Lignocellulose is the principal constituent of the biomass and is the most abundant bio-renewable organic resource on earth and therefore is of significant interest to governments, researchers, and industries. Lignocellulosic biomass is composed of three biopolymers, hemicellulose, cellulose and lignin which, depending on the type of the biomass, are intermeshed and organized into complex structures to varying degrees and different compositions.
Annually, processing of raw material gives rise to copious amounts of agricultural and industrial waste. From the quantity perspective, rice straw, wheat straw, corn straw, and sugarcane bagasse are the chief available agricultural wastes. Among them, rice straw (RS) has the most abundance over the world, with approximately 600 to 900 million tons being produced yearly. However, only a small portion of this great resource is being used as animal feed, and the rest is squandered commonly by burning, which contaminates the air and causes various environmental perils.[3,4].
Naturally, complete hydrolysis of cellulose is achieved through the cooperative action of enzymes including cellobiohydrolase (EC 126.96.36.199), endo-β-1,4-glucanase (EC 188.8.131.52) and β-glucosidase (EC 184.108.40.206). Among the three types of cellulose-degrading enzymes, endo-β-1,4-glucanase is the crucial enzyme that first acts on the cellulose polymers’ amorphous sites and randomly cleaves the internal β-1,4-bonds and releases smaller fragments of varying random lengths. Due to the sequence similarities of the catalytic domains of endo-β-1,4-glucanase enzymes were classified into glycoside hydrolase (GH) families of 5, 6, 7, 8, 9, 12, 44, 45, 48, 51 and 74[7,8].
Alongside with the vast involvement and importance in bio-ethanol production technologies, endo-β-1,4-glucanases are also applicable in various other industries such as pulp and paper, textile, food and feed processing, detergents.
The bioconversion process of lignocellulosic biomass consists of three steps, namely pretreatment of biomass, enzymatic hydrolysis of the polysaccharides, and fermentation of fermentable sugars. The densely packed structure of cellulose and hemicellulose together with lignin, which essentially serves as a protection for plants, highlights the importance of pretreatment strategies to solubilize and separate one or more of these components of biomass in order to make the remaining solid matter more easily accessible for the enzymatic hydrolysis[10,11]. Several pretreatment methods have been designed and used all of which attempt to accomplish main purposes including increasing the digestibility of solid biomass to augment the sugar yield during enzymatic hydrolysis step, evading the degradation of released sugars, and minimize the risk of emergence of inhibitors which could potentially interfere with subsequent steps. It is reported that alkali pretreatment, results in more reducing sugar yield for agricultural waste in comparison to acid pretreatment.
Although, desired enzymatic attributes vastly differ for different applications, high catalytic activity, stability at extreme environmental conditions and tolerance to end-product inhibition and high concentrations of salts and ions are generally among the most favorable characteristics of industrial cellulases[14–16].
Generally, the enzymatic degradation of cellulose takes place at 40 °C to 50 °C at slow rates with incomplete hydrolysis, low sugar yield, and high probability of microbial contamination. Thermostable enzymes that are produced by thermophilic or thermotolerant microorganisms can address the limitations as mentioned earlier. Thus, thermostable cellulases with better catalytic activity at elevated temperatures offer several advantages including decreased hydrolysis time, reduced susceptibility to microbial contamination, easier recovery of volatile products such as ethanol, and a lower necessity for cooling after thermal pretreatment.
The pretreatment of biomass often takes place in the presence of acids or bases and at high temperatures. Nonetheless, the pH must be neutralized before the initiation of enzymatic hydrolysis, and the neutralization of these acids or bases generates salts. The process of removing the formed salts before further downstream steps requires tons of water and energy; hence, halotolerant and halophile enzymes that are stable and operational in the presence of salts are in great demand[9,22].
The utilization of culture-independent approaches such as metagenomics permits access to almost all genetic information embedded within an environmental sample through DNA extraction followed by either cloning or sequencing[23,24]. Ample invaluable information about composition, function, organization, and hierarchy of microbial communities in various habitats has already been collected employing sequence-based metagenomics. Any particular microbial habitat hosts a distinct microbial community with specific characteristics and capabilities aimed at accomplishing certain objectives within their environment. As an instance, the rumen microbiota possesses a vigorous hydrolyzing enzyme profile adapted to enhance the digestion and exploitation of lignocellulosic biomass, which dominates the ruminant diet[26,27]. Metagenomic screening provides the opportunity to explore the extensive biodiversity of nature and thus enabling the isolation of several novel enzymes from different environments.
Numerous studies introduced novel cellulases with unique attributes, making them suitable for particular applications. Some of the endo- β-1,4-glucanases were directly isolated from microorganisms. For instance, a new thermostable and halotolerant endoglucanase was produced from Botrytis ricini URM 5627. In another research, a thermo-halotolerant and alkali Stable GH6 endoglucanase from T. halotolerans were discovered that displays high catalytic activity in alkaline pH conditions and the presence of NaCl. Furthermore, pretreatment with high concentrations of NaCl improved the enzyme’s activity. Many studies have focused on the isolation of novel endo- β-1,4-glucanases from metagenomic sources. For example, a novel halo-ionic liquids tolerant thermoacidophilic endo- β-1,4-glucanase was isolated from saline-alkaline lake soil microbial metagenomic DNA which could be applied in the hydrolysis of acid, and ionic liquid (IL) pretreated biomass. Moreover, Song et al. isolated and characterized a new endo-β-1,4-glucanase from black-goat rumen metagenome and Narra et al. mined a novel GH12 endo-glucanase which was applied in the enzymatic degradation of alkali-treated and delignified RS. In contrast to alkaline or alkali-tolerant cellulases, which have found use in laundry detergents around the world, only a few halophilic or halotolerant cellulases have been reported[34–36].
In the current study, the sheep rumen metagenomic data was explored and screened, and several target candidates were found, one of which, PersiCel4, was cloned, expressed, purified, and characterized. PersiCel4, a novel halotolerant endo-β-1,4- glucanase, demonstrated high thermal and alkaline stability. This enzyme was then utilized to enhance the hydrolysis of pretreated RS and substantially increased the reducing sugar yield.