Researchers study efficient enzyme that can break down woody biomass to bioethanol

The reported enzyme can have extended application in food medicine.

Indian Institute of Technology Guwahati researchers have studied the efficacy of a specific bacterial endoglucanase enzyme, RfGH5_4 from Ruminococcus flavefaciens in breaking down woody biomatter into a simple sugar that can be fermented efficiently to produce bioethanol – a promising renewable fuel that can replace petroleum-based fuel systems.

A team led by Prof. Arun Goyal, Dept. of Biosciences and Bioengineering, IIT Guwahati, in collaboration with researchers from the University of Lisbon, Portugal have recently published the discovery and observations of the research in the International Journal of Biological Macromolecules. The published paper is the research work carried out by Parmeshwar Vitthal Gavande, a Doctoral student of Prof. Arun Goyal, as a part of the PhD thesis work.

The production of fuel from renewable biological sources has elicited significant scientific interest in recent years because of the problems of dwindling fossil fuel reserves and the environmental pollution associated with their generation and use. Of the many biofuels known, ethanol (or ethyl alcohol) is widely studied because of its positive impact on the environment. This intoxicating component of spirits and drinks that can also be used to fuel vehicles, is commonly produced by the fermentation of sugar and starch-containing raw materials – grapes, barley, and potato among others. However, there is interest in developing methods to extract bioethanol for fuel from agricultural and forestry residues and crops that are rich in carbohydrate polymers (lignocellulose) – the plant dry matter that constitutes the woody part of plants.

For industrial production of bioethanol as fuel, the lignocellulose extracted from plants is deconstructed by using the biological catalysts (enzymes) called cellulases and subsequently fermented. Endoglucanase is one such cellulase enzyme. The bottleneck to the conversion of lignocellulosic biomass to bioethanol is the poor efficiency of these enzymes. Furthermore, the lignocellulosic biomass contains hemicellulose along with cellulose, which cannot be broken down by many endoglucanases.

The IIT Guwahati scientists have shown the efficacy of a new type of endoglucanase called RfGH5_4 in breaking down lignocellulosic and hemicellulosic biomass for the eventual conversion to bioethanol fuel. The enzyme was derived from a bacterium called Ruminococcus flavefaciens.

The IIT Guwahati team chose Ruminococcus flavefaciens because this bacterium is found in the gut of cows and other cud-chewing animals which have faced cellulosic pressure for millions of years. The particular gene encoding the cellulase enzyme, RfGH5_4 was fished-out from R. flavefaciens. The researchers have thus developed this efficient machinery of RfGH5_4 to break down cellulose and cellulosic structures into simple sugars.  The bacterium harbours a cohort of at least 14 different multimodular enzymes that can break down cellulose, one of which is RfGH5_4.

Explaining the research work, Prof. Arun Goyal, Department of Biosciences and Bioengineering, IIT Guwahati, said, “We characterized Endoglucanase, RfGH5_4 and found that it hydrolysed carboxymethyl cellulose (a lab-scale analog of cellulose) as well as normal amorphous cellulose with greater catalytic efficiency. Our studies also showed that this enzyme acted on lignocellulosic substrates from different agricultural residues such as cotton stalk, sorghum stalk, sugarcane bagasse, etc. and had good affinity for hemicellulosic substrates as well which include β-glucan, lichenan, xyloglucan, konjac glucomannan, xylan and carob galactomannan”.

The IITG team’s earlier work on cloning, expression and biochemical characterization of RfGH5_4 revealed that this particular endoglucanase is multifunctional and catalytically efficient.  With this knowledge, they characterized the structure of this enzyme, its reaction mechanism and the structural basis of its multi-functionality. Adding to the characteristics of RfGH5_4 they further emphasized that its multi-functionality makes RfGH5_4 stand out from the plethora of other cellulases that are present in nature and available commercially.”.

Elucidating the structural basis, Parmeshwar Gavande, PhD Research Scholar and the First author, elaborated, “The structure of RfGH5_4 was deciphered using extensive molecular dynamics and computational approaches at Param-Ishan Supercomputer facility of IITG. RfGH5_4 was found to contain some highly flexible loops in its core structure making room for different carbohydrate polymers during the reaction, thus imparting the multifunctionality to RfGH5_4”

“Agricultural residual biomasses are wasted or burned causing various environmental hazards including global warming and climate change. Their deconstruction by RfGH5_4 might extend its usage in food medicine as well”, Prof. Goyal foresees employing RfGH5_4 to serve humanity. The presented research constructively tries to lend a helping hand in addressing the Sustainable Development Goals 2030 (SDGs) of the United Nations”, Prof. Goyal further added.

RfGH5_4 is sufficiently active at ambient temperatures. Therefore, it might be suitable for the Simultaneous Saccharification and Fermentation (SSF) of lignocellulosic biomass, a process greatly admired in the industry.

The research has shown that multifunctional RfGH5_4 cellulase might be used for the efficient and cost-effective production of lignocellulosic bioethanol on an industrial scale. Thus, its ability to break down cellulose and hemicellulose makes it possibly useful in various other applications such as the textile, food and pulp industry, synthesis of prebiotics, and pharmaceuticals, among others.

The findings of this research work were also recently presented by Gavande at the International Conference of Biotechnology for Better Tomorrow (ICBBT-2022) at Bali, Indonesia, and received the Best Oral Presentation Award for it.

The reported enzyme can have extended application