A novel α-L-arabinofuranosidase of family 43 glycoside hydrolase (Ct43Araf) from Clostridium thermocellum

Abstract

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 Ca(2+) and Mg(2+) 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 Ca(2+) 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.

Figure 1. The molecular architecture of Ct43Araf shows modular structure with an N-terminal family 43 glycoside hydrolase (CtGH43) catalytic module (903 bp), a C-terminal family 6 carbohydrate binding module (CtCBM6B, 402 bp) and another family 6 Carbohydrate binding module (CtCBM6A, 405 bp) sandwiched between these two modules.

Figure 2. A) SDS-PAGE (13%) showing over-expression and purification of Ct43Araf.

Lane 1: Page Ruler protein marker, Lane 2: uninduced Ct43Araf cells, Lane 3: Induced Ct43Araf cells, Lane 4: Cell free extract, Lanes 5: Purified Ct43Araf (63 kDa approx.), B) Effect of pH and temperature on Ct43Araf activity, where (•) represents pH profile and (▾) represents temperature profile, C) pH and thermal stability analysis of Ct43Araf, where (▾) represents pH stability and (•) represents thermal stability profile.

Figure 3. Thin layer chromatography analysis of reaction products of Ct43Araf.

Dark spots on TLC plate show the standards L-arabinose, D-xylose and cellobiose (spots 1, 2 and 3, respectively) while spots 4, 5 and 6 represent hydrolyzed products from rye arabinoxylan, wheat arabinoxylan and oat spelt xylan, respectively, showing that only L-arabinose is released as the breakdown product.

Figure 4. HPAEC analysis of Ct43Araf reaction mixture showing released sugars.

A) L-arabinose, B) D-xylose, C) rye arabinoxylan, D) wheat arabinoxylan and E) oat spelt xylan. The reaction was carried out at pH 5.7, 50°C for 30 min.

Figure 5. Protein-melting analysis displaying normal melting curve (–), melting curve in presence of 10 mM Ca²⁺ ions (–), and melting curve in presence of 10 mM Ca²⁺ ions and 10 mM EGTA (–•–), A) Melting-profile of Ct43Araf and B) melting profile of truncated derivative, CtGH43.

Figure 6. Far-UV CD spectra of truncated CtGH43 (15 µM) from Clostridium thermocellum in 20 mM sodium phosphate buffer, pH 7.0.