Biochemical and Thermodynamic Studies on a Novel Thermotolerant GH10 Xylanase from Bacillus safensis

Xylanases have a broad range of applications in agro-industrial processes. In this study, we report on the discovery and characterization of a new thermotolerant GH10 xylanase from Bacillus safensis, designated as BsXyn10. The xylanase gene (bsxyn10) was cloned from Bacillus safensis and expressed in Escherichia coli. The reduced molecular mass of BsXyn10 was 48 kDa upon SDS-PAGE. Bsxyn10 was optimally active at pH 7.0 and 60 °C, stable over a broad range of pH (5.0–8.0), and also revealed tolerance toward different modulators (metal cations, EDTA). The enzyme was active toward various xylans with no activity on the glucose-based polysaccharides. KM, vmax, and kcat for oat spelt xylan hydrolysis were found to be 1.96 g·L−1, 58.6 μmole·min−1·(mg protein)−1, and 49 s−1, respectively. Thermodynamic parameters for oat spelt xylan hydrolysis at 60 °C were ΔS* = −61.9 J·mol−1·K−1, ΔH* = 37.0 kJ·mol−1 and ΔG* = 57.6 kJ·mol−1. BsXyn10 retained high levels of activity at temperatures up to 60 °C. The thermodynamic parameters (ΔH*D, ΔG*D, ΔS*D) for the thermal deactivation of BsXyn10 at a temperature range of 40–80 °C were: 192.5 ≤ ΔH*D ≤ 192.8 kJ·mol−1, 262.1 ≤ ΔS*D ≤ 265.8 J·mol−1·K−1, and 99.9 ≤ ΔG*D ≤ 109.6 kJ·mol−1. The BsXyn10-treated oat spelt xylan manifested the catalytic release of xylooligosaccharides of 2–6 DP, suggesting that BsXyn10 represents a promising candidate biocatalyst appropriate for several biotechnological applications.


Introduction
Hemicelluloses, the second most abundant biopolymer group in nature after cellulose, are mainly composed of xylan, an amorphous structural polysaccharide that encompasses a xylose backbone, decorated with a source-dependent variety of side-chain substitutions [1]. The exact structure and abundance of hemicelluloses are widely diversified among the different plant species and cell types, and are also present in most agricultural waste such as wheat straw, rice straw, corn stover, and sugar cane bagasse, among others [2].
The GH10 family includes endo-1,4-b-xylanases originating from all three domains of life (e.g., bacteria, archaea, and prokaryotes). Bacteria and fungi are widely used for the industrial production of xylanases [3]. Bacteria have an advantage over fungi for xylanase production as the optimum pH for bacterial xylanases lies in the neutral or alkaline range, whereas for fungal xylanases, it is in the acidic range [4]. Among the xylanolytic microorganisms, Gram-positive Bacillus tends to be marked as a significant industrial workhorse due to its fast growth and innate capacity to produce large amounts of extracellular enzymes [8][9][10][11][12][13].
Microbial xylanases are used in various industrial applications such as in the food industry (production of wine and beer, fruit juice, bread), for animal feed, biofuel production, in the pulp and paper industry, and pharmaceutical industry for the production of xylooligosaccharides (XOs) [14]. Xylooligosaccharides (XOs) are oligomers made up of xylose units with a DP of 2 to~6 and are considered as prebiotic compounds presenting both technical and health claims [15]. The worldwide market for XOs is expected to grow from USD 93 million in 2017 to USD 130 million in 2023 [16].
For many industrial applications, high temperatures in the range from 50 to 80 • C are often an integral part of the process. The thermal tolerance of the biocatalyst offers several advantages such as increased reaction rates, higher substrate solubility, decreased risk of microbial contamination, etc. [17].
The present work describes the isolation, overexpression, and biochemical characterization as well as the determination of certain thermodynamic properties of a novel thermotolerant 1,4-endo-β-D-xylanase (BsXyn10) belonging to the glycoside hydrolase family 10, produced by Bacillus safensis ATHUBA63. This analysis provides an excellent tool that will help optimize the operating range of the 1,4-endo-β-D-xylanase under study and to fully exploit its possible industrial applications.

Microorganism Strain and Characterization
Source DNA was obtained from a Bacillus sp. strain that was isolated from soil in Attica, Greece and deposited in the ATHUBA culture collection under accession number ATHUBA63. The strain has previously been assigned as Bacillus pumillus and was shown to excrete an alkali-resistant endo-1,4-β-glucanase upon growth in an oat spelt xylan-based medium [18]. The 16S rDNA sequence of the strain was isolated and sequenced using standard 27F and 1492R primers [19] and the resulting sequences were aligned with those from the type strains available in GenBank (Supplementary Figure S1). Based on this analysis, the strain was re-assigned as Bacillus safensis ATHUBA 63.

Plasmid Construction
Primers were constructed through the gene sequence alignment of annotated gh10 xylanase genes from the available genomes of Bacillus safensis and related species using Clustal Omega and optimized by modifying multiple alignment parameters [20]. This allowed us to design the following primer set: bsXyn10-F-NotI (5 -GCGCGCGGCCGCATGGTTAAAGAA AGAAGCTTTCTTCATC-3 ) and bsXyn10-R-SpeI (5 -GCGCACTAGTCTATCCTTCTTTAGG CAAGATTACGTC-3 )-restriction enzyme sites NotI and SPeI are underlined. The genomic DNA of Bacillus safensis ATHUBA 63 was used for PCR amplification of the GH10 enzyme encoding gene (bsXyn10, GenBank accession number ON462269) via the above-mentioned primers. Purified PCR fragments were introduced into the pET15b16S vector between the NotI and SpeI restriction sites. The pET15b16S vector was based on the pET-15b plasmid (Addgene), in-house modified on its multi-cloning site in order to incorporate the above restriction sites (Supplementary Figure S2). E. coli DH5α competent cells were transformed with the ligation mixture and spread onto selective LB agar plates (100 µg·mL −1 ampicillin). Positive transformants were initially identified by colony PCR, and further verified by plasmid extraction and sequencing. The pET15b16S plasmid harboring the bsXyn10 gene was used to transform the E. coli BL21(DE3) competent cells.

Protein Expression and Purification
Escherichia coli BL21(DE3) cells producing BsXyn10 were grown in Luria-Bertani (LB) broth containing 100 µg·mL −1 ampicillin at 37 • C, under constant shaking, until A 600 = 0.6-0.8 and induced by adding 0.1 mM (final concentration) isopropyl-1-thio-β-D-galactosidase (IPTG). Induced cultures were allowed to grow overnight at 20 • C. For BsXyn10 purification, cells were harvested by centrifugation at 9000 rpm for 20 min at 4 • C. Cell pellets were resuspended in equilibration buffer NPI10 and lysed by sonication on ice. The His-tagged enzyme was purified on a Ni-NTA column (Qiagen, Hilden, Germany) following the manufacturer's protocols. Imidazole was subsequently removed by gel filtration using a Sephadex G25M PD10 column (CYTIVA, Life Sciences, Manassas, VA, USA). The estimation of protein concentration was performed by measuring the absorbance at 280 nm using the predicted extinction coefficient of the protein, and acrylamide gels SDS-PAGE (10%) served to assess the purity of the recombinant protein.

Endo-1,4-xylanase Activity Determination
Endo-1,4-xylanase (xylanase) activity was determined as described previously [21]. Briefly, 50 µL of properly diluted enzyme was added to 450 µL of 20 g·L −1 oat spelt xylan (Sigma, St. Louis, MO, USA) in 50 mM citrate-phosphate buffer (pH 7.0) and placed in a thermoshaker (TS-100, BOECO, Hamburg, Germany) operating at 900 rpm and 60 • C for 15 min. Samples were placed into an ice bath and 500 µL of 3,5-dinitrosalicylic acid (DNS) reagent was added to terminate the reaction. Following the removal of residual xylan by centrifugation (3 min at 9000 rpm), the supernatant was boiled for 5 min. The concentration of the reducing sugars was estimated by measuring the absorbance at 540 nm using a xylose standard curve [22].
One unit of xylanase activity was defined as the amount of enzyme that produced 1 µmol of reducing sugar per minute, determined as xylose equivalents.

Biochemical Characterization of BsXyn10
The optimum pH of BsXyn10 was determined using the standard assay described above, using the following buffer systems (50 mM each): citrate-phosphate (pH 2.5-7.5), Tris-HCl (pH 7.5-9.0), and glycine-NaOH (pH 9.0-10.0). The stability was tested after incubation of the enzyme at the above buffers at 4 • C for 24 h and the determination of the residual activity.
The optimum temperature of BsXyn10 was determined using the standard assay described above, at temperatures ranging from 40 to 80 • C. The thermal stability of BsXyn10 was determined at temperatures ranging from 40 to 80 • C at pH 5.0 (50 mM citratephosphate buffer). Aliquots were withdrawn at different time intervals, cooled immediately on ice, and assayed for BsXyn10 activity under standard assay conditions. The effect of various modulators (e.g., metal ions (Na + , K + , Cu 2+ , Ca 2+ , Ba 2+ Mn 2+ , Zn 2+ , Mg 2+ , Co 2+ and Fe 3+ ), SDS (anionic surfactant) and EDTA (chelator)) on BsXyn10 activity at a concentration of 5 mM was investigated. BsXyn10 was pre-incubated with the metal ions, SDS, and EDTA at 25 • C for 30 min [11], followed by activity determination under the standard assay described above. Enzyme activity in the absence of a modulator was considered to be 100%. The substrate specificity of BsXyn10 was determined toward the xylan (oat spelt, birchwood, beechwood, and arabinoxylan, at an initial concentration of 20 g·L −1 ) and cellulose substrates (carboxymethyl cellulose-CMC, Avicel, 20 g·L −1 ). The enzymatic activity was measured at 60 • C according to the standard assay condition.
The kinetic constants (V max , K M , k cat , and k cat /K M ) were determined by incubating (15 min) a fixed amount of BsXyn10 with varied concentrations of oat spelt xylan (0 to 20 g·L −1 ) at 60 • C and pH 7.0. The data obtained were fitted to the standard Michaelis-Menten model.

Thermodynamics of Oat Spelt Xylan Hydrolysis
Thermodynamic analysis of oat spelt xylan hydrolysis was performed as described elsewhere [11,24]. The Arrhenius plot of lnV max against 1/T was applied to calculate the activation energy (E a ) of the hydrolysis of oat spelt xylan. The effect of temperature on the reaction rate was expressed in terms of the temperature quotient (Q 10 ). Q 10 is the factor by which the reaction rate is increased by raising the temperature 10 • C, and can be calculated by Equation (1) [25]: where E a is the activation energy (kJ·mol −1 ); T is the absolute temperature ( • K); and R is the universal gas constant (8.314 J·mol −1 ·K −1 ). The thermodynamic parameters, namely, ∆H*, ∆G* and ∆S*, for oat spelt xylan hydrolysis were calculated by rearranging the Eyring's absolute rate equation derived from the transition state theory [26]: where k cat is the turnover number (s −1 ); k is the Boltzmann's constant (1.38 × 10 −23 J·K −1 ); T is the absolute temperature (K); h is the Planck's constant (6.626 × 10 −34 J·s); ∆H* is the enthalpy of activation (J·mol −1 ); R is the universal gas constant (8.314 J·mol −1 ·K −1 ); and ∆S* is the entropy of activation (J·mol −1 ·K −1 ). For ∆H*, ∆G*, and ∆S*, the following equations apply: ∆G * (free energy of activation) = −R·T· ln k cat ·h k·T (4) The free energy of substrate binding and transition state formation were calculated using the following equations [11,24]: where K M is the Michaelis-Menten constant.

Thermodynamics of BsXyn10 Stability
Enzyme inactivation can be described by a first-order kinetic model as described by the following equation (Equation (8)): where A t (Units/mg protein) is the enzyme activity at time t; A 0 (Units/mg protein) is the initial enzyme activity; t (min) is the incubation time; and k d (min −1 ) is the first-order inactivation rate constantThe slope of the plot of ln A t A 0 versus t, at every temperature tested, gives the value of the inactivation rate constant k d . Thermal deactivation energy (E (a)d ) was estimated by the slope of the straight line resulting from the Arrhenius plot, that is, the plot of lnk d versus 1/T.
The half-life (t 1/2 ) (min) value of inactivation, which is defined as the time needed to reduce to 50%, the initial enzyme activity at a given temperature, can be calculated by the following expression (Equation (9)): The D-value is the time (min) needed for a 90% reduction in the initial activity and it can be calculated by the following equation (Equation (10)): where ∆H* D (enthalpy of inactivation), ∆G* D (Gibbs free energy of inactivation), and ∆S* D (entropy of inactivation) were calculated by applying Equations (3)-(5) with the following modifications: in Equation (3), E (a)d was used instead of E a and in Equation (4), k d was used in place of k cat [11,24].

Data Analysis
All data analyses were performed using linear and nonlinear regression fitting through application of the SigmaPlot software, version 12.0 (Copyright 2011 Systat Software, Inc., San José, CA, USA) for Windows.

Selection and Analysis of BsXyn10 Sequence
Following the identification (through 16S rRNA sequencing) of our strain as Baciilus safensis, a NCBI search on the specific species for xylanases resulted in the identification of an annotated and uncharacterized endo-xylanase gene product in the genome of the Bacillus safensis strain: PgKB20 (Gene ID: 61770491 (FX981_RS18680)). Based on the above sequence as well as on additional conserved sequences from the available genomes of Bacillus safensis and related species, a set of primers was constructed (see Materials and Methods) that allowed us to isolate BsXyn10 from the genomic DNA of B. safensis ATHUBA63. The amino acid sequence of BsXyn10 was analyzed with BlastP [27] against the Non-Redundant (NR), UniProtKB/SwissProt, and Protein Data Bank (PDB) sequence databases. When the BsXyn10 sequence was analyzed against the NR database, a 99% identity (100% query coverage) with a hypothetical 1,4-β-xylanase from Bacillus safensis [NCBI Reference Sequence: WP_095408496.1] was detected. This result matched our original taxonomic analysis, which assigned the 16S sequencing reads of the isolate to the genus Bacillus. BlastP analysis against the UniProtKB/SwissProt database revealed that the closest characterized homolog of BsXyn10 is an endo-1,4-β-xylanase from Acetivibrio thermocellus ATCC 27,405 [NCBI Reference Sequence: A3DH97.1] [28] with a sequence identity of 48% (query coverage 91%). The second-closest sequence according to the BlastP results was another endo-1,4-β-xylanase from A. thermocellus ATCC 27,405 [Uniprot accession no. P10478.3] with a 29% sequence identity (query coverage 59%) [29]. Furthermore, a homology search of the Protein Data Bank (PDB) revealed that BsXyn10 shares the highest sequence similarity (89% identity) with an endo-1,4-β-xylanase from B. safensis [PDB entry number: 7D88_A] [30]. Like BsXyn10, all of the aforementioned endo-xylanases originated from thermophilic bacteria and belong to the GH10 family (Supplementary Figure S3).

Purification of BsXyn10
In order to study the biochemical properties of the new xylanase, BsXyn10 was recombinantly produced and purified to homogeneity, as confirmed by the single band on the SDS-PAGE, (Figure 1b), by applying affinity chromatography, followed by gel filtration chromatography. Results of the purification procedure in the cell lysates and isolated form indicate that its apparent molecular mass is~48 kDa (Figure 1b), which is in accordance with its calculated size (47.89 kDa). The molecular mass of BsXyn10 is in the range of molecular masses reported for GH10 xylanases of different origin (Table 1).

Effect of pH on BsXyn10 Activity and Stability
BsXyn10 exhibited its maximum hydrolytic activity at pH 7.0 in the cit- The molecular mass of BsXyn10 is in the range of molecular masses reported for GH10 xylanases of different origin (Table 1).

Effect of pH on BsXyn10 Activity and Stability
BsXyn10 exhibited its maximum hydrolytic activity at pH 7.0 in the citrate-phosphate buffer system. In addition, the enzyme retained more than 70% of its highest catalytic activity at pH 6.0-8.0. At pH < 6.0 as well as pH > 8.0, a considerable drop in activity was detected, indicating only a moderate alkalophilic profile of the enzyme (Figure 2a). According to Chakdar et al., [4] bacterial xylanases, the majority of which belong to the GH10 family, acts optimally at neutral to alkaline pH. For instance, xylanases of the GH10 family such as Xyn10 isolated from Bacillus sp. N16-5 [8], Xyn10A of Bacillus sp. SN5 [9], Xyn30Y5 of Bacillus sp. 30Y5 [13], and r-XynA of Sorangium cellulosum So9733-1 [31] were optimally active at neutral pH, while iXylC of Cohnella laeviribosi HY-21 [32], Xyn10A of Flavobacterium johnsoniae [33], CbXyn10B of Caldicellulosiruptor bescii [34], and XynDZ5 of Thermoanaerobacterium sp. [35] were at slightly alkaline pH (7.5-8.0) ( Table 1). The stability of BsXyn10 was studied at different pH and the results are presented in Figure 2b. The enzyme retained more than 60% of its initial activity at pH 5.0-8.0 after 24 h incubation at 4 °C, while at pH = 9.0, the residual activity dropped to 39%. Similar results were obtained for xylanases of different bacterial species that were stable in wide pH ranges including acidic, neutral, and alkaline conditions (Table 1). For example, Xyn10 retained more than 60% of its initial activity at pH 5.4-10.6 after incubation at 4 °C for 16 h [8]. rXynAHJ2 of Bacillus sp. HJ2 [10] and Xyn30Y5 [13] exhibited over 50% and 80% residual activity, respectively, when incubated at a pH range of 6.0-10.0, but at different incubation conditions (Table 1). The stability of BsXyn10 was studied at different pH and the results are presented in Figure 2b. The enzyme retained more than 60% of its initial activity at pH 5.0-8.0 after 24 h incubation at 4 • C, while at pH = 9.0, the residual activity dropped to 39%. Similar results were obtained for xylanases of different bacterial species that were stable in wide pH ranges including acidic, neutral, and alkaline conditions (Table 1). For example, Xyn10 retained more than 60% of its initial activity at pH 5.4-10.6 after incubation at 4 • C for 16 h [8]. rXynAHJ2 of Bacillus sp. HJ2 [10] and Xyn30Y5 [13] exhibited over 50% and 80% residual activity, respectively, when incubated at a pH range of 6.0-10.0, but at different incubation conditions (Table 1).

Substrate Specificity, Kinetic Parameters, and Thermodynamics of Xylan Hydrolysis
BsXyn10 revealed higher hydrolytic activity on arabinoxylan followed by xylans from oat spelt, beech wood, and birchwood. No activity was recorded on CMC and Avicel ( Table 2). The substrate spectrum studies suggested that the substrate binding domain of xylanase has an equally high affinity for different xylans. Xyn10 [8], Xyn10A of Bacillus sp. SN5 [9], Xyn30Y5 [13], r-XynA [31], and Xyn10A of F. johnsoniae [33] were also active on different xylans with no activity toward glucose-based polysaccharides, while the XynA of Bacillus sp. KW1 [12], iXylC [32], and CbXyn10B [34] exhibited activity toward CMC and aryl-glycosides. The enzyme was incubated at 2% (w/v) of the various substrates at pH 7.0 and 60 • C. 1 Activity with oat spelt xylan was taken as 100%. ± indicates standard deviation among three independent readings.
The K M and V max values of BsXyn10 toward oat spelt xylan, determined from the Michaelis-Menten plot, at 60 • C were 1.96 g/L and 58.6 µmole·min −1 ·(mg protein) −1 , respectively ( Figure 3). The k cat value of BsXyn10 was found 49 s −1 and the catalytic efficiency (k cat /K M ) was 25 (L·s −1 ·g −1 ). The K M value of BsXyn10 was significantly lower than that reported for r-XynA, Xyn10A of F. johnsoniae, and XynZD5 [31,33,35], indicating a higher affinity for the substrate (Table 1). A higher k cat /K M value compared to rXynAHJ2, r-XynA, Xyn10A of F. johnsoniae, and XynZD5 indicates that BsXyn10 had a greater hydrolytic efficiency [10,31,33,35] (Table 1). The optimal reaction temperature of BsXyn10 was 60 °C, while a sharp decline in activity was observed at higher temperatures (Figure 4a). Thermotolerant xylanases active at a high temperature of 60-75 °C have been reported from different bacterial strains (Table 1). Low-temperature active xylanases are not very common, but have been iso- The optimal reaction temperature of BsXyn10 was 60 • C, while a sharp decline in activity was observed at higher temperatures (Figure 4a). Thermotolerant xylanases active at a high temperature of 60-75 • C have been reported from different bacterial strains (Table 1). Low-temperature active xylanases are not very common, but have been isolated from several bacteria such as Bacillus sp. HJ2, S. cellulosum So9733-1, and F. johnsoniae [10,31,33]. The optimal reaction temperature of BsXyn10 was 60 °C, while a sharp decline in activity was observed at higher temperatures (Figure 4a). Thermotolerant xylanases active at a high temperature of 60-75 °C have been reported from different bacterial strains (Table 1). Low-temperature active xylanases are not very common, but have been isolated from several bacteria such as Bacillus sp. HJ2, S. cellulosum So9733-1, and F. johnsoniae [10,31,33]. The activation energy (E a ) of BsXyn10 for oat spelt xylan hydrolysis, calculated by the Arrhenius plot (Figure 4b) was found to be 39.8 kJ·mol −1 . This value is comparable to that reported for the GH10 xylanases isolated from Arthrobacter sp. GN16 (29.74 kJ·mol −1 ) [38] and B. halodurans TSEV1 (30.51 kJ·mol −1 ) [11]. Higher activation energy (65.5 kJ·mol −1 ) was recorded for the Thermotoga naphthophila RKU-10T xylanase [39]. The low E a value estimated for BsXyn10 indicates that less energy is required to form the activated complex, thus highlighting an effective hydrolytic capacity.
The temperature quotient (Q 10 ) for the BsXyn10 was found to be 1.5 (Table 3). The Q 10 value for the hydrolysis of birchwood xylan by a GH10 xylanase isolated from Arthrobacter sp. GN16 was found to be 1.44 [38], while the corresponding value for the xylanase of B. halodurans TSEV1 was 1.29 [11]. The Q 10 value suggests whether or not the metabolic reaction is mainly controlled by temperature or by other factors. In general, for enzymatic reactions, Q 10 values range between 1 and 2 and any deviation from this value indicates the influence of other factors [40]. Table 3. A summary of the thermodynamic parameters (calculated at optimum temperature for activity, 60 • C) for oat spelt xylan hydrolysis by BsXyn10.
The ∆S*, ∆G*, and ∆H* values for xylan hydrolysis by the GH10 xylanases of Arthrobacter sp. GN16 and B. halodurans TSEV1 were −127.53 and −198.5 J·mol −1 ·K −1 , 67.65 and 197.65 kJ·mol −1 , and 27.09 and 27.56 kJ·mol −1 , respectively [11,38]. Furthermore T. naphthophila RKU-10T xylanase exhibited ∆S*, ∆G*, and ∆H* values equal to −22.88 J·mol −1 ·K −1 , 70.86 kJ·mol −1 , and 62.44 kJ·mol −1 , respectively [39]. In general, low ∆H* values such as those recorded for BsXyn10 and the negative values of ∆S* may suggest the formation of a more efficient and ordered transition state complex between the enzyme and substrate. The feasibility and extent of an enzyme-catalyzed reaction is best determined by measuring the change in ∆G* for substrate hydrolysis (i.e., the conversion of the enzyme-substrate complex into the product(s)). Low ∆G* values suggest that the conversion of a transition state complex into a product was more spontaneous. The low ∆G* value for BsXyn10 indicated that the conversion of its transition complex into products was more spontaneous compared to the xylanases produced from Arthrobacter sp. GN16, B. halodurans TSEV1, and T. naphthophila RKU-10 T [11,38,39].
The free energy of substrate binding (∆G * E−S ) and transition state formation (∆G * E−T ) for BsXyn10 were 6.0 and −18.2 kJ·mol −1 (Table 3), respectively; the values for B. halodurans TSEV1 xylanase were 21.15 and −24.84 kJ·mol −1 , respectively [11]. The negative value of ∆G * E−T indicates the spontaneous formation of products after substrate binding. The lower values of free energy for substrate binding (∆G * E−S ) and transition state formation (∆G * E−T ) confirmed the higher affinity of BsXyn10 for xylan hydrolysis compared to B. halodurans TSEV1 xylanase [11].

Effect of Various Modulators on BsXyn10 Activity
The metal ions are known to be involved in enzyme catalysis through a variety of methods such as accepting or donating electrons, electrophiles, nucleophiles, a coordinating group between the enzyme and substrate, or they may simply stabilize a catalytically active conformation of the enzyme [11]. The effect of metal ions (Na + , K + , Cu 2+ , Ca 2+ , Ba 2+ Mn 2+ , Zn 2+ , Mg 2+ , Co 2+ and Fe 3+ ) as well as of SDS and EDTA on BsXyn10 activity was studied at a concentration of 5 mM (Table 4). The BsXyn10 activity was suppressed in the presence of Na + and Mn 2+ , resulting in more than 65% loss of activity. The presence of Ca 2+ , Zn 2+ , and Fe 3+ ions marginally affected the BsXyn10 activity (approximately 97% residual activity), while the addition of K + , Cu 2+ , Mg 2+ , and Co 2+ enhanced its activity. The enzyme was incubated with 5 mM of the various modulators for 30 min at 25 • C and the remaining activity was determined at pH 7.0 and 60 • C. The enzyme activity of the control sample without any additives was taken as 100%; ± indicates the standard deviation among the three independent readings.

Thermodynamics of BsXyn10 Stability
BsXyn10 was stable at 40 • C as it retained more than 60% of its initial activity after 72 h of incubation (Figure 5a), while at 50 • C, the residual activity was 53% after 5 h of incubation. There was a reduction of 50% in the enzyme activity at 60 • C after 25 min. BsXyn10 lost all activity after 15 and 5 min at 70 • C and 80 • C, respectively (Figure 5b). The deactivation rate constant (k d ) at various temperatures was obtained from the slopes of the straight lines by plotting ln[A t /A 0 ] vs. time (Figure 5a,b). As shown in Table 5, the kd value of the BsXyn10 at 40-80 °C ranged from 2 × 10 −4 to 7.2 × 10 −1 ·min −1 . The inactivation constant gradually increased with increasing temperature, so the irreversible thermal inactivation became progressively more significant. The lower the value of kd at higher temperatures, the more stable the enzyme. The half-life (t1/2) and decimal reduction time (D value) are important parameters in industrial applications because the higher its value, the higher the enzyme thermostability. The t1/2 and D values for BsXyn10 over the temperature range tested are presented in Table 5. BsXyn10 exhibited t1/2 and D values of 5.3 h and 17.4 h, respectively, at 50 °C, while at 60 °C, the values were 25 and 83 min.  As shown in Table 5, the k d value of the BsXyn10 at 40-80 • C ranged from 2 × 10 −4 to 7.2 × 10 −1 ·min −1 . The inactivation constant gradually increased with increasing temperature, so the irreversible thermal inactivation became progressively more significant. The lower the value of k d at higher temperatures, the more stable the enzyme. The half-life (t 1/2 ) and decimal reduction time (D value) are important parameters in industrial applications because the higher its value, the higher the enzyme thermostability. The t 1/2 and D values for BsXyn10 over the temperature range tested are presented in Table 5. In general, the thermal inactivation of enzymes is a two-step process: Native enzyme (N) → Unfolded enzyme (U) → Inactive enzyme (I). Upon exposure of the native enzyme at elevated temperatures, an unstable intermediate (U) is formed. Thermal inactivation energy E (a)d is the minimum energy that must be acquired before enzyme unfolding takes place. If the input energy is less than the thermal inactivation energy E (a)d , the unstable intermediate can be refolded upon cooling. Irreversible unfolding of the enzyme and concomitant inactivation occurs upon prolonged exposure to heat (input energy higher than E (a)d ) [11,39]. The thermal inactivation energy E (a)d for BsXyn10 was estimated by the plot of lnk d vs. 1/T (Arrhenius plot) at 195.4 kJ·mol −1 (Figure 6). This value was higher than that reported for B. halodurans TSEV1 (98.8 kJ·mol −1 ) [11], much lower than that of T. naphthophila RKU-10 T (516.3 kJ·mol −1 ) [39] and comparable to that of XynB-A01 isolated from Cohnella sp. A01 (232.8 kJ·mol −1 ) [41].  The thermal inactivation of enzymes is also accompanied by the disruption of non-covalent linkages (hydrophobic interactions). Enthalpy change (ΔH*D) is a measure of the number of non-covalent linkages broken in forming a transition state for enzyme inactivation while entropy change (ΔS*D) indicates the enzyme disorder due to disruptions of the enzyme structure [42].
The thermodynamic parameters of BsXyn10 were calculated in the temperature range of 40-80 °C and presented in Table 5. The ΔH*D values in the temperature range studied (192.8-192.5 kJ·mole −1 ) were fairly stable when the temperature increased, as also reported for the B. halodurans TSEV1 GH10 xylanase (ΔH*D = 251.7 kJ·mole −1 ) at a temperature range of 65-80 °C [11] while a slight decrease in ΔH*D values (230.07-229.86 kJ·mole −1 ) at a temperature range of 55-80 °C was recorded for XynB-A01 [40]. The energy required to remove a -CH2 moiety from a hydrophobic bond is approximately 5.4 kJ·mole −1 , and thus the formation of the transition state leading to inactivation of the BSXyn10 implies the disruption, as an average, of 35.7 non-covalent bonds [43].
Positive ΔS* values at each temperature clarified that no noteworthy aggregation processes occurred; a similar trend has previously been reported [11,41].
The Gibbs free energy of inactivation (ΔG*D) includes both the entropic and enthalpic contributions and thus is considered as a more accurate and reliable variable to assess the enzyme's stability. The value of ΔG*D for BsXyn10 ranged from 109.6-99.9 kJ·mole −1 at 40-80 °C ( Table 5). The lower value of ΔG*D indicates that the reaction is more spontaneous, which means that the enzyme stability is reduced, thus readily undergoing denaturation. Comparable values of ΔG*D were reported for XynB-A01 (103.42-95.26 kJ·mole −1 ) at a temperature range of 55-80 °C [41]. The B. halodurans TSEV1 GH10 xylanase seemed less thermodynamically stable compared to BsXyn10 as ΔG*D decreased The thermal inactivation of enzymes is also accompanied by the disruption of noncovalent linkages (hydrophobic interactions). Enthalpy change (∆H* D ) is a measure of the number of non-covalent linkages broken in forming a transition state for enzyme inactivation while entropy change (∆S* D ) indicates the enzyme disorder due to disruptions of the enzyme structure [42].
The thermodynamic parameters of BsXyn10 were calculated in the temperature range of 40-80 • C and presented in Table 5. The ∆H* D values in the temperature range studied (192.8-192.5 kJ·mole −1 ) were fairly stable when the temperature increased, as also reported for the B. halodurans TSEV1 GH10 xylanase (∆H* D = 251.7 kJ·mole −1 ) at a temperature range of 65-80 • C [11] while a slight decrease in ∆H* D values (230.07-229.86 kJ·mole −1 ) at a temperature range of 55-80 • C was recorded for XynB-A01 [40]. The energy required to remove a -CH 2 moiety from a hydrophobic bond is approximately 5.4 kJ·mole −1 , and thus the formation of the transition state leading to inactivation of the BSXyn10 implies the disruption, as an average, of 35.7 non-covalent bonds [43].
Positive ∆S* values at each temperature clarified that no noteworthy aggregation processes occurred; a similar trend has previously been reported [11,41].
The Gibbs free energy of inactivation (∆G* D ) includes both the entropic and enthalpic contributions and thus is considered as a more accurate and reliable variable to assess the enzyme's stability. The value of ∆G* D for BsXyn10 ranged from 109.6-99.9 kJ·mole −1 at 40-80 • C ( Table 5). The lower value of ∆G* D indicates that the reaction is more spontaneous, which means that the enzyme stability is reduced, thus readily undergoing denaturation. Comparable values of ∆G* D were reported for XynB-A01 (103.42-95.26 kJ·mole −1 ) at a temperature range of 55-80 • C [41]. The B. halodurans TSEV1 GH10 xylanase seemed less thermodynamically stable compared to BsXyn10 as ∆G* D decreased from 98.8 to 21.95 kJ·mole −1 when the temperature increased from 65 to 70 • C, respectively [11].

Hydrolytic Properties of BsXyn10
The main xylooligosaccharides produced after 24 h of oat spelt xylan hydrolysis by BsXyn10 were xylotriose (X3 approximately 32%), followed by xylotetraose (X4, approximately 29%). Xylobiose (X2) and xylopentaose (X5) accounted for 18 and 12%, respectively, while smaller amounts of xylose (X1, approximately 5%) and xylohexaose (X6, approximately 3%) were detected (Supplementary Figure S4). An almost linear increase in the X2 and X3 concentration was observed in the first 8 h of hydrolysis, while xylose started to accumulate after the first 6 h of hydrolysis. The X4 and X5 concentration increased up to 6 h and remained almost constant after that, while the X6 concentration was constant after the third hour of hydrolysis ( Figure 7). in the X2 and X3 concentration was observed in the first 8 h of hydrolysis, while xylose started to accumulate after the first 6 h of hydrolysis. The X4 and X5 concentration increased up to 6 h and remained almost constant after that, while the X6 concentration was constant after the third hour of hydrolysis ( Figure 7). BsXyn10 exhibited the same hydrolytic pattern as Xyn10A of F. johnsoniae, which produced X2-X6 xylooligosaccharides and a low amount of xylose [33] (Table 6). According to Collins et al. [44], lower DP oligosaccharides are frequently found in the hydrolytic products of xylan degraded by GH10 xylanases, which is the characteristic BsXyn10 exhibited the same hydrolytic pattern as Xyn10A of F. johnsoniae, which produced X2-X6 xylooligosaccharides and a low amount of xylose [33] (Table 6). Table 6. The XO production of BsXyn10 and other GH10 xylanases of bacterial origin.

Conclusions
Xylanases are important biocatalysts in biomass processing, used for the hydrolysis of xylan, the planet's most abundant hemicellulose. They have received significant attention in several industrial biotransformations related to food, animal feed, paper, and pulp processing; thus, the discovery of novel glycoside hydrolases is pivotal to developing the potential of the existing processes. In the present study, we reported the discovery, heterologous expression, and characterization of a GH10 xylanase (BsXyn10), a novel thermotolerant xylanolytic enzyme with great potential for various industrial biotransformations due to its high specific activity and significant thermal tolerance, as corroborated by the thermodynamic and kinetic analysis of the enzyme. The hydrolytic efficacy of BsXyn10 on different xylan types is consistent with the broad substrate specificity of the GH10 family xylanase. BsXyn10 produces X2-X6 xylooligosaccharides and a low amount of xylose. The production of XOs of similar DP at moderate temperatures indicated the suitability of BsXyn10 in the bioprocesses, preferably performed with less (heat) energy input. Overall, this study illustrates the discovery of a novel member of the GH10 family of xylanases where BsXyn10 has emerged as a promising candidate for use in various applications as it features characteristics favorable for different industrial setups.

Conflicts of Interest:
The authors declare no conflict of interest.