A Novel α-L-Arabinofuranosidase of Family 43 Glycoside Hydrolase (Ct43Araf) from Clostridium thermocellum

The study describes a comparative analysis of biochemical, structural and functional properties of two recombinant derivatives from Clostridium thermocellum ATCC 27405 belonging to family 43 glycoside hydrolase. The family 43 glycoside hydrolase encoding α-L-arabinofuranosidase (Ct43Araf) displayed an N-terminal catalytic module CtGH43 (903 bp) followed by two carbohydrate binding modules CtCBM6A (405 bp) and CtCBM6B (402 bp) towards the C-terminal. Ct43Araf and its truncated derivative CtGH43 were cloned in pET-vectors, expressed in Escherichia coli and functionally characterized. The recombinant proteins displayed molecular sizes of 63 kDa (Ct43Araf) and 34 kDa (CtGH43) on SDS-PAGE analysis. Ct43Araf and CtGH43 showed optimal enzyme activities at pH 5.7 and 5.4 and the optimal temperature for both was 50°C. Ct43Araf and CtGH43 showed maximum activity with rye arabinoxylan 4.7 Umg−1 and 5.0 Umg−1, respectively, which increased by more than 2-fold in presence of Ca2+ and Mg2+ salts. This indicated that the presence of CBMs (CtCBM6A and CtCBM6B) did not have any effect on the enzyme activity. The thin layer chromatography and high pressure anion exchange chromatography analysis of Ct43Araf hydrolysed arabinoxylans (rye and wheat) and oat spelt xylan confirmed the release of L-arabinose. This is the first report of α-L-arabinofuranosidase from C. thermocellum having the capacity to degrade both p-nitrophenol-α-L-arabinofuranoside and p-nitrophenol-α-L-arabinopyranoside. The protein melting curves of Ct43Araf and CtGH43 demonstrated that CtGH43 and CBMs melt independently. The presence of Ca2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed 48% β-sheets, 49% random coils but only 3% α-helices.


Introduction
Plant cell wall is mainly composed of complex structural polysaccharides like cellulose and hemicellulose [1,2]. The heteropolymers of pentoses like D-xylose, L-arabinose and hexoses viz. D-mannose, D-glucose and D-galactose constitutes the hemicellulose. Often, xylans are hetero-polysaccharides with 1,4-linked-b-D-xylopyranose backbone chains containing arabinose, glucuronic acid, or its 4-O-methyl ether, acetic, ferulic, and p-coumaric acids side chains depending mainly on the source of xylans [3]. Rye arabinoxylans contain arabinose and xylose in the A/X ratio of 0.49-0.82 and also ferulate residues attached to arabinose as esters at its O-5 position [4] but in wheat arabinoxylans the arabinose to xylose ratios [A/X] varies from 0.47 to 0.58 [5]. The L-arabinosyl residues are often found in hemicelluloses, such as arabinan, arabinoxylan, gum arabic and arabinogalactan. The cereal arabinoxylans are composed majorly of a backbone of 1,4linked-b-D-xylopyranosyl residues substituted with single aarabinofuranosyl substituents attached to the O-2, O-3 or to both O-2,3 of the xylose residues [6,7]. It has been documented that a-L-arabinofuranosyl and to a lesser extent a-L-arabinopyranosyl side chains are attached to the b-D-galactopyranose main chain by 1,3-and 1,6-linkages in type II arabinogalactans [4,6,8,9,10]. The a-L-arabinofuranosidase (EC 3.2.1.55) are enzymes known to release terminal a-1,2-, a-1,3and a-1,5 a-L-arabinofuranosyl residues from hemicellulose such as arabinoxylan and other Larabinose containing polysaccharides [6,9]. Arabinofuranosidase have been reported from a few glycoside hydrolase families (GHs) viz., GH30 [11], GH43 [12,13], GH51 [14,15,16], GH54 [17] and GH62 [18,19]. The GH43 arabinoxylan arabinofuranohydrolase from Bacillus subtilis reported by Bourgois et al., 2007 [12] specifically released arabinofuranosyl residues from the monosubstituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. Whereas, Cartmell et el., 2011 [13] reported an arabinan-specific GH43 (a-1,2-arabinofuranosidase) from Cellvibrio japonicus capable of cleaving the a-1,2-arabinofuranose decorations in single or double substitutions. The arabinofuranosidase (Arf51B) from Clostridium stercorarium as reported by Aldesberger et al., (2004) was able to release a-L-arabinose residues from deesterified arabinoxylan [15]. The arabinofuranosidase (Araf51) from Clostridium thermocellum as described by Taylor et al., (2006) catalyzed the hydrolysis of both a-1,5-linked arabino-oligosaccharides and the a-1,3 arabinosyl side chain of xylan with equal efficiency [16]. a-L-Arabinofuranosidases have been used syner-gistically with other cellulose degrading enzymes in agro-industrial processes [6,9]. Lignocelluloses are known to have cellulose as major content but they also contain 20% hemicellulose which mainly consists of pentose such as xylose and arabinose [1,2]. In the past a-L-arabinofuranosidases received less importance in the production of bio-ethanol because pentoses are less efficiently converted to ethanol than hexose sugars [20]. But recently they have been used along with Candida shehatae that utilizes the pentose sugars for bio-ethanol production from cellulosic waste like mango and poplar leaves [21]. Also L-arabinose has been shown to inhibit intestinal sucrase and reduce the glycaemic response after sucrose ingestion in animals [22]. The carbohydrate binding modules (CBMs) are the non-catalytic modules known to help or bring the catalytic modules in close proximity to its substrates and also some CBMs are known to stabilize the enzyme structure and increase its temperature resistance [23,24]. The CBMs may be found to contain up to 200 amino acids and can be found attached as single, double or triple domains in one protein, located at both Cor N-terminal within the parental protein [23,24].
Biochemical, structural and functional characterization of Ct43Araf and CtGH43 is essential as all the a-L-arabinofuranosidases have the same inverting mechanism of catalysis but the enzyme activities are different (http://www.cazy.org/GH43.html). In the present study the recombinant proteins Ct43Araf and its truncated derivative CtGH43 were investigated and biochemically, structurally and functionally characterized. To our knowledge this is the first report of any a-L-arabinofuranosidase from Clostridium thermocellum showing the ability to hydrolyze both 4-nitrophenyl-aarabinofuranoside (pNPAf) and 4-nitrophenyl-a-arabinopyranoside (pNPAp).

Bacterial Strains and Plasmid
The genomic DNA of Clostridium thermocellum was a gift from Professor Carlos Fontes, Faculdade de MedicinaVeterinaria, Lisbon, Portugal. Escherichia coli (DH5a) cells were used for cloning, whereas, E. coli BL-21 (DE3) and E. coli BL-21 (DE3) pLysS cells were used as expression host. The plasmids used for cloning and expression were pET-21a(+) and pET-28a(+). All the above mentioned items were procured from Novagen (Madison, USA).

Gene Amplification and Cloning
Oligonucleotide primers containing NheI and XhoI restriction sites were designed and the DNA encoding Ct43Araf (GenBank Accession No: ABN52503.1; comprising of CtGH43, CtCBM6A and CtCBM6B domains, sequence range 1545911 to 1547651) and truncated gene CtGH43 were amplified from genomic DNA of C. thermocellum (7.7 ng) using 2.5 U of Pfu DNA polymerase (Stratagene, USA). A 50 ml PCR reaction mixture contained Mg 2+ ions (2.5 mM), dNTPs (1.6 mM) forward and reverse primer (0.45 mM) and PCR-grade water (Sigma, USA). The primers used for amplifying Ct43Araf were forward 59-ctcgctagcgctgccgattatccg-39, reverse 59-cacctcgagaattatgccactactgc-39. Primers for CtGH43 have been reported previously [21]. The PCR amplification cycles used were: denaturation at 94uC for 4 min followed by 30 cycles of i) denaturation at 94uC for 30 s, ii) annealing at 55uC for 60 s and iii) extension at 72uC for 2 min followed by a final extension at 72uC for 10 min. The PCR amplified DNA of Ct43Araf was cloned in NheI-XhoI digested pET-21a(+) expression vector while PCR DNA of CtGH43 was cloned earlier in pET-28a(+) vector [21], resulting in respective cloned plasmids. The positive clones were confirmed by NheI-XhoI digestion and DNA sequencing of recombinant plasmid. Thereafter, E. coli (DH5a) cells were transformed with above recombinant plasmids (pCt43Araf and pCtGH43). The transformed cells were grown on LB-agar plates supplemented with ampicillin (100 mg ml 21 ) in case of Ct43Araf and kanamycin (50 mg ml 21 ) in case of CtGH43 at 37uC. Positive clones were screened by restriction digestion analysis of the isolated recombinant plasmids DNAs from the cells. The truncated enzyme CtGH43 was amplified, cloned and expressed earlier and was reported [21].
Expression and Purification of Recombinant a-Larabinofuranosidase E. coli BL-21 (DE3) pLysS cells were used for expressing CtGH43 as described earlier [21], whereas, E. coli BL-21 (DE3) cells were transformed with recombinant plasmid pCt43Araf. The cells were grown in 100 ml LB medium containing ampicillin (100 mg ml 21 ) for growing Ct43Araf and kanamycin (50 mg ml 21 ) for growing CtGH43 at 37uC with 180 rpm till mid-exponential phase (A 550< 0.6), the cells were then induced with isopropyl-1thio-b-D-galactopyranoside (1.0 mM) for over-expression of recombinant proteins by incubation at 24uC with 180 rpm for 24 h [25]. The cells were harvested at 10,000 g at 4uC for 15 min using centrifuge (Sigma, 4 K 15) and the resulting cell pellets were re-suspended in 20 mM sodium phosphate buffer pH 7.4. Then the cells were sonicated (Sonics, Vibra cell) on ice for 8 min (5 s on/15 s off pulse; 33% amplitude) and again centrifuged at 19,000 g at 4uC for 30 min to get the cell free extract. The recombinant proteins from the cell free extracts were purified in a single step by immobilized metal ion affinity chromatography (IMAC) using sepharose columns (GE Healthcare, HiTrap chelating) and further dialyzed against 20 mM sodium phosphate buffer pH 5.7 for Ct43Araf and 20 mM sodium acetate buffer pH 5.4 for CtGH43 following the method as reported previously [21]. The purity and molecular mass of recombinant proteins were verified by SDS-PAGE [26].

Substrate Specificity of Ct43Araf and CtGH43 against Natural Substrates
The enzyme assay for Ct43Araf was performed using 20 mM sodium phosphate buffer (pH 5.7), whereas, the assay for CtGH43 was carried out in 20 mM sodium acetate buffer (pH 5.4). The 100 ml reaction mixture contained 1.0%, w/v substrate, 10 ml of enzyme (Ct43Araf 0.45 mg ml 21 or CtGH43 0.5 mg ml 21 ) and in both cases the reaction mixture was incubated at 50uC for 15 min. 'The enzyme activity was determined by measuring the reducing sugar by the method of Nelson and Somogyi [27,28]. The concentration of reducing sugar was estimated using a standard curve of L-arabinose as both Ct43Araf and CtGH43 predominantly showed a-L-arabinofuranosidase activity. One unit of activity was defined as the amount of enzyme which produced 1 mmole of L-arabinose per min under the optimized condition of temperature and pH. For studying the pH profile of Ct43Araf, 20 mM sodium acetate buffer, pH 4.0-5.6, 20 mM sodium  phosphate buffer, pH 5.7-7.5, 20 mM Tris/HCl, pH 7.5-8.0, buffer were used in enzyme assays that employed 1.0 (%, w/v) rye arabinoxylan, similar to the method employed for optimization of CtGH43 [21]. The optimum temperature and thermostability of Ct43Araf was determined by performing assay at different temperatures following the method reported earlier [21]. The kinetic parameters of Ct43Araf and CtGH43 were determined by performing assays at varying concentrations of the soluble substrates such as rye arabinoxylan, wheat arabinoxylan, oat spelt xylan, beechwood xylan and birchwood xylan and insoluble wheat arabinoxylan under the optimized condition of temperature and pH. The optimum pH and pH stability profile was generated by performing the enzyme assays at the optimum temperature of 50uC. The experiments were performed in triplicate.
Substrate Specificity of Ct43Araf and CtGH43 with Synthetic p-nitrophenyl-glycosides The assays of Ct43Araf and CtGH43 with synthetic pnitrophenyl glycosides (pNP-glycosides) viz., p-nitrophenyl-a-Larabinofuranoside (pNPAf) and p-nitrophenyl-a-L-arabinopyranoside (pNPAp) were carried out by estimating the release of 4nitrophenol (pNP) at 405 nm using a UV-Visible spectrophotometer (Varian, Cary 100 Bio) following the method described by Cartmell et al. 2011 [13]. The enzyme reaction was performed in 1.0 ml reaction mixture containing pNPAf or pNPAp in 50 mM sodium phosphate buffer (pH 6.0), 20 ml of enzyme (Ct43Araf 0.45 mg ml 21 or CtGH43 0.5 mg ml 21 ) incubated for 15 min at 50uC in a peltier temperature controller (Varian, Cary 100 Bio). The kinetic parameters of Ct43Araf and CtGH43 with pNPAf or pNPAp were determined by varying their concentrations in the range 20 to 500 mM. The initial (pseudo-first order) rates of Ct43Araf and CtGH43 with both the pNP-glycosides were measured in order to calculate the kinetic parameters. Continuous readings were recorded every one second for the initial linear absorbance range (0-15 mins) with an array of concentrations (20-500 mM) of pNP-glycosides. The reaction was stopped after 15 min by the addition 0.5 M sodium carbonate to make the reaction mixture highly alkaline (around pH 11.0). The assays were performed in triplicates. The released p-nitrophenol was quantified using the molar extinction coefficient of 24150 litre mole 21 cm 21 as reported by Cartmell et al. 2011 [13].

Effects of Metal Ions and Chemical Agents
The effects of different metal cations on the activity of Ct43Araf (0.45 mg ml 21 ) and CtGH43 (0.5 mg ml 21 ) were determined using 100 ml reaction mixture (in duplicates) with oat spelt xylan (1%, w/v) as the substrate and adding respective metal salt at low molar concentrations (up to 20 mM). Assays for Ct43Araf using 20 mM sodium phosphate buffer (pH 5.7) and for CtGH43 using 20 mM sodium acetate buffer (pH 5.4) were performed at 50uC. The reaction mixtures in both the cases were subjected to 15 min of incubation. The blank with substrates having the respective salts were also assayed in parallel. The effects of various salts of Na + , Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Co 2+ , Ni 2+ , Al 3+ and chelating agents like disodium EDTA and disodium EGTA were studied by varying their concentrations from 2.0-20.0 mM in the enzymesubstrate reaction mixture. The enzyme activity was calculated by estimating the reducing sugars as described above.

Thin Llayer Chromatography of Ct43Araf Hydrolyzed Products
The qualitative analysis of Ct43Araf hydrolyzed products of natural substrates was performed by thin layer chromatography (TLC) on silica gel-coated aluminium plate (TLC Silica gel 60 F 254, 20620 cm, Merck) for detecting the released sugars. The enzyme catalyzed reactions with 1% (w/v) substrate (rye arabinoxylan, wheat arabinoxylan or oat spelt xylan) were carried out in 100 ml reaction mixture maintaining the optimized condition of temperature and pH as mentioned above. The 100 ml reaction mixture was then precipitated with 2 volumes of acetone and centrifuged at 4uC at 13,000 g for 5 min [29]. The supernatant was transferred to another micro-centrifuge tube and the reaction product precipitate was concentrated by evaporating the acetone. Then 2.0 ml of sample (enzyme-substrate reaction product) as well as of standard (L-arabinose and D-xylose) solutions (1.0 mg ml 21 ) were loaded on the TLC plate. The plate was dried for few min and kept in the developing chamber saturated with the developing solution (mobile phase). The mobile phase consisted of acetic acid-n-propanol-water-acetonitrile in the ratio 4:10:11:14 [29]. At the end of the run, migrated sugars were visualized by immersing the chromatogram in a solution (sulphuric acid: methanol 5:95, v/v; and a-napthol 5.0%, w/v). The TLC plates were then dried in a hot-air oven at 80uC for 20 min. The migrated sugars appeared as blue spots on the TLC plate.

HPAEC Analysis of Polysaccharide Hydrolysis Product by Ct43Araf
Ct43Araf (4.7 U/mg, 0.5 mg/ml) catalyzed reactions with 1% (w/v) substrate (rye arabinoxylan, wheat arabinoxylan and oat spelt xylan) was carried out in 100 ml reaction mixture maintaining the optimal assay conditions as mentioned above. The 100 ml reaction mixture was incubated for 30 min at 50uC. The reaction was stopped by boiling the reaction mixture in a boiling water bath for 5 min. The 100 ml reaction mixtures were treated with 2 volumes of acetone to precipitate the remaining polysaccharides (substrates) and then centrifuged at 13,000 g for 10 min at 4uC. The supernatant containing the liberated sugar was transferred to another micro centrifuge tube and the acetone was removed by evaporation. The supernatant (50 ml) was diluted to 500 ml by adding ultra-pure (MilliQ) water and filtered through syringe filter using 0.2 mm membrane. The liberated sugars released due to the polysaccharide hydrolysis by enzyme reaction were analysed by high pressure anion exchange chromatography (HPAEC) using ion chromatography system (Dionex, ICS-3000). From the filtered 500 ml, 25 ml of sample (liberated sugars) was run on CARBO-PACK TM PA-20 column (15063 mm, Dionex), attached with CarboPac TM PA20 guard column (3063 mm, Dionex) with Borate and Amino trap columns which removed impurities and provided high resolution. The instrument (Dionex, ICS-3000) was kept at constant temperature of 30uC during the analysis. The sample loop (sample loaded) size was kept to 25.0 ml and the flow rate was maintained at 0.3 ml min 21 . The elution of liberated sugars released due to enzymatic reaction was carried out with 50.0 mM sodium hydroxide (Sigma, USA) using pulsed amperometric detector (PAD). L-arabinose and D-xylose (10 mg ml 21 ) were used as standards. The solutions of standards as well as samples were filtered by passing through 0.2 mm filter before loading on the column.

Protein Melting Study of Ct43Araf and CtGH43
Protein melting curves were generated by subjecting recombinant proteins (Ct43Araf and CtGH43) to varying temperatures and measuring the change in the absorbance at 280 nm (tryptophan absorption maximum) by a UV-Visible spectrophotometer (Varian, Cary 100-Bio) following the method of Dvortsov et al. [30]. The column (IMAC) purified Ct43Araf was dialyzed against 20 sodium phosphate buffer, pH 5.7, while, purified CtGH43 was dialyzed against 20 mM sodium acetate buffer, pH 5.4. The protein concentration for both Ct43Araf and CtGH43 were kept at 0.4 mg ml 21 . The absorbance at 280 nm was measured at different temperatures varying from 40-90uC using a peltier temperature controller. The protein solutions (1 ml) of Ct43Araf  and CtGH43 were kept at the particular temperature for 10 min to attain the equilibrium. Similar experiment was carried out; with the addition of 10 mM CaCl 2 in the 1 ml enzyme (0.4 mg/ml) solution and the temperature was then varied. The experiment was repeated with the addition of CaCl 2 and EGTA to 1 ml enzyme solution (0.4 mg ml 21 ) in equimolar concentrations of 10 mM, and finally the change in absorbance at 280 nm was measured. The relative derivative absorption coefficient is the normalization of melting points calculated at each increasing temperature repeated twice with an error of 5%. The relative derivative absorption coefficients were calculated using the Agilent ChemStation for UV-Visible Spectroscopy software (Agilent Technologies, USA) as described by Dvortsov et al., [30]. A curve of relative derivative absorption coefficient versus temperature was plotted to display the melting profile of Ct43Araf and CtGH43.

Circular Dichroism Analysis of Truncated Catalytic Derivative CtGH43
Far-UV Circular dichroism (CD) spectra of CtGH43 were recorded on a spectropolarimeter (Jasco Corporation, Tokyo, JASCO J-815), equipped with a peltier system for temperature control at 25uC using a cell with a path length of 0.1 cm. The   spectral accumulation parameters were carried out using a scanrate of 50 nm min 21 , a 1 nm bandwidth in the wavelength range of 195-250 nm with an average of six scans for each far-UV spectrum. The CD spectra of CtGH43 is presented in terms of mean residue ellipticity (MRE, expressed as deg cm 2 dmol 21 ) as a function of wavelength, calculated following the procedure described earlier [31] using a protein concentration of 15 mM in 10 mM Tris-HCl, pH 7.5. The CD spectra were corrected for buffer contributions and secondary structures were calculated by using web based K2d neural network software package (http:// www.ogic.ca/projects/k2d2/) as described by Perez-Iratxeta and Andrade-Navarro [32].

Expression and Purification of Recombinant Ct43Araf and CtGH43
After confirming the positive clones by restriction enzyme digestion, E. coli BL-21(DE3) cells were transformed with recombinant plasmid of Ct43Araf. CtGH43 was transformed in E. coli BL-21 (DE3) pLysS cells [21]. The expression of recombinant proteins were analysed by SDS-PAGE. The His 6tagged clostridial recombinant proteins were purified by immobilized metal ion affinity chromatography (IMAC) from the cell free extracts. The SDS-PAGE analysis of purified recombinant protein Ct43Araf is displayed in Fig. 2A. The recombinant protein Ct43Araf displayed molecular size of 63 kDa, whereas, CtGH43 showed a size of 34 kDa as reported previously [21]. The above recombinant proteins were expressed as soluble proteins. The ability of Ct43Araf and CtGH43 to hydrolyze the arabinosecontaining polysaccharides was explored and compared.

Effect of pH and Temperature on Ct43Araf and CtGH43
Ct43Araf showed maximum enzyme activity (4.7 U/mg) at a pH of 5.7 and was observed to be stable in the pH range of 5.0-6.5 (Fig. 2B). The maximum enzyme activity of Ct43Araf was observed at 50uC and the enzyme was found to be stable up to 50uC (Fig. 2C) which is consistent with the pH and temperature profiles of previously reports of recombinant proteins from Clostridium thermocellum by Taylor et al. 2005 [33] and Lee et al. 2013 [34].

Substrate Specificity and Kinetic Parameters of Recombinant Ct43Araf and CtGH43
Substrate specificity and kinetic parameters with natural substrates. The assays for natural substrates were carried out using the pH, buffers and other conditions as described in methods section. The activities of Ct43Araf and its truncated derivative CtGH43 with various polysaccharides are reported in Table 1. The maximum specific activity (U mg 21 ) of Ct43Araf and CtGH43 were found to be 4.7 and 5.0, respectively, with rye arabinoxylan which was followed by the decreasing order of activities against wheat arabinoxylan, oat spelt xylan, beechwood xylan and birchwood xylan as illustrated in Table 1. Both the catalytic enzymes showed low activity (less than 1.0 Umg 21 ) with arabinogalactan and rhamnogalactouronan (a-L-arabinopyranosyl side chain containing polysaccharides) [ Table 1]. Ct43Araf and CtGH43 did not show any considerable increase in activity with 1,5-a-L-arabinobiose and 1,5-a-L-arabinotetraose (the enzymatically hydrolyzed de branched sugar beet arabinan) as compared to complex sugar beet arabinan which indicated that the above enzymes lack specificity for 1,5-a-L-arabinosyl linkages [ Table 1]. The above results also indicated that both Ct43Araf and CtGH43 do not possess a-L-arabinanase type of activity. The kinetic properties and catalytic efficiency of both the enzymes were determined with the natural substrates [ Table 2]. Ct43Araf and CtGH43 showed highest turnover number (k cat ) of 280 min 21 and 298 min 21 , respectively, and also highest catalytic efficiency (k cat / K m , min 21 mg 21 ml) of 3.4610 3 and 3.6610 3 with rye arabinoxylan. Both the enzymes were able to act on insoluble wheat arabinoxylan showing catalytic efficiencies (k cat /K m ) of 7.1610 2 min 21 mg 21 ml and 6.1610 2 min 21 mg 21 ml for Ct43Araf and CtGH43, respectively. The catalytic efficiencies (k cat /K m ) observed with other soluble substrates like beechwood and birchwood xylans were found to be comparatively less.
Substrate specificity and kinetic parameters with synthetic substrates. The catalytic efficiencies (k cat /K m ) of Ct43Araf and CtGH43 with pNP-a-L-arabinofuranoside (pNPAf) were found to be 5.6610 3 min 21 mg 21 ml and 7.16 10 3 min 21 mg 21 ml, respectively, while with pNP-a-L-arabinopyranoside (pNPAp), the k cat /K m value for both the modules was 2.2610 3 min 21 mg 21 ml [ Table 2]. Therefore, we can say that the Ct43Araf and CtGH43 were able to release p-nitrophenol from pNP-a-L-arabinofuranoside (pNPAf) as well as from pNP-a-Larabinopyranoside (pNPAp), but the catalytic efficiencies of both modules were approximately, 3-fold higher with pNPAf as compared to pNPAp. Based on the catalytic efficiencies observed for Ct43Araf and CtGH43 with natural as well as synthetic substrates, it is evident that both these proteins are predominantly a-L-arabinofuranosidase. Effects of Metal Ions and Chemical Agents on Ct43Araf and CtGH43 Activity The enzyme activities of Ct43Araf and CtGH43 increased by more than 2-fold with Mg 2+ and Ca 2+ salts at low concentrations (5-10 mM) [ Table 3]. A slight increase in activity of Ct43Araf and CtGH43 were also observed with Ni 2+ salts (15% and 47%), Mn 2+ (20% and 21%) and Zn 2+ (8% and 24%) salts [ Table 3]. The enzyme activity of Ct43Araf and CtGH43 were adversely affected at higher concentrations (20 mM) of Co 2+ (75% in both enzymes), Hg 2+ (80% and 70%), Cu 2+ (70% in both enzymes) and Ag + (80% and 75%), respectively [ Table 3]. The enzyme activity of both Ct43Araf and CtGH43 decreased by more than 90% in presence of EDTA (10 mM) or 10 mM EGTA [ Table 3]. The decrease in activity in presence of EGTA indicated that Ca 2+ ions may be essential for enzyme activity as EGTA specifically binds and chelates calcium ions in 1:1 molar ratio [35]. The catalytic activity was noticeably increased in the presence of Ca 2+ and Mg 2+ salts elucidating the fact that these metal cations may be needed as cofactors while the heavy metals especially Co 2+ , Hg 2+ , Cu 2+ and Ag + caused decrease in enzyme activity as shown for recombinant cellulases [36,37].

TLC Analysis of Enzyme Reaction Products
The TLC analysis of the enzyme reaction products (sugars) indicated that Ct43Araf releases only arabinose from rye arabinoxylan, wheat arabinoxylan and oat spelt xylan [ Fig. 3]. The similar results were obtained with beechwood xylan and insoluble wheat arabinoxylan (data not shown). The above result and the results obtained from the assays with pNP-glycosides indicated that Ct43Araf could be a-L-arabinofuranosidase since the above mentioned polysaccharides contain a-L-arabinofuranosyl residues. The relative migration of Ct43Araf hydrolyzed sugars with commercially available standards clearly indicated that Larabinose could possibly be the major sugar released as no spots for xylose was observed on the TLC plate [ Fig. 3]. This was in agreement with the previous reports on a-L-arabinofuranosidases [38].

HPAEC Analysis of Enzyme Reaction Products
The reaction products of Ct43Araf with rye arabinoxyalan, wheat arabinoxylan (soluble) and oat spelt xylan analyzed by HPAEC showed only arabinose as the released sugar [ Fig. 4]. L-Arabinose and D-xylose used as standards for the HPEAC analysis of enzyme-substrate reaction products showed peaks at 10.4 min and at 13.6 min, respectively [ Fig. 4A and 4B]. The chromatograms of hydrolysis products (rye arabinoxylan, wheat arabinoxylan and oat spelt xylan) by both Ct43Araf and CtGH43 showed only the peak corresponding to L-arabinose at 10.4 min and no peak for xylose was observed [Fig. 4C, 4D and 4E]. Based on HPAEC and TLC analysis and the results obtained with p-NPglycoside assays we can conclude that both Ct43Araf and CtGH43 exhibit a-L-arabinofuranosidase activity as also reported earlier [38]. The HPAEC analysis of the hydrolyzed products of a-Larabinofuranosidase Ct43Araf from C. thermocellum supported the observation that both the modules released L-arabinose from arabinoxylans.

Protein Melting-curve Analysis Ct43Araf and CtGH43
The recombinant protein Ct43Araf showed two separate melting peaks at 53uC and 78uC [ Fig. 5A], whereas, CtGH43 displayed a single melting peak at around 78uC [ Fig. 5B]. This suggested that the peak at 53uC is associated with CBMs (CBM6A-CBM6B) and the peak at 78uC was due to CtGH43.
This type of melting curve indicated that CtGH43 and noncatalytic CBMs (CBM6A-CBM6B) are melting independently [ Fig. 5A]. The presence of Ca 2+ ions (10 mM) caused significant changes in Ct43Araf as well as in CtGH43 protein-melting profiles. The peak for CtGH43 shifted towards higher temperature i.e. 83uC from 78uC but the peak for corresponding to CBMs (CBM6A-CBM6B) of Ct43Araf was masked in presence of Ca 2+ ions [ Fig. 5 A & 5B]. When EGTA salt was added (10 mM) to the enzyme-substrate reaction mixture containing Ca 2+ (10 mM), there was shifting back of the melting peaks to the original temperature of 78uC of catalytic CtGH43 as evident from Fig. 5 A & B (small dotted lines).

Structural Analysis of CtGH43 by Circular Dichroism
The analysis of CD spectra of CtGH43 for detecting the secondary structural elements was based on the previous reports of CD spectra analysis of proteins by Kelly et al. [31] which showed that it mostly contained b-sheets and random coils [ Fig. 6]. The CD spectra CtGH43 was analysed using K2d as described by Perez-Iratxeta and Andrade-Navarro, [32] revealed that it contains 48% b-sheets, 49% random coils and only 3% a-helices [ Table 4].

Discussion
In recent past, few family 43 glycoside hydrolases have been reported in the CAZy database (www.cazy.org) from C. thermocellum (galactanase), B. thetaiotaomicron (a-1,2-arabinofuranosidase), C. japonicas (a-1,5-exoarabinanase) B. adolescentis (only other known arabinofuranosidase with ability to hydrolyze doubly substituted xylans) [6,9,13,14]. CtGH43 unlike other member of the family 43 glycoside hydrolase showed significant homology with CBM6s from different bacterial sources [39]. The sequence and phylogenetic tree analysis of CtGH43 has been reported by Ahmed et al., 2012 [39]. Ct43Araf and CtGH43 showed maximum activity against rye arabinoxylan; however, significant activities were also observed with wheat arabinoxylan, oat spelt xylan, birchwood xylan and beech wood xylan. It has been previously reported that the rye arabinoxylans have nearly 50% of the xylose residues substituted at the terminal by L-arabinose at O-3 and around 2% at both O-2 and O-3. This suggested that Ct43Araf displays a-1,3arabinofuranosidase type of activity in exo-acting manner similar to the previous report by Bengtsson et al. [4]. Ct43Araf and CtGH43 also showed lesser but considerable activity with water soluble wheat arabinoxylan, which are rich in 2-mono and disubstituted xyloses and low in 3-mono and di-substituted xylose [4,6,9], indicating that they also act at O-2 substituted xylose. Further, Ct43Araf and CtGH43 displayed noticeable activities with beechwood and birchwood xylans comprising of O-2 and O-3 substituted xylose. The above observations and previous report by Bourgois et al. [12] indicated that both the recombinant enzymes have the ability to catalyze the hydrolysis of terminal non-reducing a-L-1,2as well as a-L-1,3arabinosyl residues in exo-acting manner. The activity with oat spelt xylan can be attributed to the presence of arabinose (10%, w/v). The low activity with arabinogalactan and rhamnogalactouronan was mainly due to the fact that similar to b-1,4-xylopyranose, the the b-1,4-galactans too, are poorly substituted with a-L-arabinopyranose side-chains as reported by Øbro et al. [8]. Therefore, we can conclude that enzymes (Ct43Araf and CtGH43) acted mainly on the glycosidic linkage of a-arabinfuranosyl substituted to the main chain b-1,4xylose occurring predominantly in the arabinoxylans. Both Ct43Araf and CtGH43 were able to act on and degrade synthetic substrates pNP-a-L-arabinofuranoside as well as pNP-a-L-arabi-nopyranoside. Both Ct43Araf and CtGH43 showed high catalytic efficiencies against pNPAf and pNPAp, elucidating their bifunctional nature. But close inspection of catalytic efficiency data revealed that Ct43Araf is primarily a-L-arabinofuranosidase as the k cat /K m was 3-fold higher in case of pNPAf as compared to pNPAp.
The enzyme activity of Ct43Araf and CtGH43 increased significantly by more than two-fold in presence of Ca 2+ and Mg 2+ salts, implying that these ions are needed as cofactors. However, the enzyme activity was unaffected by lower concentrations of Cu 2+ , Co 2+ , Hg 2 or Ag + ions but it decreased at higher concentrations. The enzyme activity of Ct43Araf and CtGH43 decreased sharply in the presence of EGTA. This implied that Ca 2+ ions might be involved in the catalysis and imparting stability to the structures of Ct43Araf and CtGH43. A few recombinant family 43 glycoside hydrolases have been reported in the past by Sanctis et al., [40], Morais et al., [41] and Jordan et al., [42] which showed enhanced enzyme activity in the presence of Ca 2+ ions.
The TLC and HPAEC analyses indicated that both Ct43Araf and CtGH43 released L-arabinosyl side chain sugars from arabinoxylans. The TLC analysis of the hydrolysis products of rye arabinoxylan, wheat arabinoxylan and oat spelt xylan with Ct43Araf indicated that L-arabinose is the main sugar that was released as a result of enzyme substrate reaction. HPAEC analysis of the hydrolysis products of Ct43Araf with rye arabinoxylan, wheat arabinoxylan and oat spelt xylan further corroborated the above observation that L-arabinose is the only monosaccharide released after the hydrolysis. However, close inspection of the TLC and HPAEC results combined with the results obtained with synthetic pNP-glycosides confirmed that both Ct43Araf and CtGH43 are predominantly a-L-arabinofuranosidase since both showed higher catalytic efficiency with pNPAf.
Protein-melting curves of Ct43Araf and CtGH43 showed that the CtGH43 and CBMs (CBM6A-CBM6B) melt independently of each other. The protein-melting peaks of CtGH43 and CBMs shifted to higher temperature in the presence of Ca 2+ ions. However, on addition of equimolar concentration of EGTA and Ca 2+ ions to the solutions of Ct43Araf and CtGH43, the melting temperature peaks were shifted back to the original positions. The presence of Ca 2+ ions stabilized both the CBMs and CtGH43 module and prevented unfolding or denaturation. This enhanced stability of the CBM modules is observed through the absence of a 53uC denaturation event.. Similar observations were also reported with a b-1,3-glucanase and associated CBMX module [30]. The independent melting of protein modules was deduced by comparing with the previously reported melting profile of protein modules [30,43]. The shifting back of melting peaks in the presence of EGTA can be explained by the highly specific chelation of calcium ions by EGTA, making them unavailable to stabilize the enzyme CBM modules.
The CD analysis confirmed the fact that b-sheets and random coils were the main secondary structural elements present in the recombinant protein CtGH43. Only 3% a-helices were present in the CtGH43 structure. The results of CD analysis of CtGH43 were in agreement with the secondary structure prediction of the same protein using PSIPRED VIEW software [44] with slight difference in number of a-helices [ Table 4]. However, it is important to consider that the presence of aromatic residues can significantly affect the far-UV CD spectrum of peptides as reported by Pace and Scholtz [45] and Fujiwara et al., [46]. Such interactions sometimes could produce a significant increase in the helical population.

Conclusions
The Ct43Araf and its truncated derivative CtGH43 possess a-Larabinofuranosidase type of activity as analyzed by pNP-glycoside based assays, TLC and HPAEC analysis. The presence of CBMs did not affect the a-L-arabinofuranosidase activity of Ct43Araf and CtGH43. The enzyme activity of both Ct43Araf and CtGH43 was significantly enhanced in the presence of Ca 2+ and Mg 2+ salts. The Ct43Araf and CtGH43 showed ability to degrade both pnitrophenol-a-L-arabinofuranoside and p-nitrophenol-a-L-arabinopyranoside. The presence of Ca 2+ ions imparted thermal stability to both the enzymes. The circular dichroism analysis of CtGH43 showed that it is mostly composed of b-sheets and random coils.