A novel high performance metagenome-derived alkali-thermostable endo-β-1,4-glucanase for lignocellulosic biomass hydrolysis in the harsh conditions

Background: Lignocellulosic biomass, is a great resource for the production of bio-energy and bio-based material since it is largely abundant, inexpensive and renewable. The requirement of new energy sources has led to a wide search for novel effective enzymes to improve the exploitation of lignocellulose, among which the importance of thermostable and halotolerant cellulase enzymes with high pH performance is signi�cant. Results: The primary aim of this study was to discover a novel alkali-thermostable endo-β-1,4-glucanase from the sheep rumen metagenome. Using a multi-step in-silico analysis, primary candidates with desired properties were found and subjected to cloning, expression, and puri�cation followed by functional and structural characterization. The enzymes' kinetic parameters, including V max , Km, and speci�c activity, were calculated. The PersiCel4 demonstrated its optimum activity at pH 8.5 and a temperature of 85°C and was able to retain more than 70% of its activity after 150 hours of storage at 85°C. Furthermore, this enzyme was able to maintain its catalytic activity in the presence of different concentrations of NaCl, MgCl 2 , CaCl 2 , and MnCl 2 . Our results showed that treatment with MnCl 2 could enhance the enzyme’s activity by 89%. PersiCel4 was ultimately used for enzymatic hydrolysis of autoclave pretreated rice straw, the most abundant agricultural waste with rich cellulose content. In autoclave treated rice straw, enzymatic hydrolysis with the PersiCel4 increased the release of reducing sugar up to 260% after 72 hours in the harsh condition ( T= 85°C, pH = 8.5). Conclusion


Background
Lignocellulose is the principal constituent of the biomass and is the most abundant bio-renewable organic resource on earth and therefore is of signi cant 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 [1].
Annually, processing of raw material gives rise to copious amounts of agricultural and industrial waste.
Alongside with the vast involvement and importance in bio-ethanol production technologies, endo-β-1,4glucanases are also applicable in various other industries such as pulp and paper, textile, food and feed processing, detergents [9].
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 [12].It is reported that alkali pretreatment, results in more reducing sugar yield for agricultural waste in comparison to acid pretreatment [13].
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][15][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 [17], reduced susceptibility to microbial contamination [18], easier recovery of volatile products such as ethanol [19], and a lower necessity for cooling after thermal pretreatment [20].
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 [21].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 [25].Any particular microbial habitat hosts a distinct microbial community with speci c characteristics and capabilities aimed at accomplishing certain objectives within their environment.As an instance, the rumen microbiota possesses a vigorous hydrolyzing enzyme pro le 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 [28].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 [29].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 [30].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-1,4glucanase 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 [31].Moreover, Song et al. isolated and characterized a new endo-β-1,4-glucanase from black-goat rumen metagenome [32] and Narra et al. mined a novel GH12 endo-glucanase which was applied in the enzymatic degradation of alkali-treated and deligni ed RS [33].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][35][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, puri ed, 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.

Results And Discussion
As the necessity for new alternative energy sources grows and given the amplitude of agricultural waste which is being produced and squandered every year, enzymatic hydrolysis of these feedstocks gains increasing attention and importance.Bio-conversion-based industries demand hydrolytic enzymes with high catalytic e ciency and tolerance to extreme conditions such as pH, temperature, and presence of different salts.Endo-β-1,4-glucanase is one of the major enzymes involved in the degradation process of cellulose.Moreover, ruminants' rumen enzymatic diversity is known to be rich and capable of degrading recalcitrant lignocellulosic biomass, the primary constituent of most ruminants' diet.Hence, sheep rumen metagenome was screened with the focus to nd halotolerant endo-β-1,4-glucanases which are particularly stable at alkaline pH and elevated temperatures.This enzyme could improve the sugar yield of the enzymatic hydrolysis phase.

Prediction of thermostable endo-glucanase sequences
One of the contigs assembled from the sheep rumen metagenome, named PersiCel4, passed all lters applied for identifying thermostable endo-glucanase.The PersiCel4 is an enzyme with amino acid length of 339.The blast results show that it is most similar to an endo-glucanase enzyme from GH5 family with Endohydrolysis of (1-4)-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans catalytic activity and UniProt accession number B7UAM4 with E-Value of 3e-112 (Table 1).
Based on the CDD results, the PersiCel4 best matches the Pssm-ID 333879 with Bit Score 212.21 and Evalue 2.57e-67 (Fig. 1A).
Phyre2 suggests that the most similar structure to the PersiCel4 with 100% con dence and 52% identity belongs to an endo-1,4-beta-glucanase from Bacillus subtilis 168 with 3PZV PDB code (Fig. 1B).

Expression and puri cation for recombinant endo-β-1,4glucanase
From the metagenomic DNA of sheep rumen with degenerate primers and over-expressed under the control of the T7 promoter of pET-28a vector in E. coli BL21 (DE3), the sequence of PersiCel4 fragment was ampli ed.The puri cation step was checked by SDS-PAGE and using Ni-NTA Fast Start Kit for puri ed the N-terminus His-tagged protein.The single PersiCel4 band was visible with a molecular weight corresponding to the calculated 38 kDa (Fig. 1C).

Properties of puri ed PersiCel4
The kinetic parameters (K m and V max ), of PersiCel4, was evaluated.The enzyme obeys based on a Lineweaver-Burk map in Michaelis-Menten kinetics concerning CMC as substrate, K m and V max values of PersiCel4 were 0.58 µmol ml − 1 , 1.95 µmol min − 1 mg − 1 .The calculated speci c activities for puri ed PersiCel4 was 22.425 Umg − 1 .
Optimum pH was determined in room temperature.Figure 2A shows that the optimal pH value for PersiCel4 was 8.5, and it preserved more than 60% of its maximum activity in the pH range between 5 up to 10 (Fig. 2A).By xing the pH for enzyme, the maximum activity was observed at 85 °C in the range of temperature between 30-90 °C, and the activity of the enzyme was decreased just 8% of its maximum value at 95 °C (Fig. 2B).However, PersiCel4 was also highly stable when stored for prolonged periods.
The PersiCel4 retained 70% of its relative activity after being stored for 168 hours at 85 °.The enzyme activity was diminished drastically after 288 hours of storage (Fig. 2C).
Because of slow pace and incomplete hydrolysis of cellulose in moderate temperatures, the susceptibility of the microbial contamination on one hand and the easier recovery of ethanol at high temperatures and decreased importance of cooling after thermal pretreatment, on the other hand, thermostable enzymes nd great importance and value [17,20].The competence of cellulases to operate in extreme pH conditions is another desirable attribute.Alkaline pretreatments on agricultural wastes such as RS have shown better sugar yield results compared to acid pretreatments [13] and alkali-stable cellulases have been reported to be suitable potential enzymes for lignocellulosic sacchari cation at such conditions [37].As presented in Table 2, PersiCel4 depicted stability and functionality at more extreme pH and thermal conditions in comparison to some other known endo-β-1,4-glucanase representatives.PersiCel4's prolonged storage stability in addition to its optimal activity at 85 °C and pH 8.5, which are considerably higher than other endo-glucanase representatives, clearly distinguish this enzyme from many other reported endo-glucanases for industrial waste purposes.

Effects of salts on enzyme activity
As described before, since several pretreatment methods take place at either acidic or alkaline conditions and due to formation of various salts at the pH neutralization step, an important challenge is the removal of such salts that consumes an abundance of water and energy [21].Given this fact, cellulolytic-enzymes that can tolerate the presence of different salts without reduction in their catalytic ability nd attraction [9,22].
The PersiCel4 activity was investigated in the presence of different concentrations of NaCl (1 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM), MgCl

Effect of different pretreatment and enzymatic hydrolysis of rice straw
Lignocellulosic materials constitute economically renewable raw materials and are available in large quantities [38].Several researchers have addressed the use of agricultural waste in the feasibility, sustainability, and economics of biofuels production [39][40][41][42][43].
One of the most abundant residues of lignocellulose in the world is RS [2,44,45].Due to the complex structure of lignin and hemicelluloses, the conversion of RS to reducing sugar is a very complicated process [46].Generally, by pretreatment of brous biomass through mechanical, physical, thermal, chemical or enzymatic pathways, it results in the conversion of complex cellulose and hemicellulose into reducing sugars.In the pretreatment process, cellulose and hemicellulose can be removed, and cellulose crystallization reduced, and porosity of the materials increased use recent reference.An effective pretreatment method is the one which maximizes sugar productivity during the enzymatic hydrolysis step while preventing the degradation or loss of carbohydrates [12].The effects of different preparations before enzymatic hydrolysis produced the highest sugar production.
Figure 3 shows the percentage of reduced sugar in each pretreatment obtained from untreated and different treated RS.The highest yield of reducing sugar observed when the RS (2%w/v) in sodium phosphate buffer (50 mM, pH 7) had done with an incubator at 100˚C, 30 min by thermal pretreatment.
In this study, it observes that autoclave pretreatment RS improves enzymatic hydrolysis resulting in a higher yield of reducing sugar than untreated RS.This may be due to the de ciency caused by the swelling of RS particles and an increase in the inner surface, which in turn has led to access of more enzymes to the internal structure, and thereby, increasing sugar yields [47,48].
In the next step, the enzymatic hydrolysis of thermal treated RS was evaluated using screening of the reducing sugar concentrations against time, at 85˚C (pH 8.5) (Fig. 4).
The results showed that PersiCel4 signi cantly enhanced the sacchari cation yield in 96 hours, speci cally after 72 hours, which con rmed the boosting effect PersiCel4.In autoclave treated RS, the comparison between enzymatic hydrolysis with the PersiCel4 and without it, revealed a 260% increase in the released reducing sugar after 72 hours.In this study, it observes that the enzymatic hydrolysis of thermal pretreated RS was the most e cient method of converting the RS into reducing sugars.This study highlights the competence of the combined implementation of metagenomic screening and insilico analysis in order to e ciently discover and isolate particular enzymes of interest.As described, PersiCel4 depicted various characteristics that are essential for enhanced hydrolysis of cellulose.

Conclusions
Waste management and energy crisis are among the most important issues that the world faces today.Such concerns have inspired the extensive search for capable novel enzymes to improve the recalcitrant lignocellulosic biomass conversion, such as agricultural wastes.
In this study, the sheep rumen metagenome was explored using in-silico analysis to nd a novel alkalithermostable halotolerant endo-β-1,4-glucanase. Ultimately, PersiCel4 was identi ed and isolated and characterized by multiple experiments, all of which veri ed the computationally predicted properties.PersiCel4 was successfully utilized for the hydrolysis of autoclave pretreated RS in extreme temperature and pH, which is the most plentiful agricultural waste.Regarding several proposed distinctive characteristics of PersiCel4, it can potentially nd commercial applications the harsh conditions of the industrial bioconversion process.

Methods
Computational prediction of thermostable endo-β-1,4glucanase enzyme sequences The primary and preprocessing operations, including quality control and short read assembly, carried out on sheep rumen metagenomics data using FastQC and MEGAHIT assembler respectively.After that, the assembled contigs were explored for putative thermostable endo-glucanase sequences by the stages described below.In the rst step, MetaGeneMark was employed for predicting potential microbial genes.
The predicted endo-glucanase enzymes were separated from assembled contigs for next step analysis.Six thermostable endo-glucanase sequences from different GH families were identi ed from previous works and their sequences were downloaded from UniProt (Table 1).Using standalone NCBI BLAST, the mined enzymes from metagenome were aligned against the list of enzymes mined from literature.The most similar predicted metagenomics endo-glucanases to the known enzymes were determined based on pairwise blast with appropriate E-value.
The resulting PCR products were detected on agarose gel 1.5% (w/v) and for puri ed using the gel extraction kit (BioRon, Germany).Puri ed DNA fragments were cloned and digested into the pET28a.The resulting plasmids were then transformed into the E. coli BL21 (DE3) and correct insertion was con rmed by sequencing.In the Luria-Bertoni (LB) medium, the recombinant strain pET28an was cultivated at the temperature of 37 ˚C.Adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a nal concentration of 0.4 mM for 20 h at 20 °C, expression of enzymes were induced.By utilizing Ni-NTA Fast Start Kit (Qiagen, Hilden, Germany), N-terminal Histidine-tagged recombinant protein was puri ed and evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
The candidate enzyme produced from successful cloning, expression, and puri cation were named PersiCel4.This endo-β-1,4-glucanase was subjected to further biophysical experiments.Nucleotide sequence of PersiCel4 was submitted to Genbank Database with accession number Mn016942.Protein concentrations were determined through the Bradford method using the bovine serum albumin as the standard.By measuring the optical density (OD) of chromatography eluent at 280 nm, the protein concentration was estimated.

Spectroscopy studies
Synergy™ HTX Multi-Mode Microplate Reader was used for UV-vis spectrophotometry.For matured protein concentration using the Bradford method, and bovine serum albumin (BSA) as the protein standard was used.The CMC substrate and reducing sugars amount was used for enzyme activity measurement by DNS method at 540 nm.

Measurement of and kinetic parameters
The DNS method was used for determination of cellulase activity.After the occurrence of enzymatic hydrolysis of cellulose, the resulting reducing sugars can reduce the nitro of 3,5-dinitrosalicylic acid (DNS) into amino, forming a reddish-brown color due to the presence of amino compounds [53].The intensity of brown color and the quantity of reducing sugar present a positive correlation in a particular concentration range which is diagnosable through the spectrophotometric method.
The cellulose substrate carboxymethyl cellulose (CMC) was used in the cellulose assay [54].The intensity of brown color and the quantity of reducing sugar present a positive correlation in a particular concentration range which is diagnosable through the spectrophotometric method.20 µL enzyme solution was added to 60 µL of 0.5% solubilized CMC in a test tube.Then 1.5 mL, 3,5-dinitrosalicylic acid (DNS) reagent was added and incubated in the water bath at 80˚C for 5 min.After that the optical absorbance was determined at 540 nm.
In the assay conditions described by Kamble RD, et al, the amount of enzyme that can 1 µmol of reduced sugars released per minute per millilitre is a unit of cellulose activity [55].Ultimately, enzymes were incubated with CMC concentrations ranging from 0 to 10 mg/ml in 10 mM phosphate buffer at pH 8.
Using Michaelis-Menten plots at room temperature, K m and V max and Michaelis constant were determined.

Determination of optimum pH and temperature and storage
To determine the optimum pH of enzyme activity, 10 mM phosphate buffer was prepared at different pH (4 to 11), and after added enzyme was incubated with a substrate for 20 min at room temperature.The DNS was used to measure activity [53].To determine the optimum temperature of enzyme activity, enzyme solution in 10 mM phosphate buffer (pH 8.5) with substrate was incubated in the different temperature (30-95 °C) for 20 min and DNS was used to measure activity.
For reporting PersiCel4 activity, relative activity was considered as the percentage of highest activity.The methods for determining storage stability was used by incubation of the puri ed enzymes for 12 days in kit elution buffer at 85 °C and the enzymes' activities were examined in 24 h time intervals.

Effects of salts on enzyme activity
The PersiCel4 was pre-incubated at various concentrations of NaCl (1, 2.5, 5, 7.5, 10 mM) at 40˚C for 30 min without the substrate and the activity was assayed according to the standard conditions.The activity of the enzyme not reacted with any NaCl concentrations was assessed as the control (%100).
To evaluate the effect of ionic liquid on enzyme activity the enzyme was pre-incubated with 1, 5 and 10 mM of MgCl 2 , CaCl 2 and MnCl 2 at 40 °C for 30 min before adding the substrate.After pre-incubation, remaining activity was measured by the standard method described above and the effect of different ionic liquid concentration was tested.The enzyme activity without any chemicals was considered to be 100% activity and compared with the other values.

Rice straw (RSH)
Rice straw (RS) collected from a local farm and washed with water and then dried at 70 ˚C in a hot air oven.By mixer grinder, size of dried RS has been reduced.The RS with suitable particle size (2-3 mm) pretreated by ve different methods (alkaline, thermal, autoclave, microwave).

Alkaline pretreatments
In the RS (2%w/v), the pH was set at 12 by the addition of 2M NaOH, then mixed with magnetic stirrer for 30 min [56].
The microwave pretreatments   Comparative analysis of reducing sugar release of RS after pretreatment by four different methods.

Figures
Figures

Figure 1 A
Figure 1

Table 3 ,
besides the depicted tolerance to presence of NaCl, the presence of MgCl 2 , CaCl 2 , and MnCl 2 resulted in a signi cant increase in PersiCel4's activity.This feature is yet to be added to other mentioned notable characteristics of PersiCel4 to nominate it for biofuel industries.

Table 1
Six endo-glucanase sequences with thermostability properties obtained from literature mining