobifida fusca by Empirical Domain Engineering

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.

A Combined Study on Optimization, In Silico Modeling, and Genetic Modification of Large Scale Microbial Cellulase Production

“…Their research suggests that cellulose hydrolysis can be improved for larger bioethanol outputs. Ali et al [ 94 ], also found that uncovering Cel6A variations from Thermobifida fusca utilizing protein domain engineering and molecular dynamics investigations improved their enzymatic activity. Computer-based different microbial cellulase enzyme is given in Figure 2 .…”

The Effects of Carbon Source and Growth Temperature on the Fatty Acid Profiles of Thermobifida fusca

“…The readily available T. fusca genome sequence reveals that it has the capacity to express many enzymes useful for hydrolyzing biomass, including numerous cellulases, xylanases, and carbohydrate transporters for sugar uptake ( Lykidis et al, 2007 ). Many of these thermally stable enzymes have been heterologously expressed in alternative microbial hosts and analyzed for their applicability to biomass conversion ( Ghangas and Wilson, 1987 ; Ali et al, 2015 ; Klinger et al, 2015 ; Saini et al, 2015 ; Zhao et al, 2015 ; Setter-Lamed et al, 2017 ; Yan and Fong, 2018 ; Ali et al, 2020 ).…”