Characterization of Thermostable Cellulase from Bacillus licheniformis PANG L Isolated from the Himalayan Soil

This study aimed to isolate, purify, and characterize a potential thermophilic cellulase-producing bacterium from the Himalayan soil. Eleven thermophilic bacteria were isolated, and the strain PANG L was found to be the most potent cellulolytic producer. Morphological, physiological, biochemical, and molecular characterization identified PANG L as Bacillus licheniformis. This is the first study on the isolation of thermostable cellulase-producing Bacillus licheniformis from the Himalayan soil. This bacterium was processed for the production of cellulase enzyme. The optimum conditions for cellulase production were achieved at 45°C after 48 h of incubation at pH 6.5 in media-containing carboxymethyl cellulose (CMC) and yeast extract as carbon and nitrogen sources, respectively, in a thermo-shaker at 100 rpm. The enzyme was partially purified by 80% ammonium sulphate precipitation followed by dialysis, resulting in a 1.52-fold purification. The optimal activity of partially purified cellulase was observed at a temperature of 60°C and pH 5. The cellulase enzyme was stable within the pH ranges of 3–5 and retained 67% of activity even at 55°C. Cellulase activity was found to be enhanced in the presence of metal ions such as Cd2+, Pb2+, and Ba2+. The enzyme showed the highest activity when CMC was used as a substrate, followed by cellobiose. The Km and Vmax values of the enzyme were 1.8 mg/ml and 10.92 μg/ml/min, respectively. The cellulase enzyme obtained from Bacillus licheniformis PANG L had suitable catalytic properties for use in industrial applications.


Introduction
Cellulose is a fbrous, tough, crystalline, and linear polymer of D-glucose units linked by β-1, 4-glycosidic bonds [1,2].It is a major component of plant material and the most abundant renewable source of energy [3,4].Tis cellulose is of major economic value in developing methods for successfully treating and using cellulosic wastes as cheap carbon sources [5].Cellulases, a group of glycosyl hydrolases, can efciently hydrolyze cellulose into fermentable sugar through the synergistic action of endoglucanase, exoglucanase, and β-glucosidase [6,7].
Te Himalayas are highly diversifed regions and have multiple stress conditions such as temperature fuctuations, excessive UV exposure, nutrient scarcity, and low oxygen and air pressure [26][27][28].Te evolution of microorganisms in response to these pressures has given rise to their unique biochemical and physiological diversity with innovation and unique traits [29].Terefore, the present study was conducted to isolate and optimize the medium for potential thermostable cellulase-producing bacteria and to purify and characterize the produced enzyme according to various parameters.

Isolation of Termophilic Bacteria.
Te soil samples were collected from three diferent areas of the Solukhumbu District, Nepal, namely, Pangboche (altitude 3450 m, latitude 27 °51.426N, and longitude 86 °47.640E), Lobuche (altitude 4960 m, latitude 27 °57.269N, and longitude 86 °48.89E), and Makalu Barun National Park (altitude 3700 m 27 °39.29 N and longitude 87 °45.52 E).One gram of soil sample was suspended in 9 ml of sterile Milli-Q water and serially diluted under sterile conditions.Te diluted cultures were evenly spread on nutrient agar (NA) plates and incubated at 55 °C for 24 h.Te pure cultures on the NA medium were transferred to a freshly prepared NA slant with 20% glycerol and stored at −20 °C [30].

Assessment of Enzymatic Production.
Te pure isolates were streaked on CMC agar plates and incubated at 55 °C for 24 h [31].Te plates were stained with 0.1% congo-red solution for 15 min and washed with 1 M NaCl for destaining [32].Te bacterial isolates were also screened for other industrially important enzymes like amylase, lipase, caseinase, pectinase, and gelatinase [33][34][35][36].Te cultures were inoculated in nutrient broth (NB) at 55 °C.For enzyme production, 1 ml of the culture was inoculated in 10 ml of CMC broth and incubated overnight at 55 °C under shaking conditions at 100 rpm [37].

Morphological, Physiological, and Biochemical
Characterization of the Isolates.Te selected cellulase-producing bacterial isolate was identifed by performing Gram staining and several biochemical and carbohydrate utilization tests [38].

Amplifcation of the 16S rDNA Gene Using PCR.
Te DNA was extracted and quantifed by NanoDrop.Te 16S rDNA gene was amplifed by PCR from the genomic DNA of the strain using universal primer pair fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (5′-ACG GCTACCTTGTTACGACTT-3′). Amplifcation of DNA was carried out under the following conditions: denaturation at 94 °C for 2 min followed by 35 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 2 min, and a fnal extension at 72 °C for 10 min.Amplifed PCR products of bacterial isolates were analyzed by electrophoresis on 1% agarose gel at 80 V for 30 min, stained with ethidium bromide, and visualized in a gel documentation system [39].Te PCR products were purifed using exonuclease shrimp alkaline phosphatase (Exo-SAP) kit protocol and then sequenced by using BigDye Terminator Cycle Sequencing kit (Applied Biosystems, CA).Te sequencing results were compared using the basic local alignment search tool (BLAST) on NCBI and 16S rRNA gene sequence homology evaluation carried out using GenBank data.A phylogenetic tree was constructed using MEGA 6.0 [40].

Optimization of Culture Conditions on Cellulase Activity.
Te optimization of various physicochemical parameters of growth conditions was carried out for maximum cellulase production.Te efect of a single parameter was determined at a time by keeping the rest of the parameters constant.Te major parameters and their efects on cellulase production were determined by measuring the incubation period (24-120 h), pH (3)(4)(5)(6)(7)(8)(9)(10)(11), temperatures (30-70 °C), and various agitation speeds (static, 50, 100, 150, and 200 rpm).Various carbon sources tested included CMC, xylose, maltose, glucose, fructose, starch, sucrose, and cellobiose.Diferent types of nitrogen sources such as potassium nitrate, ammonium sulphate, tryptone, ammonium nitrate, ammonium chloride, peptone, yeast extract, and beef extract were examined for their efects on growth and enzyme production.For each step, the enzyme activity was assayed to determine the optimal yield [41].
2.6.Extraction of Crude Enzyme.Te isolated bacterial strain was cultured in CMC broth and incubated at 45 °C for 48 h under shaking conditions (100 rpm).Te culture was centrifuged at 10,000 rpm for 10 min at 4 °C, and the supernatant was used as a crude enzyme for cellulase activity assay and partial purifcation [22].
2.6.1.Cellulase Assay.Cellulase activity was determined by measuring the amount of reducing sugar liberated from CMC using the 3, 5-dinitrosalicylic acid (DNS) method [42].Te enzyme assay mixture was prepared by mixing 500 μl of crude enzyme solution with 500 μl of 1% (w/v) CMC dissolved in 0.1 M phosphate bufer at pH 7. Te mixtures were incubated at 45 °C for 15 min.Te reactions were stopped by adding 1 ml of DNS reagent.All the mixtures were heated in boiling water at 100 °C for 5 min for color development, and the optical density was measured at 540 nm.All of the cellulase assays were performed in triplicate.Te enzyme activity was determined by using a calibration curve for glucose.One unit (U) of the enzyme activity is defned as the amount of enzyme that releases 1 μmol of glucose per minute [43].

2
International Journal of Microbiology 2.7.Partial Purifcation of Cellulase.Partial purifcation of the crude enzyme was carried out by fractionation using ammonium sulphate (20-80%) followed by dialysis Te crude enzyme was precipitated with ammonium sulphate overnight at 4 °C in a magnetic stirrer and centrifuged at 10,000 rpm for 15 min at 4 °C to collect the pellets.Te pellets were resuspended in a small amount of 0.1 M phosphate bufer, pH 7, and dialyzed overnight for 24 h at 4 °C against the same sample bufer by using snakeskin-pleated dialysis tubing [22].Protein concentrations in the crude sample were estimated by using the biuret method with bovine serum albumin as a standard [44].Te heat stability of the enzyme was determined by incubating the enzyme in 0.1 M phosphate bufer and pH 7 for 60 min at temperatures ranging from 30 °C to 90 °C for a period of 1 h.Te residual activity of each sample for the hydrolysis of CMC was then calculated under assay conditions [22].Te optimum pH of the cellulase was determined by incubating the mixture of the enzyme and 1% CMC in the presence of bufers: 0.1 M acetate bufer (pH 3-5), 0.1 M phosphate bufer (pH 6-8), and 0.1 M glycine NaOH bufer (pH 9-11).Te reaction mixtures were incubated at 60 °C for 15 min.For the determination of pH stability, the enzyme was incubated in diferent bufers at diferent pH ranges (pH 3-11) for 1 h at 60 °C [22].

Efect of Incubation Time, Various Metal Ions, Diferent
Substrates, and Time Stability on Enzyme Activity.Te optimum incubation time of the enzyme was determined by incubating the mixture of the enzyme and 1% CMC in 0.1 M phosphate bufer and pH 7 at optimum temperatures (60 °C) for 15, 30, 45, 60, 75, and 90 min.Te enzyme activity at each incubation time was monitored using the DNS assay [22].Te efect of various metal ions on the enzyme activity was determined by the presence of Na + , K + , EDTA, Mn 2+ , Ca 2+ , Ba 2+ , Fe 2+ , Fe 3+ , Zn 2+ , Mg 2+ , Ni 2+ , Co 2+ , Pb 2+ , and Cd 2+ metal ions.Te concentration of each metal ion was 10 mM in 0.1 M acetate bufer pH 5 [22].
Te specifcity of the cellulase substrate was determined by testing diferent substrates, namely, CMC, flter paper, and cellobiose substrate [45].Te enzymes were kept at 25 °C, 4 °C, and −20 °C for 25 days, and the residual cellulase activities were measured at intervals of fve days [46].

Enzyme Kinetics.
Te K m and V max values were determined by plotting velocity against substrate concentration CMC (5-30 mg/ml).Te data were plotted and kinetic constants were calculated.Calculations were also performed using the Lineweaver-Burk plot [47].
2.9.Data Analysis.All the measurements were conducted in triplicate, and the values were reported as mean ± S.D. GraphPad Prism 8.4.3 and MS-Excel 2013 were used for data analysis and graphical illustrations.

Isolation and Screening for Cellulase Production.
Eleven thermophilic bacterial strains were isolated from the Himalayan soil.All the isolates produced cellulase, amylase, gelatinase, and lipase enzymes.Nine isolates produced caseinase, while pectinase was not reported in any of the isolates.Te isolate coded PANG L showed maximum cellulase activities (0.044 ± 0.004 U/ml) and was processed further (Table 1).

Characterization and Identifcation of PANG L Bacterial
Isolate.Te morphological, physiological, and biochemical test results of PANG L are shown in Tables 2-4 as well as in Figure 1.Te concentration of DNA for the PANG L was 269 μg/ml with purity (A 260 /A 280 ) value of 1.79.Te amplifed PCR product was l.5 Kb which was further purifed and sequenced (Figure 2(a)).Te 16S rDNA gene sequence of PANG L exhibited maximum homology (99%) with strain B. licheniformis.Te 16S rDNA sequence was submitted to Gene bank with the accession number OQ455938.According to the phylogenetic tree, the isolate PANG L was closely related to the B. licheniformis strain ATCC 14580 (Figure 2(b)).Based on these results, the isolate was designated as B. licheniformis strain PANG L.

Optimization of B. licheniformis PANG L Culture Conditions and Enzyme Activity.
Te optimum incubation period, pH, temperature, agitation, carbon, and nitrogen sources were determined to improve the overall growth and enzyme production (Figure 3).Te enzyme-producing ability of the isolate increased with the fermentation period up to 48 h (0.034 ± 0.002 U/ml) thereafter, it declined (Figure 3(a)).Te optimum enzyme production (0.058 ± 0.008 U/ml) was recorded at pH 6.5.A sharp decrease in the cellulase activity was observed below and above this pH (Figure 3(b)).Te maximum cellulase activity (0.083 ± 0.001 U/ml) was found to be at 45 °C (Figure 3(c)).Te optimum agitation rate for higher cellulolytic enzyme production (0.089 ± 0.003 U/ml) was observed at 100 rpm while the production was the least at 200 rpm (Figure 3(d)).CMC was found to be the most suitable carbon source, which recorded a maximum enzyme activity of (0.085 ± 0.004 U/ml) followed by cellobiose (0.072 ± 0.008 U/ml) (Figure 3(e)).Among all nitrogen sources, the maximum cellulase activity (0.103 ± 0.005 U/ml) was observed when yeast extract was used as a source of nitrogen.Potassium nitrate (0.012 ± 0.002 U/ml) was found to be the least efective nitrogen source (Figure 3(f)).
International Journal of Microbiology 3.4.Partial Purifcation of Cellulase.Ammonium sulphate precipitation of the crude enzyme was standardized, and the maximum activity was observed at 80% saturation.Terefore, 80% ammonium sulphate was used and no other concentration was applied.Te crude enzyme exhibited a specifc activity of 0.271 U/mg, whereas the ammonium sulphate precipitated and dialyzed enzyme showed a specifc activity of 0.344 U/mg with 1.26 and 0.413 U/mg with 1.52fold enhancement, respectively (Table 5).

Efect of Temperature and pH on Enzyme Activity and
Stability.Te optimum cellulase activity was observed at an incubation temperature of 60 °C but gradually declined this temperature.Regarding the thermal stability of the cellulase enzyme, it retained 67% of its activity after preincubating at 55 °C for 1 h.When the temperature was increased to 75 °C, the activity reduced by 35% (Figure 4(a)).Based on the results of the pH activity and stability in Figure 4(b), it is observed that the maximum cellulase activity was maintained at pH 5 in 0.1 M sodium acetate bufer and the activity decreased as the pH increased towards alkalinity.Te enzyme retained more than 70% of its activity over the pH range of 3-6.More than 50% of the activity of the cellulase was maintained at a broad pH range, ranging from pH 7 to 10 after 1 hr.

Efect of Incubation Time, Metal Ions, Substrates, and
Storage Stability on Enzyme Activity.Te optimum incubation time with the substrate 1% CMC in 0.1 M phosphate bufer, pH 7 was found to be 45 min.Further incubation for more than 45 min resulted in a gradual loss of the enzymatic activity (Figure 5(a)).Te activity of the enzyme increased in the presence of Cd 2+ followed by Pb 2+ and Ba 2+ , respectively, whereas the presence of manganese and calcium ions signifcantly decreased the activity of the enzyme (Figure 5(b)).
Te enzyme showed the highest activity towards CMC (100%) and moderate activity towards cellobiose (77.48%) and the least activity towards flter paper (36.32%) (Figure 5(c)).Te enzyme retained 72% of activity after storing at 25 °C for 25 days, while 98.8% of the activity was recorded for the enzyme stored at −20 °C.About 8% loss of activity was recorded at 4 °C (Figure 5(d)).6).

Discussion
Te demand for microbial cellulase enzymes is continuously increasing due to their tremendous importance in the bioenergy and bioprocessing industries.Te use of novel cellulase from various thermophiles could improve thermophilic cellulase production in the industrial process [48].
Te present study focused on the search for high cellulaseproducing thermophilic bacterial isolates from the Himalayan soil.Te studies conducted by Marchant et al. [49] and Takur et al. [50] reported thermophilic microorganisms from cold environments.Eleven isolates were able to grow at 55 °C.Each thermophilic isolate produced thermostable hydrolytic enzymes such as lipase, cellulase, amylase, caseinase, and gelatinase.Tis showed that soil-derived bacterial isolates were the source of extracellular enzymes.
Other studies have also reported hydrolytic activities in several thermophilic bacterial strains [51,52].Based on the quantitative cellulase assay, isolate PANG L exhibited higher cellulase activity among the isolated strains.Te obtained cellulase activity was lower than that of other studies conducted by Ladeira et al. [14] and Kazeem et al. [53], indicating that the PANG L strain is a moderate cellulase producer.Te morphological, physiological, biochemical, Optimization of fermentation conditions can improve cellulase production and play a signifcant role in an industrial bioprocess [56].Te fermentation process was carried out for up to 120 h.Te results revealed that maximum enzyme production was obtained at 48 h.Te slight reduction in the cellulolytic activity after 48 h might be due to either unavailability of nutrients or inhibition by toxic components present in the medium [57].Te study conducted by Shahid et al. [58] reported a similar trend in cellulase production by Bacillus sp.Temperature is a crucial factor that controls bacterial physiology and enzyme     International Journal of Microbiology production ability [59].Te optimum cellulase production was obtained at 45 °C.However, several other studies revealed that the cellulase production was achieved at 50 °C by Bacillus subtilis K-18 [18] and at 40 °C by B. subtilis [60].In the case of pH, maximum cellulase production was observed at pH 6.5.A similar result was also found for B. licheniformis MVS1 by previous researchers [61].
Agitation is another factor that plays an important role in the transfer rate of nutrients and oxygen, the increased aerobic metabolism of microbes, and cell aggregate dispersion [62].Te current study showed that the maximum enzyme production was observed at an agitation rate of 100 rpm.At increased agitation, the enzyme may get inactivated due to the shearing of the bacterial cell [63].Te choice of the cheapest substrate is of great importance for the production of enzymes.CMC was found to be the best carbon source for the maximum production of cellulase, which is similar to other studies [64,65].Tis might be due to the activation of regulatory mechanisms responsible for higher cellulase production [48].Organic nitrogen sources result in better cellulase production as compared to inorganic sources.Tis enhancement may be due to other nutrients and growth stimulators in the organic nitrogen source besides nitrogen [66].Te fndings agreed with the results of Shahid et al. [58], who reported that organic nitrogen sources were more suitable for optimizing cellulase production by B. megaterium than inorganic sources.Trough this successive selection of incubation time,  8 International Journal of Microbiology temperature, pH, agitation, carbon, and nitrogen source, a 1.7-fold increase in the cellulase activity was obtained for the strain PANG L. Te efect of temperature on the cellulase activity was determined at diferent temperatures ranging from 30 °C to 90 °C.Te maximum enzyme activity was observed at 60 °C indicating that the enzyme is moderately thermostable.Te fndings were also consistent with those obtained from Bacillus strains M-9 [67], Bacillus subtilis [68], and B. megaterium BM05 [58] lower than that for B. vallismortis RG-07 65 °C [24] and B. licheniformis JK7 70 °C [55].Te concern of thermal inactivation of the enzyme is often experienced in industrial processes [46].Hence, enzyme stability is a critical issue on an industrial scale.Te thermal stability of the cellulase indicated that the enzyme retained 67% activity up to 55 °C.Terefore, it was concluded that the enzyme was moderately thermostable and could have a promising industrial application.In the case of pH, the optimal enzyme activity was found at pH 5 representing the acidophilic nature of the enzyme.Te same trend was also obtained from Bacillus strain M-9 [67].On the contrary, Kim et al. [69] reported that Bacillus sp.HSH-910 was optimally active at alkaline pH.Te enzyme showed good stability toward acidic pH ranges of 3-6.Bischof et al. [70] reported that cellulase from B. licheniformis was more stable under acidic conditions.Te optimum incubation time with the substrate was found to be 45 min and declined thereafter.Te decrease in the cellulase activity beyond 45 min may be due to the thermal denaturation of the enzyme.
Metal ions can form complexes with proteins and other molecules related to enzymes and act as donors or acceptors of electron as structural regulators [71].Te cellulase activity was stimulated in the presence of metal ions such as Pb 2+ , Cd 2+ , and Ba 2+ .Te partial inhibition was observed in Ni 2+ and Zn 2+ .In accordance with the study conducted by Gaur and Tiwari [24], there is partial inhibition of cellulase observed in the presence of Ni 2+ and Zn 2+ .However, the activity was strongly inhibited by Mn 2+ and Ca 2+ in the study reported by Bacillus strain [64].Te enzyme exhibited a high activity toward CMC.Te substrate specifcity of CMC indicates that the enzyme might be an endo-β-1, 4-glucanase [14].Islam and Roy [41] also found the same characteristics of enzymes produced by Paenibacillus sp.Te K m and V max values were found to be 1.8 mg/ml and 10.92 μg/ml/min, indicating that the enzyme has a high afnity towards CMC due to its low K m value.Te results difer from some earlier studies in which the K m value was higher at 7.2 mg/ ml [45] and 1.923 mg/ml [24].Te important and desirable quality for industrial applications is the capacity to retain enzymes at room and/or refrigerated temperatures without signifcant loss of activity.From this study, it was observed that the enzyme was stable at room temperature for less than 10 days after which a slight decline was observed.Te enzyme was stable at −20 °C.Tis implies that the room temperature was less suitable for enzyme storage, resulting in a decrease in the enzyme activity.However, Islam et al. [72] reported that 68% of activities were retained at room temperature after 28 days for cellulase from a Bacillus sp.

Conclusions
A cellulase-producing bacterium, B. licheniformis strain PANG L, showed an optimum activity at a temperature of 60 °C and pH 5, with good stability at pH ranges of 3-5, indicating its thermo-acidic nature.Considering its stability at elevated temperatures and acidic conditions, the cellulase from B. licheniformis strain PANG L could be desirable for bioconversion processes and industrial applications.Besides cellulase, B. licheniformis strain PANG L also produced important hydrolytic enzymes for various substrates such as lipids, proteins, and starch indicating its potential for various industrial applications.

Figure 4 :
Figure 4: Efects of temperature and pH on cellulase activity and stability (a) temperature and (b) pH.
Enzyme 2.8.1.Efect of Temperature and pH on Enzyme Activity and Stability.Te optimum temperature of the enzyme was determined by incubating the mixture of the enzyme and 1% CMC in 0.1 M phosphate bufer and pH 7 for 15 min at diferent temperatures ranging from 30 °C to 90 °C.

Table 1 :
Quantitative screening of cellulase enzyme.

Table 2 :
Morphological and physiological characteristics. °C