Three-Step Puriﬁcation and Characterization of Organic Solvent-Tolerant and Alkali-Thermo-Tolerant Xylanase from Bacillus paramycoides T4 [MN370035]

: In the present study, an extracellular alkali-thermo-tolerant xylanase from Bacillus paramycoides was produced in the presence of an organic solvent. The enzyme was puriﬁed by ammonium sulphate precipitation, gel ﬁltration, and ion exchange chromatography, with an overall recovery of 25.9%. The puriﬁed enzyme hada 70 kDa molecular weight (MW) conﬁrmed by SDS-PAGE gel analysis. The maximum enzyme activity was reported at 55 ◦ C and pH 7.0. Xylanase activity and stability were improved in the presence of 30% ( v / v ) n-dodecane, iso-octane, n-decane, and cyclohexane (7 days). The enzyme activity was improved by Co 2+ , EDTA, and Triton-X-100 while vigorously repressed by Hg 2+ and Cu 2+ . The puriﬁed enzyme showed 1.473 mg/mL K m and 654.017 µ g/mL/min V max values. The distinctive assets of the isolate veriﬁed the potential application in the ﬁeld of biomass conversion into fuel and other industrial processes. Organic solvent-tolerant xylanases can be used for concurrent sacchariﬁcation and bioethanol production, the ampliﬁcation of intoxicating beverages, and the fermenting industry.


Introduction
Xylan is the most important renewable hemicellulose and is a complex polysaccharide of the plant cell wall. Because hardwoods contain 25-32% hemicelluloses, and softwood includes just 15-25% hemicelluloses, they constitute a considerable reservoir of utilizable biomass. Undoubtedly, hemicelluloses are the second most abundant renewable resource, only exceeded by cellulose. Xylan is a complex structure having β-1, 4-linked xylose residues in the backbone, to which short side-chains of O-acetyl, α-L-arabinofuranosyl, D-α-glucuronic, and phenolic acid residues are bound [1]. The structure of xylan varies,

Results
This study involves the purification and characterization of organic solvent-tolerant xylanase from alkali-thermophilic B. paramycoides T4. Heavy metal tolerance in xylanaseproducing bacteria has not been reported so far. The isolate T4 showed high tolerance for different heavy metals, suggesting that it may be used in the bioremediation of such metals from natural contaminated sites. The purified and biochemical characterized enzyme was significantly used for bio-ethanol production.

Heavy Metal Resistance Pattern
Various heavy metals under investigation had varying levels of toxicity on the isolate. Strain T4 was able to tolerate extremely high concentrations of several heavy metals, which were arranged in the following order of tolerance: chromium (3000 µg/mL), arsenic (2800 µg/mL), cesium (1500 µg/mL), and lead (950 µg/mL). It was likewise resistant to relatively high amounts of cobalt (325 µg/mL), nickel (300 µg/mL), and selenium (325 µg/mL), but sensitive to even lower mercury quantities (90 µg/mL) ( Table 1).  (***) 2600 to 2900 µg/mL concentrations were used only for As (Arsenic). This concentration was used for further examinations.

Purification of Xylanase
The ammonium sulfate precipitation method was used to concentrate the raw/crude enzyme extract initially. The results showed that 80% ammonium sulfate precipitation reported maximum activity with a 19.54 mg/mL protein content. The enzyme fraction had 904.59 U/mg of specific activity with a 68.8% recovery rate. This fraction showed a 3.6-fold purification after the process ( Table 2). Each value presented here is an average of triplicates of three independent trials. The mean standard deviation for each value is <±5.0%.
The concentrated enzyme (1 mL) was loaded onto the Q-sepharose column and almost all the xylanase activity was detected in the unbound fraction. There was no xylanase activity in the fraction, while 1.32 mg/mL of protein was estimated. The xylanase activity was detected in the fraction released by the addition of 0.5 M NaCl, and an anion-exchange chromatography of xylanase resulted in one prominent peak at the 21st fraction ( Figure 1a).
Each value presented here is an average of triplicates of three independent trials. The mean sta ard deviation for each value is ˂±5.0%.
The concentrated enzyme (1 mL) was loaded onto the Q-sepharose column and most all the xylanase activity was detected in the unbound fraction. There was no xylan activity in the fraction, while 1.32 mg/mL of protein was estimated. The xylanase acti was detected in the fraction released by the addition of 0.5 M NaCl, and an anion change chromatography of xylanase resulted in one prominent peak at the 21st frac ( Figure 1a).  Further purification of active fraction from ion exchange chromatography xylanase purification profile using Sephadex G-75 gel filtration chromatography. Sodium phosphate buffer was used to equilibrate the column (100mM, pH 7.0). The same buffer was used to load and elute the sample.
Further purification of the active fraction was performed by size-exclusion chromatography on a Sephadex G-75 column. Figure 1b represents the fractionation chart of xylanase on the Sephadex G-75 column with one protein peak that overlapped the xylanase activity. The purification technique yielded a purity factor of 13.51 and a final recovery of 38.5% of the enzyme, with a specific activity of 3321.71 U/mg (Table 2).

Electrophoretic Analysis
A single band on SDS-PAGE was the confirmation of the pure enzyme of the isolate, and its molecular weight was determined as approximately 70 kDa ( Figure 2).
Further purification of the active fraction was performed by size-exclusion chromatography on a Sephadex G-75 column. Figure 1b represents the fractionation chart of xylanase on the Sephadex G-75 column with one protein peak that overlapped the xylanase activity. The purification technique yielded a purity factor of 13.51 and a final recovery of 38.5% of the enzyme, with a specific activity of 3321.71 U/mg (Table 2).

Electrophoretic Analysis
A single band on SDS-PAGE was the confirmation of the pure enzyme of the isolate, and its molecular weight was determined as approximately 70 kDa ( Figure 2).

Effect of Temperature on Enzyme Activity and Stability
The effect of temperature on enzyme activity and stability was studied by varying the temperature range from 35 to 105 °C. The purified enzyme showed its activity in wideranging temperatures (40-95 °C) with the maximum activity at 55 °C ( Figure 3). From the result, it was clear that the enzyme activity was 150.23% at 55 °C and thereafter the activity decreased gradually with the increase in the temperature ( Figure 3). Interestingly, the enzyme retained 100 and 80% activities even at 90 and 100 °C temperatures, respectively. The effect of temperature on enzyme activity and stability was studied by varying the temperature range from 35 to 105 • C. The purified enzyme showed its activity in wideranging temperatures (40-95 • C) with the maximum activity at 55 • C ( Figure 3). From the result, it was clear that the enzyme activity was 150.23% at 55 • C and thereafter the activity decreased gradually with the increase in the temperature ( Figure 3). Interestingly, the enzyme retained 100 and 80% activities even at 90 and 100 • C temperatures, respectively.
The thermal stability profile of the enzyme showed initial retention of 100% activity in the wide temperature range (45-90 • C) throughout the incubation period (60 min). However, with a further rise in temperature, there was a gradual decline in enzyme stability (20-21%) upto 105 • C ( Figure 3). Above 105 • C, a sharp decrease in enzyme stability was observed (data not shown) ( Figure 3).

Effect of pH on Enzyme Activity and Stability
The observations for the effect of pH and buffer system on the activity suggested that the composition of buffer noticeably affected the enzyme activity. The enzyme was active in the broad range of pH 5.0-9.0 and had optimum activity at pH 7.0 (148.3%) followed by pH 7.5 (129.1%) with the sodium phosphate buffer system. The minimum activity was observed at pH 4.0 (sodium acetate, 100 mM) and pH 10.0 (Tris-HCl, 100 mM), which was only 32.0 and 35.0%, respectively, when compared to the control ( Figure 4). From pH 4.0, the increase in pH value increased the enzyme activity, which was maximum at pH 7.0; however, at the same pH of different buffer systems, the activity was different.
Catalysts 2022, 12, x FOR PEER REVIEW 6 of 20 Figure 3. Effect of temperature on enzyme activity and stability. The reaction mixture was incubated at various temperatures (35-105 °C) for enzyme activity. For enzyme stability, the enzyme was preincubated at respective temperatures for 1 h, and the reaction was carried out as per the standard test method.
The thermal stability profile of the enzyme showed initial retention of 100% activity in the wide temperature range (45-90 °C) throughout the incubation period (60 min). However, with a further rise in temperature, there was a gradual decline in enzyme stability (20-21%) upto 105 °C ( Figure 3). Above 105 °C, a sharp decrease in enzyme stability was observed (data not shown) ( Figure 3).

Effect of pH on Enzyme Activity and Stability
The observations for the effect of pH and buffer system on the activity suggested that the composition of buffer noticeably affected the enzyme activity. The enzyme was active in the broad range of pH 5.0-9.0 and had optimum activity at pH 7.0 (148.3%) followed by pH 7.5 (129.1%) with the sodium phosphate buffer system. The minimum activity was observed at pH 4.0 (sodium acetate, 100 mM) and pH 10.0 (Tris-HCl, 100 mM), which was only 32.0 and 35.0%, respectively, when compared to the control ( Figure 4). From pH 4.0, the increase in pH value increased the enzyme activity, which was maximum at pH 7.0; however, at the same pH of different buffer systems, the activity was different.  . The impact of pH on enzyme activity and stability. For enzyme activity, the reaction was measured at the appropriate pH, and for stability, the enzyme was pre-incubated for 1 h at 55 °C with buffers (100 mM, in a 1:1 ratio) of various pH (sodium acetate (pH 4.0-6.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 8.0-10.0) and then measured using a standard assay method. The enzyme assay performed at pH 7.0 (sodium phosphate buffer) and 55 °C was served as control (100%). For enzyme stability, the residual activities were compared with enzyme activity measured at pH 7 (sodium phosphate buffer) and 55 °C (100%). buffers (100 mM, in a 1:1 ratio) of various pH (sodium acetate (pH 4.0-6.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 8.0-10.0) and then measured using a standard assay method. The enzyme assay performed at pH 7.0 (sodium phosphate buffer) and 55 • C was served as control (100%). For enzyme stability, the residual activities were compared with enzyme activity measured at pH 7 (sodium phosphate buffer) and 55 • C (100%).
The enzyme exhibited considerable stability with all the buffers tested. However, maximum stability (100%) was recorded in the enzyme sample incubated with buffers having pH 6.0-9.0. The enzyme was almost unstable at pH 4.0; however, a marginal decrease in the stability was observed at pH 9.5 and 10.0 with Tris-HCl buffer ( Figure 4).

Substrate Specificity
The mechanism actions of the purified xylanase for different substrates were examined. Xylanase showed maximum activity in the presence of birchwood xylan followed by oat speltxylan and p-nitrophenyl xylopyranoside. When Avicel, carboxymethyl cellulose, cellobiose, and starch were utilized as substrates, the enzyme showed no activity (Table 3). Even when the enzyme concentration was six times higher than in the standard assay and the incubation period was 30 min rather than 10 min, purified xylanase was not active on Avicel, carboxymethyl cellulose, cellobiose, or starch (data not shown). Table 3. Substrate specificity of purified enzyme.

Kinetic Analysis
Different concentrations of birchwood xylan (0.25 to 4.0 mg/mL) were used to study the enzyme kinetics of purified enzyme. The Lineweaver-Burk plots revealed that the K m and V max for birchwood xylan were 1.473 mg/mL and 654.017 g/mL/min, respectively ( Figure 5).

Effect of Inhibitors and Surfactants on Enzyme Stability
The data suggested that the enzyme activity was induced by all the inhibitors investigated. When the purified enzyme was mixed with EDTA, PMSF, dithiothreitol (DTT), urea, and β-mercaptoethanol, it retained its activity at 145.5, 105, 123.9, 104, and 171% of the original activity at a concentration of 10 mM, respectively (Table 5). The mean and standard error values are represented by each value.

Kinetic Analysis
Different concentrations of birchwood xylan (0.25 to 4.0 mg/mL) were used to s the enzyme kinetics of purified enzyme. The Lineweaver-Burk plots revealed that th and Vmax for birchwood xylan were 1.473 mg/mL and 654.017 g/mL/min, respectively ure 5).

Effect of metal ions on activity and stability
B. paramycoidesT4 xylanase was activated by 10 mM Co 2+ , Ca 2+ , Mg 2+ , Zn 2+ , and but was inhibited by all other metal ions to a variable extent. The results indicate xylanase had the highest relative activity (284.3, 256.7, 245.1, 234.5, and 229.4%) and bility (234.7, 216.1, 213.5, 205, and 206.5%) in the presence of Co 2+ , Ca 2+ , Mg +2 , Zn +2 Fe +2 ions, respectively. Metal ions such as Ni 2+ , Mn 2+ , and Na 2+ all decreased xylanas tivity marginally (Table 4). In the presence of Cu 2+ and Hg 2+ , xylanase activity was h repressed.   Enzyme activity was measured directly at 55 • C in the presence of metal ions in the reaction mixture, and for stability, the enzyme was pre-incubated with various metal ions at 55 • C for 1 h before being tested using the standard assay method. The enzyme activity without metal was considered as a control (100%) against which residual activity (%) was calculated. The control for the metallic ions was measured against the buffer. For all of the values, the mean standard deviation is ≥5.0 percent.
From Table 5, it is clear that the enzyme was significantly stable with nonionic surfactants such as Tween-40, Tween-60, Tween-80, Triton-X-100, and detergent SDS. Conversely, at higher concentrations (1.0% v/v), these substances vaguely repressed the xylanase activity with 93.6, 95, 97.2, 115.8, and 98.4% of residual activity. Therefore, xylanase of B. paramycoides T4 was resistant with 1% SDS (95%) and could be applicable as an efficient stabilizer in detergents. The enzyme was pre-incubated for 1 h at 55 • C with various inhibitors and surfactant agents before being analyzed using a standard test method. Without incubation with surfactants and commercial detergents, the enzyme activity was assumed to be 100%. For all of the values, the mean standard deviation is ≥5.0 percent.

Effect of Organic Solvents on Xylanase Stability
The influence of several organic solvents (30%, v/v) on xylanase stability was also tested for 7 days, and the results are mentioned in Table 6. The xylanase of B. paramycoides T4 in the present study revealed significant stability with both hydrophobic (log p > 2.0) and hydrophilic (log p < 2.0) organic solvents. Most hydrophobic organic solvents tended to increase enzyme stability, whereas hydrophilic solvents tended to reduce enzyme stability ( Table 6). Other solvents, aside from benzene, propanol, and ethanol, were shown to increase xylanase activity. The xylanase activity increased to 300.9, 177.5, 279.3, 137, 250.2, 231, 239.3, 119.9, 129.8, and 140% after incubation with n-dodecane, iso-octane, n-decane, xylene, toluene, n-hexane, n-butanol, acetone, methanol, and cyclohexane, respectively. Benzene, ethanol, and propanol all lowered the xylanase activity slightly, with residual activities of 86.9%, 81.5, and 83.9%, respectively (Table 6). The enzyme was pre-incubated with various organic solvents at a concentration of 30% (v/v) at 55 • C for various periods before being analyzed using a standard assay technique. The enzyme activity without any organic solvent was considered as control (100%) against which residual activity (%) was calculated. Each value shown here is the average of three separate trials in duplicate. For all of the values, the mean standard deviation is ≥5.0%.

Analysis of Hydrolytic Products
In this experiment, xylotriose and xylotetraose were found as the main products after hydrolysis (4 h of incubation) of birchwood xylanby xylanase of B. paramycoides; nevertheless, a small amount of xylobiosewas also produced ( Figure 6). From the above results, it is clear that this xylanase is an endoxylanase because it hydrolyzed the substrate and produced xylooligosaccharides, although it is not suitable to work on the resulting oligosaccharides to form xylose.

Discussion
This study comprises the three-step purification and characterization of organic solvent-tolerant xylanase from alkali-thermophilic B. paramycoides T4. This is the first report on organic-solvent-tolerant xylanase from B. paramycoides. Furthermore, heavy metal tolerance in xylanase-producing bacteria has not been studied. The isolate T4 showed high tolerance for chromium, arsenic, lead, and cesium ( Table 1), suggesting that it may be used in the bioremediation of such metals from natural contaminated sites. Xylanase-producing organisms showing heavy metal tolerance could be applicable for the management of multi-metal-polluted sludge produced from wastewater treatment. Pharmaceutical and metal industries increase a load of antibiotics/disinfectants and heavy metals, which generate discriminating stress for the survival of microorganisms in a polluted atmosphere. Hence, in various harassed situations, microbial cells acquire resistance/tolerances by the variation in the genetic framework also by mutation or the transfer of resistant genes among the microbial cell.
The chromatography results showed that a 41.1-fold purification and 25.9% final recovery of the enzyme with 101,007.14 U/mg of specific activity was achieved during the purification process (Table 2). Sanghi et al. [29] reported that purified xylanase of Bacillus subtilis achieved a 10.5-fold purification factor with 43.05% recovery after gel filtration chromatography. From Figure 3, it is clear that the enzyme was purified up to homogene-

Discussion
This study comprises the three-step purification and characterization of organic solvent-tolerant xylanase from alkali-thermophilic B. paramycoides T4. This is the first report on organic-solvent-tolerant xylanase from B. paramycoides. Furthermore, heavy metal tolerance in xylanase-producing bacteria has not been studied. The isolate T4 showed high tolerance for chromium, arsenic, lead, and cesium ( Table 1), suggesting that it may be used in the bioremediation of such metals from natural contaminated sites. Xylanase-producing organisms showing heavy metal tolerance could be applicable for the management of multimetal-polluted sludge produced from wastewater treatment. Pharmaceutical and metal industries increase a load of antibiotics/disinfectants and heavy metals, which generate discriminating stress for the survival of microorganisms in a polluted atmosphere. Hence, in various harassed situations, microbial cells acquire resistance/tolerances by the variation in the genetic framework also by mutation or the transfer of resistant genes among the microbial cell.
The chromatography results showed that a 41.1-fold purification and 25.9% final recovery of the enzyme with 101,007.14 U/mg of specific activity was achieved during the purification process (Table 2). Sanghi et al. [29] reported that purified xylanase of Bacillus subtilis achieved a 10.5-fold purification factor with 43.05% recovery after gel filtration chromatography. From Figure 3, it is clear that the enzyme was purified up to homogeneity and had a molecular weight of approximately 70 kDa, confirmed by the presence of a single band on SDS-PAGE. Our observations are in good agreement with the study of Kamble and Jadhav [30] who also purified xylanase from an alkalophilic Cellulosimicrobium sp. MTCC10645 with a molecular weight of 78 kDa [30], but different from Bacillus pumilus xylanase (60 kDa) [31].
The xylanase of strain T4 was completely stable in the broad temperature range of 45-90 • C during 1 h of incubation, while, with a further temperature increase at every 5 • C, there was a gradual decrease in enzyme stability ranging between 20-21% and 105 • C. The enzyme retained 90, 80, and 71% activities at 95, 100, and 105 • C, respectively (Figure 4). Similarly, alkalophilic xylanase of Bacillus subtilis, B. subtilis ASH, and Caldicoprobacter algeriensis TH7C1 T showed a 100% enzyme activity and stability at 55 • C [14,29,32,33]. In another study, Kamble and Jadhav [30] also reported the xylanase from Cellulosimicrobium sp. MTCC 10645 showed optimum activity at 50 • C with a gradual decrease in enzyme activity at 80 • C. The xylanase of strain T4 is more thermostable than the xylanase studied by several other researchers. Purified xylanases from Bacillus sp. and Cellulosimicrobium cellulans CKMX1 have been reported to have an optimal temperature of 50-55 • C [4,13,34]. The activity pattern of pure enzymes is confirmed by the fact that increasing temperature increases the rate of an enzymatic reaction, but at temperatures around the optimal value, enzyme denaturation occurs, resulting in inactivation. However, some xylanases showed higher-temperature optima [35][36][37][38][39]. Its industrial application would require significant enzyme stability at higher temperatures. Hence, from the result, it is clear that the xylanase of strain T4 is thermostable and could be used for numerous industrial and biotechnological applications.
The effect of pH on enzyme activity and stability was examined by evaluating the enzyme activity at different pH values. From the result, it is clear that maximum activity resulted at pH 7.0. However, in the case of stability, it showed its best result at pH 7.0-8.0 ( Figure 5). Similar results were also found in Bacillus sp. NTU-06 [40], Bacillus sp. [41], Cellulosimicrobiumcellulans CKMX1 [4], and Streptomyces olivaceus MSU3 [16]. It is well established that purified xylanases are usually steady over a broad range of pH from 6.0 to 11.0 [14,33,39,41]. The enzymes that remained stable in alkaline environments had fewer acidic residues and more arginines [42].
The enzyme showed maximum activity in the presence of birchwood xylan followed by oat speltxylan and p-nitrophenyl xylopyranoside. The enzyme reported zero activity when Avicel, CMC, cellobiose, and starch were used as substrates (Table 3). Similar to our results, several works have also been reported where purified xylanase of Bacillus cereus, Bacillus arseniciselenatis, Trichoderma inhamatum, and Caldicoprobacteralgeriensis were active on birchwood xylan and oat spelt xylan while reporting zero activity for glycans such as starch, carboxymethyl cellulose, cellobiose, and Avicel [14,23,30,43,44].
The K m and V max for birchwood xylan were recorded as 1.473 mg/mL and 654.017 g/mL/min, respectively ( Figure 6). The K m for birchwood xylan was reported to be 2.3 mg/mL, while the V max for protein from Aspergillus versicolor was 233.1 µmol/mg/min [45]. Other similar studies, conducted by Wang et al. [40] and Boonchuay et al. [46], reported a comparatively higher value of K m (3.45 mg/mL and 37.6 uM) and lower values of V max (387.3 µmol/min/mg and 303 U/mg) from Bacillus sp. and Streptomyces spp. using birchwood xylan as a substrate. When the K m value is low, it indicates that the enzyme has a high affinity for the substrate [44].
Different inhibitors such as EDTA, PMSF, dithiothreitol (DTT), urea, and β-mercaptoethanol enhanced the enzyme activity (Table 5). Similarly, Lv et al. [48] and Hwang et al. [52] also reported that microbial community EMSD5 and Paenibacillus sp. retained full activity in the presence of EDTA, DTT, PMSF, and β-mercaptoethanol. Contrastingly, some other works reported that EDTA [38,53] and DTT [49] slightly inhibit the xylanase activity of Bacillus sp. In the presence of EDTA, the enzyme's activity is inhibited, demonstrating that xylanase contains any form of metal ion in its active site. The substantial increase in enzyme activity in the presence of thiol group-reducing chemicals such as β-mercaptoethanol can be explained by the prevention of sulfhydryl group oxidation [39,54].
The nonionic surfactants such as Tween-40, Tween-60, Tween-80, Triton-X-100, and detergent SDS stimulate the enzyme activity (Table 5). Similarly, Lv et al. [48] also reported that alkaliphilic xylanase from a microbial community EMSD5 exhibited a stimulatory effect on enzyme activity and stability in the presence of nonionic detergents such as Triton X-100 and Tween-20 and was slightly inhibited in the presence of SDS at a 0.1% concentration. Meanwhile, xylanase from alkalophilic bacteria Bacillus pumilus, Trichoderma inhamatum, and Paecilomyces variotii lost their total activity in the presence of SDS [23,31,38]. In another study, xylanase of Simplicillium obclavatum showed maximum enzyme activity in the presence of SDS while inhibiting its activity in the presence of Triton X-100 [43]. Therefore, xylanase of B. paramycoides T4 was resistant with 1% SDS (95%) and could be applicable as an efficient stabilizer in detergents.
The ability to withstand organic solvents could be a paramount property of xylanases as several industrial and biotechnological applications are carried out in the presence of solvents. For instance, ethanol-tolerant xylanases are employed in bioethanol production; solvent-tolerant xylanases are used in the bioremediation of solvent-contaminated industrial wastewaters; solvent-and surfactant-tolerant xylanases are used in the deinking of recycled paper; solvent tolerance makes it easier to selectively precipitate, recover, and use enzymes [55][56][57][58], as well as in the bioconversion of lignocellulose [59]. The xylanase under the current study revealed significant stability with hydrophobic (log p > 2.0) and hydrophilic (log p < 2.0) organic solvents. Generally, the enzyme stability was improved by various hydrophobic organic solvents, whereas the stability declined with hydrophilic organic solvents (Table 6). It was observed that the xylanase activity was increased by n-dodecane, iso-octane, n-decane, xylene, toluene, n-hexane, n-butanol, acetone, methanol, and cyclohexane except for benzene, propanol, and ethanol, which decreased the xylanase activity. The improved stability of xylanase in nonpolar solvents may be due to hydrophobic solvents' failure to extract the necessary water linked with the protein, allowing the enzyme to maintain its active conformation, which is required for the process. Similarly, Eltaweel et al. [60] also stated that the protease enzyme showed maximum stability from the hydrophobic solvent due to similar reasons discussed above. The reduced xylanase activity in hydrophilic solvents, on the other hand, could be owing to these solvents stripping the critical water layer around the enzyme, which is required for enzyme catalysis [61,62]. Alkaline cellulase of Bacillus halodurans CAS 1 showed enhanced activity by various organic solvents (25%, v/v) reported by Annamalai et al. [63]. Organic solvents may boost enzyme activity because residues of the carried-over nonpolar hydrophobic solvent provide an interface, retaining the enzyme in an open conformation and resulting in accelerated activation, according to Zaks and Klibanov [64]. As a result of our findings, it is clear that B. paramycoides T4 xylanase is exceptionally stable in the presence of a wide range of hydrophilic and hydrophobic organic solvents.
In this experiment, it is clear that this xylanase is an endoxylanase because it hydrolyzed the substrate and produced xylo-oligosaccharides, although it is not suitable for work on the resulting oligosaccharides to form xylose ( Figure 6). A similar result has been reported from endoxylanase of alkalophilic Bacillus sp. no. C-125 by high-pressure liquid chromatography (HPLC) analysis [65]. The thin-layer chromatography analysis for hydrolytic products of birchwood xylan by the xylanase of Cellulosimicrobium sp. reported that xylan was degraded to several intermediate products (xylobiose and xylotriose) without a significant accumulation of xylose [30]. Similarly, Lv et al. [48] and Amel et al. [14] also reported that xylooligosaccharides were the main hydrolytic products when xylanase of microbial community EMSD5 and Caldicoprobacter algeriensis reacted with birchwood xylan.
From the above study, it is clear that the purified xylanase from the B. paramycoides T4 showed some different and novel properties with heavy metal, surfactant, and organic solvent when compared with other Bacillus strains reported earlier [11,32,35,66]. The pure enzyme also showed high temperature and pH activity and stability [12,36,66], which could be applicable for different industrial applications.

Materials
All analytical-grade reagents and media components were purchased from Hi-Media (Mumbai, India) and Merk (Mumbai, India). Column chromatography materials and a protein ladder for electrophoresis were procured from Sigma-Aldrich Pvt. Ltd., St. Louis, MO, USA.

Microorganisms
The strains of B. paramycoides T4, isolated from the soil sample of different sites in Varanasi, were used in this study. The B. paramycoides T4 culture was maintained on xylan agar slants at 4 • C and sub-cultured monthly.

Inoculum Preparation
The mother culture was prepared by inoculating one full loop of the 24 h grown culture of B. paramycoides T4 (MN370035) from the xylan agar plate into a 50 mL xylan broth and incubated at 55 ± 1 • C overnight to achieve the active exponential phase. A suitable amount of cell suspension was used to inoculate the test flasks.

Enzyme Production
The culture was grown in a 150 mL Erlenmeyer flask containing a 50 mL basal medium containing 1% birchwood xylan and 0.05% ammonium sulfate for xylanase production. The pH of the medium was adjusted to 7.0 before sterilization. The flasks were inoculated and incubated at 55 ± 1 • C for 24-48 h. The crude enzyme was filtered and centrifuged at 10,000 rpm for 10 min at 4 • C, and an enzyme assay was carried out. Enzyme activity was measured by the Nelson-Somogyi method [67,68]. One unit of enzyme activity is defined as 1 mg of the reducing end group (glucose) released per minute at 55 ± 1 • C.

Heavy Metal Resistance Studies on Efficient Bacterial Strain T4
In this experiment, 0.1 mL of bacterial culture of 1.0 OD (A 620 nm; 1cm cuvette) was spread on nutrient agar plates, supplemented with various concentrations (µg/mL) of the various heavy metals such as Pb (0.0-1000 µg/mL), As

Extraction and Purification of Xylanase
A three-step purification process was used to purify the thermo-tolerant xylanase produced by the B. paramycoides. All the purification steps were performed at temperatures between 0 and 4 • C unless otherwise stated.

Enzyme Extraction
To achieve raw enzyme, 24 h old isolates (xylan broth containing 1.0%; xylan (Birchwood), 0.5%; ammonium sulfate) were centrifuged at 10,000 rpm for 15 min. After centrifugation, bacterial cells were removed and the supernatant was used as the raw/crude enzyme for different enzyme activities.

Ammonium Sulfate Precipitation
The culture supernatant of B. paramycoides was subjected to cooling centrifugation at 10,000 rpm for 15 min at 4 • C. The supernatant containing xylanase and other soluble proteins was treated to ammonium sulfate precipitation for partial purification, and the pellets containing cells and other debris were discarded. Saturation was achieved in the range of 30 to 80%. The ammonium sulfate was slowly added to the supernatant, which was then gently agitated for 60 min with a magnetic stirrer. The pellets were collected after 60 min by centrifugation at 12,000 rpm for 15 min at 4 • C, and the pellets of different fractions were re-suspended separately in a small amount of 100 mM phosphate buffer (2 mL, pH 7.0). By placing the re-suspended pellets in a nitrocellulose dialysis membrane with a molecular weight cut-off value of 10kDa, they were dialyzed against the phosphate buffer (pH7.0) to remove the salt. The buffer was stirred continuously for 10-12 h and the buffer was changed regularly. The dialyzed sample's xylanase activity and protein content were measured using different methods.

Ion exchange Chromatography
The dialyzed material was put into a Q-Sepharose (15 × 70 mm) column (Sigma-Aldrich, St. Louis, MO, USA) equilibrated with sodium phosphate buffer (100 mM, pH 7.0). For 2 h at 4 • C, the required enzyme fraction was allowed to bind to the matrix. The unbound fraction was collected and the enzyme activity and protein content were determined. At a flow rate of 1.0 mL/min, the bound fractions were eluted with a linear gradient of NaCl (0.1-0.5 M, 10 mL each) in the same buffer.

Gel filtration Chromatography
The active fractions were collected and dialyzed against sodium phosphate buffer (pH 7.0) and were further purified by gel-filtration chromatography. The Sephadex G-75 column (1.5 × 40 cm) was equilibrated with sodium phosphate buffer (100 mM, pH 7.0) and 1 mL of concentrated sample was applied to the column. The flow rate was adjusted to 5-6 mL/h and a fraction of 2 mL each was collected.

Enzyme Assay and Protein Estimation
Xylanase was assayed by measuring the reducing sugar released by the reaction on xylan. A xylanase assay was performed by Nelson [67] and Somogyi [68] methods. Enzyme activity was defined as the amount of enzyme that liberates 1.0 µg of glucose/min/mL. Quantitative estimation of protein content was performed by the method of Lowry et al. [69] using bovine serum albumin (BSA) as a standard and expressed as mg/mL. The protein content of individual fractions of purification steps was monitored by measuring the relative activity at 280 nm.

Polyacrylamide Gel Electrophoresis
The active fraction with the highest specific activity was electrophoresed in a 12.5% polyacrylamide gel using Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), according to the Laemmli method [70]. SDS-PAGE was used to determine the approximate molecular weight of the xylanase by comparing it to molecular mass markers such as lysozyme ( The optimum temperature of the enzyme was determined by incubating the reaction mixture in sodium phosphate buffer (100 mM; pH 7.0) at various temperatures (30-105 • C) and measuring xylanase activity. The enzyme was pre-incubated for 1.0 at various temperatures (35-105 • C) to determine the thermal stability, and the residual activity was determined under standard assay conditions [67,68]. The non-heated enzyme was considered as control (100%).

Determination of Optimum pH and Stability
The effect of pH on purified xylanase was calculated by assaying its activity in the pH range of 4.0-10.0 using xylan as a substrate. The following buffers (100 mM), viz., sodium acetate (pH 4.0-6.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 8.0-10.0) were applied for the analysis. The enzyme was diluted in de-ionized water to create appropriate enzyme units and pre-incubated with different buffers (in a 1:1 ratio) at 55 • C for 1 h to ensure pH stability [67,68]. The residual activity was measured under standard assay conditions.

Substrate Specificity
The purified xylanase substrate specificity was tested using 1% (w/v) birchwood xylan, oat spelt xylan, cellobiose, starch, carboxymethyl cellulose (CMC), p-nitrophenyl xylopyranoside, and Avicel as substrates. Substrates were prepared in sodium phosphate buffer (50 mM, pH 7.0) and the activity was assayed by following the standard method.

Kinetic Analysis (Determination of V max and K m Values)
The purified enzyme was used for kinetic analysis toward determining the K m and V max values with birchwood xylan as a substrate by following the method of Lineweaver and Burke [71]. The xylanase was incubated with different concentrations of substrate (2.5-4.0 mg/mL) and the activity was assayed by the standard assay method. The Lineweaver-Burk double reciprocal plot was used to represent the Michaelis-Menten equation. The calculation of both V max and K m was performed through nonlinear adjustment by using Microsoft Excel (MS Office) with the "Solver" add-in.

Effect of Metal Ions on Activity and Stability
The FeSO 4 , CaCl 2 , CoCl 2 , NaCl, MgCl 2 , MnCl 2 , ZnSO 4 , CuSO 4 , HgCl 2 , and NiCl 2 were used to investigate the effects of different metal ions (5 mM and 10 mM) on enzyme activity. To investigate metal ion stability, the enzyme was incubated with various metals at 55 • C for 1 h before being analyzed under normal assay conditions. The enzyme activity measured without metal ions was taken as a control (100%).

Effect of Inhibitors on Xylanase Activity
To characterize the enzyme, the effects of EDTA, β-mercaptoethanol, phenylmethylsulfonyl fluoride (PMSF), and urea as inhibitors on xylanase activity were investigated at concentrations of 5 mM and 10 mM. The purified enzyme was pre-incubated for 1 h at 55 • C with these enzyme inhibitors, and the residual activity (%) was calculated using standard assay procedures. The enzyme activity without any inhibitor was considered 100% (control).

Effect of Surfactants on Enzyme Stability
The xylanase sample was incubated with surfactants, viz., Tween-40, Tween-60, Tween-80, Triton-X-100, and SDS (0.1 and 1.0%, v/v) for 1 h at 55 • C, and then the residual activity (%) was examined under standard assay conditions. The enzyme activity without any surfactants was taken as a control (100%).

Effect of Organic Solvent on Xylanase Stability
The effect of various organic solvents having log p values ranging from 0.21 to 6.6 on enzyme stability was investigated by following the method of Ogino et al. [72]. The supernatant having maximum xylanase activity was filtered with nitrocellulose film (pore size of 0.22 µm) and mixed with different organic solvents, viz., n-dodecane, n-decane, iso-octane, xylene, n-hexane, n-butanol, cyclohexane, acetone, toluene, benzene, ethanol, methanol, and propanol at 30% (v/v) for 7 days in capped culture tubes at optimal temperature. The residual xylanase activity was measured against the control (solvent-free).

Statistical Analyses
Each experiment with the required controls was performed in triplicate and the data are presented as the mean ± standard deviation (SD). The significance of differences between means was tested by analysis of variance (ANOVA) and Duncan's multiple means tests (DMMTs) on the parametric or arc-sine square-root-transformed data using the SPSS software, where avalue of less than 0.05 was considered as significant.

Conclusions
The present study is the first report of thermo-solvent-tolerant xylanase from a thermotolerant Bacillus paramycoides T4 isolate. Increased xylanase activity in the presence of several solvents with higher hydrophobicity (log P4.5) is specific to this strain. The isolate's tolerance to a variety of solvents and heavy metals makes it suitable for use in stressful situations. The thermo-alkaline character of xylanase is abundantly demonstrated by its activity in a wide pH and temperature range of 4.0-10.0 and 30-105 • C, respectively. Because of its stability at high temperatures and in mild alkaline environments, xylanase could be used in industry.
Author Contributions: S.T., J.S.Y. and J.P. carried out the research work. P.H. and A.S. planned the manuscript. P.K.P. planned the manuscript, critically reviewed and revised the manuscript. J.Y., R.G. and R.S. planned the experiment, added considerably to the investigation and elucidation of data, and gave final approval to the version to be published. S.K.Y. and J.D. improved the English of the manuscript. F.J. critically reviewed and revised the manuscript. All authors have read and agreed to the final version of this manuscript. Data Availability Statement: NCBI (National Center for Biotechnology Information) is the name of the repository where our data were deposited, and the dataset DOI is https://www.ncbi.nlm.nih. gov/nuccore/MN370035.1 (accessed on 24 May 2022).