*3.1. Sequence Analysis*

The gene sequence of putative xylanase (GenBank: ARA92359.1) was extracted from the complete genome of *R. sacchariphilus* RA (GenBank: CP020382.1) [20,21]. The mature xylanase gene was designated as *xynRA2*. The protein sequence similarity was assessed using NCBI BLASTp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments were performed using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). A phylogenetic tree of XynRA2 with its closest homologs was constructed with the neighbor-joining algorithm using MEGA v7.0 software [59] with a bootstrap value of 1000. The signal peptide sequence was predicted using SignalP v4.1 (http://www.cbs.dtu.dk/services/SignalP/). Conservative domains were identified using InterPro. The homology model of XynRA2 was performed using SWISS-MODEL (https://swissmodel.expasy.org/) with an evolved CBM of Xyn10A from *R*. *marinus* (PDB: 3JXS) [60] and GH10 xylanase (XynB) from *Xanthomonas axonopodis* pv *citri* (PDB: 4PN2) complexed with xylotriose as a template [61]. The predicted model and its surface electrostatic potential were assessed using APBS plugin in PyMOL (v2.2.3, Schrödinger Inc., New York, NY, USA).

### *3.2. Cloning of Xylanases*

Genomic DNA of *R. sacchariphilus* RA was extracted using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The gene sequence of *xynRA2* was amplified from the genomic DNA using a forward primer GH10F (5 -AGCCATATGCGTGCGCAGAGCAACACCA-3 ) and a reverse primer GH10R (5 -CGATGGGTACTGGTCCGCCTCGAGCACC-3 ). The underlined sequences represent *Nde*I and *Xho*I restriction sites respectively. N-terminal signal peptide was not included in the recombinant enzymes. The truncated gene *xynRA2*Δ*CBM* was amplified using primer GH10F-LC (5 -AGCCATATGCCCCTGGCGGGAGC-3 ) and GH10R.

Both the gene fragments were amplified using Q5® High-Fidelity PCR kit (NEB, Ipswich, MA, USA). The PCR products were digested with *Nde*I and *Xho*I followed by ligation into pET28a(+) (Novagen, Madison, WI, USA) at the corresponding sites. The recombinant plasmids (pET28a\_*xynRA2 and* pET28a\_*xynRA2*Δ*CBM*) were separately transformed into *E. coli* BL21 (DE3) competent cells using the heat shock method. The transformants were grown in Luria-Bertani (LB) medium containing 50 μg/mL kanamycin at 37 ◦C for 18 h. Transformants harboring the recombinant plasmid were identified by restriction digestion and DNA sequencing.

#### *3.3. Expression and Purification of Xylanases*

The transformed cells were grown in LB medium containing 50 μg/mL kanamycin at 37 ◦C to an *A*600 nm of 0.6. Protein expression was induced by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) at a final concentration of 0.4 mM at 25 ◦C for 18 h. The cells were harvested by centrifugation (6000× *g*, 4 ◦C, 10 min) and lysed using B-PER™ Direct Bacterial Protein Extraction Kit (Thermo Scientific, Waltham, MA, USA) The crude enzyme was collected (12,000× *g*, 4 ◦C, 10 min) and dialyzed against 20 mM sodium phosphate buffer (pH 7.4) at 4 ◦C overnight in a SnakeSkin™ Dialysis Tubing 10k MWCO (Thermo Scientific, Waltham, MA, USA). To purify the His-tagged proteins, the crude enzyme was loaded onto a Ni-NTA Superflow column (Qiagen, Hilden, Germany) equilibrated with 20 mM sodium phosphate buffer (pH 7.4) and 50 mM imidazole. The enzymes were eluted with a linear gradient of 50–500 mM imidazole in 20 mM phosphate buffer (pH 7.4) containing 500 mM NaCl. Upon elution, fractions containing the XynRA2 and XynRA2ΔCBM were respectively pooled and dialyzed against 20 mM sodium phosphate buffer (pH 7.4) at 4 ◦C overnight to remove the remaining salts. The purity and apparent molecular mass of XynRA2 and XynRA2ΔCBM were validated by SDS-PAGE. The activity of the purified enzymes was assayed as described below.

#### *3.4. Xylanase Assay*

The xylanase activity of XynRA2 and XynRA2ΔCBM was calculated by measuring the reducing sugars released from substrates using 3,5-dinitrosalicylic acid (DNS) method. The reaction mixtures contained 50 μL of appropriately diluted enzymes and 500 μL of 1% (*w*/*v*) beechwood xylan (Megazyme, Bray, County Wicklow, Ireland) in 0.1 M Tris-HCl buffer (pH 8.5). The enzymatic reaction was carried out at 70 ◦C for 15 min, stopped with 500 μL DNS reagent and boiled for 5 min. The absorbance at 540 nm was measured when the reaction mixture is cooled to room temperature. The amount of sugar released was estimated using a standard curve of d-xylose (Sigma-Aldrich, St. Louis, MO, USA). One unit (U) of xylanase activity was defined as 1 μmol of reducing sugars released from substrate per minute per mL of enzyme under the assay condition. The enzyme activity was calculated by this standard procedure unless otherwise noted. All reactions were performed in at least triplicate.

### *3.5. Biochemical Characterization of XynRA2 and XynRA2*Δ*CBM*

The optimum pH of XynRA2 was determined in a range of 2–11 at 50 ◦C. The buffers used were 0.1 M of glycine HCl (pH 2–3), sodium acetate (pH 4–6), Tris-HCl (pH 7–9), and glycine-NaOH (pH 10–11) containing 1% (*w*/*v*) purified beechwood xylan (Megazyme, Bray, County Wicklow, Ireland). The optimum temperature of the enzyme was determined over a range of temperature from 20 to 90 ◦C in Tris-HCl buffer (pH 8.5). The optimum pH of XynRA2ΔCBM was determined at 70 ◦C and the optimum temperature was determined in acetate buffer (pH 6.0). Thermostability of XynRA2 and XynRA2ΔCBM were determined by measuring the residual activity of the enzyme after pre-incubation in 0.1 M Tris-HCl buffer (pH 8.5) and 0.1 M acetate buffer (pH 6.0), respectively, at 70 ◦C without substrate for 2 h. The initial activity of enzymes without pre-incubation was set as 100%.

The effect of NaCl on the activity of XynRA2 and XynRA2ΔCBM was determined at 70 ◦C in 0.1 M Tris-HCl buffer (pH 8.5) and 0.1 M sodium acetate buffer (pH 6.0), respectively, in the presence of up to 5.0 M NaCl.

To determine the specific activities of purified XynRA2 and XynRA2ΔCBM, the enzyme activities were determined using a xylanase assay as described above, and the protein concentration was determined by Pierce™ BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) using BSA as a standard. To determine the turnover rate (*k*cat) of XynRA2 and XynRA2ΔCBM, the respective enzymatic reaction was carried out at 70 ◦C in 0.1 M Tris-HCl buffer (pH 8.5) and 0.1 M sodium acetate buffer (pH 6.0) containing 0.1–1.5% (*w*/*v*) of purified beechwood xylan. The *k*cat of the enzymes were determined based on non-linear regression using PRISM 7 software (GraphPad Software Inc., San Diego, CA, USA).

#### *3.6. Analysis of Substrate Specificity and Hydrolysis Products*

The substrate specificity of XynRA2 and XynRA2ΔCBM was determined in 0.1 M Tris-HCl (pH 8.5) and 0.1 M acetate buffer (pH 6.0) containing 1% (*w*/*v*) of beechwood xylan, arabinan (Megazyme, Bray, County Wicklow, Ireland), oat-spelt xylan, CMC, Avicel™, D-cellobiose (Sigma, St. Louis, MO, USA), pullulan (TCI chemicals, Tokyo, Japan), starch (QReC, Auckland, New Zealand) or 5 mM of X2–X6 xylo-oligosaccharides (Bz Oligo Biotech, Qingdao, China). The reaction mixture contained 50 μL of purified enzymes and 500 μL of the substrate solutions. The substrate hydrolysis was detected as described below.

To analyze the hydrolysis products of XynRA2 and XynRA2ΔCBM, the reaction mixtures with 3 U of purified enzymes and 1% (*w*/*v*) beechwood xylan and oat-spelt xylan were incubated at 70 ◦C in Tris-HCl buffer (pH 8.5) for 24 h. The hydrolysis products were eluted using Rezex™ RSO-oligosaccharides Ag<sup>+</sup> (4%) column (Phenomenex, Torrance, CA, USA) at a flow rate of 1.0 mL/min at 80 ◦C for 80 min and detected using 1260 Infinity ELSD (Agilent Technologies, Santa Clara, CA, USA). Xylo- oligosaccharides (X2–X6) and xylose were used as its product standards.

#### **4. Conclusions**

*Roseithermus* is a newly proposed genus in family *Rhodothermaceae* affiliated to order *Rhodothermales*. Currently, the whole taxonomic order is comprised of only 14 type strains. So far, xylanase from *Rhodothermus marinus* is the only enzyme that was well characterized. This study described for the first time the biochemical properties of a xylanase from *Roseithermus*. The native XynRA2 was active at alkaline pH and elevated temperature (pH 8.5 and 70 ◦C) while retaining excellent activity even at 5.0 M NaCl. Such properties make XynRA2 a potential candidate for applications involving an alkaline environment, elevated temperature, and high salinity. In a separate part of the study, the CBM4\_9 domain was removed. The data elucidated that CBM truncation affected enzyme specific activity, turnover rate, pH optimum, and NaCl tolerance, with an additional marginal effect on thermostability.

**Author Contributions:** Formal analysis, S.C.T.; Funding acquisition, M.S.S., C.S.C., N.C.B. and K.-G.C.; Methodology, K.J.L.; Supervision, K.M.G.; Writing—original draft, S.C.T. and K.M.G.; Writing—review & editing, K.J.L., M.S.S., C.S.C., N.C.B. and K.-G.C.

**Funding:** This work was financially sponsored by the Ministry of Education Malaysia and Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom under the program of United Kingdom-Southeast Asia Newton Ungku Omar Fund (UK-SEA-NUOF) with project number 4B297 and BB/P027717/1. This project was co-financially supported by Universiti Teknologi Malaysia RU grant (Grant number: 16H89). K-G Chan thanked University of Malaya for financial support (PPP grants: PG136-2016A, PG133-2016A, HIR grant: H50001-A-000027).

**Acknowledgments:** S.C. Teo appreciates UTM Zamalah scholarship for providing for financial support.

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

#### **Abbreviations**



#### **References**


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