Insight perspectives of thermostable endoglucanases for bioethanol production: A review

Highlights

• Bioethanol from waste biomass as an attractive renewable source of energy.

• Thermostable endoglucanases have garnered relatively more attention as compare to mesozymes.

• Some molecular features are responsible for increase thermostability of endoglucanase.

• Thermostable and novel endoglucanases can improve efficiency of biodegradation process of biomass.

Abstract

Currently, production of bioenergy has received much attention because it offers a mean to reduce dependence on natural crude oil and to reduce emissions of greenhouse gases which affect our environment globally. Thermotolerant cellulolytic enzymes can hydrolyze the cellulosic polymer chiefly for the production of bioethanol as a second generation fuel and are now being used in many biotechnological fields. Among cellulases, endoglucanase enzyme has been studied and reviewed from various sources and their expression within plants is a futuristic approach that has recently come into focus. Thermostability and solvent-tolerant endoglucanases are highly valuable tools for versatile industrial processes. The structural and sequential properties responsible for endoglucanase heat stability have been scrutinized by many researchers. Here, we have attempted to identify those factors which concern the thermophilic behavior of endoglucanases both individually and synergistically along with other factors, to increase endoglucanase expression and specific activity under favorable conditions. Additionally, we look at the prospect of in-planta expression of endoglucanase as a means to produce cheap and abundant biocatalyst for various industrial and biotechnological applications.

Introduction

The utilization of fossil fuel based petroleum products to drive the ever-growing energy demand is a vexing problem for scientists in recent time and age. The exhaustive nature of fossil fuels, combined with severe air pollution problems, and greenhouse gas emissions has been a cause of great concern. Every year world energy demand is rapidly increasing. In 2012, energy consumption has increased by 1% and by this carbon dioxide (CO₂) emissions has led to an augmented by 1.4% [1]. Globally, all known and anticipated reserves of oil will not run out for more than 100 years because of excessive production and high consumption rates [2]. As the supplies of the world's petroleum based fuels dwindle and oil prices rise, the exigent need to come up with an alternative and cheap energy source continues to grow [3]. In this regard, the use of bioenergy (biofuel) has been widely accepted to be the most appropriate replacement of fossil fuels. As the impending problem of exhausting fossil fuels looms ever larger in the distance, producing cheap bioethanol industrially with the help of cellulose degrading enzymes from lignocellulosic biomass has become a sustainable and lucrative option for the global fuel market.

Firstly, the idea of converting natural plant biomass-derived carbohydrate sugars to bioethanol was proposed in the 1970s [4]. In recent time, this idea is being considered again seriously and biofuel has received considerable attention, an environmentally sound and economically viable option that is fulfilling the world's energy demands (Fig. 1) and considerable work is being reported using various biotechnology approaches. The rising demand of bioenergy and depleting fossil fuels has led to the search and bioprospecting of such thermophilic highly active cellulolytic enzymes that can be efficiently utilized in biorefineries on large scale. Many developed as well as developing states have made huge investments in infrastructure, process development and biofuel (bioethanol) production facilities [5]. In 2008, 1.8% world transport fuel has been provided by biofuel [6].

Lignocellulosic biomass is the most abundant and renewable energy source that has great potential as a substrate for bioethanol production. The enzymatic saccharification of plant biomass to monosugars (glucose) that can be fermented to ethanol has been conceived as source of future fuel and bio-based chemicals [8]. It is a safe substitute to conventional fuels with the added benefit of being readily available, would significantly reduce the environmental pollution and the health hazards associated with burning of coal based fuels. It will also improve the economic and social structure of oil importing regions and lead to more employment generation in the rural areas, where biomass feedstock is more readily available [9]. Biomass contributes between 9 to 13% of the global energy supply [10], and offers a complex mixture of lignin, hemicelluloses, celluloses, xylans and waxes that acts as a substrate for the synergistic action of cellulolytic and xylanolytic enzymes [11].

Cellulose, an abundant natural polysaccharide, is the main component of plant cell walls that composed of D-glucose units linked together to form linear chains via β-1,4-glycosidic linkages [12]. Currently, in biofuel industries sucrose- and starch-containing agricultural crops (sugarcane, corn, maize and wheat) are used as substrates for production with conventional technologies, known as first generation (1G) biofuels. However, edible raw materials are a controversial resource for biodegradation and will not be adequately sufficient to meet the raising demand of biofuel [13,14]. According to the study of Billion Ton, 76 million tons of maize is used annually for the manufacturing of 14.2 billion gallons of bioethanol and its consumption will be raised to 103 million tons till 2017 [15]. Since 2012, more than 40% of US corn crop was being used to produce corn ethanol (biofuel.org.uk/first-generation-biofuel). Bioethanol production via crop continues to be expensive, incessant strategies and endless resolve to produce cost-effective biofuel are the central interests of biofuel industries. Therefore, there is a strong necessity to look at non-edible biomass (feedstock, municipal and agricultural wastes) that can use effectively as cellulosic substrates for the production of second generation (2G) biofuels and commodity chemicals, this renewable source will provide us a sustainable and economical alternative energy in the form of bioethanol [16]. The complete process of 2G bioethanol production from waste biomass with all steps demonstrate in Fig. 2a and Fig. 2b.

The degradation of lignocellulosic biomass through the use of thermophilic microbial enzymes has become a major research area. The third largest group of enzymes are cellulases, which are extensively used in several industries such as brewing, textile, paper and pulp, detergent, food and feed processing, biofuels production and many others [17,18]. What directs the attention of many scientists towards this subject is a host of potential applications, linked to the breakdown of complex cellulosic matter and the subsequent release of sugars [19]. Heat-stable cellulases have been brought to the forefront in the biotechnological world and wide scale production in plants is being experimented. Employing plants as a means to achieve this end is not a new discipline; in fact, in recent years, plants have been used as bioreactors for the production of various biotechnological products, especially since the inception of genetic engineering [[20], [21], [22]]. They have been employed for the production of vaccines, antibodies, vitamins, biodegradable plastics, fatty acids, carbohydrates, recombinant proteins and predominantly industrially imperative biocatalysts [23,24]. Plants are an attractive option in this regard as they essentially function as cheap and abundant chemical factories. Low production cost and rapid biomass generation are the key factors that have encouraged the use of plants for the production of stable heterologous proteins [25,26].

Thermotolerant enzymes efficiently act at elevated temperatures, their robust nature and ability to withstand harsh conditions, tolerance towards organic solvents, prolonged activity and significantly low risk of contamination even after long storage make them the preferred catalytic alternative to mesozymes. The conventional methods of biomass processing require the use of high temperatures for pre-treatment followed by incubating biomass at low temperatures which cater to the optimum temperature of endoglucanases needed to breakdown the cellulosic biomass [27]. These steps are carried out in skillful bioreactors through which the temperature and pH can be controlled with ease. These dependent parameters are critically important for bioprocess as the enzymatic conversion of complex cellulosic matter to simple sugars is very sensitive to their fluctuations, which can significantly affect the yield [28]. Despite such measures, there is a strong need to make bioethanol more economical, remains one of the chief concern of biorefineries. Therefore, an obvious and reasonable approach is to look for cellulolytic enzymes that offer the possibility of working at adequately high temperature, and altogether eliminate many of the procedural steps leading up to the sugar production through cellulosic breakdown; this will not only decrease production cost but also help to make the overall process more efficient and cost-effective [29].

The fundamental property of thermostability is governed by many factors. It is essential to have an understanding of that essential features that lend heat stability to endoglucanases (Egls), which includes protein structure (folding), function, dynamics, weak interactions (inner forces between molecules), composition and the role of amino acids. In fact, various studies have revealed that multifarious features that may be working additively and enhance heat stability including increase in hydrophobicity [30], conformational compactness, increase in positively charged residues [31], selective pressure of certain amino acids [32], Gibbs free energy change of hydration [33], and sometimes a single mutation [19]. These aspects of thermostability have been concluded through comparative studies between a thermophilic and mesophilic protein. However, these may give information on a wider scale i.e., among protein families, folds and their cellular locations, whereas cannot be applied within the same families. Specific studies regarding thermostable proteins and the factors leading to this complex but coveted property remain somewhat limited and hence much is left to be accounted for. This review aims to shed light on insight thermostability of endoglucanase and its crucial role in cellulose biosaccharification process.