Cloning of α-L-arabinofuranosidase Genes and Its Expression in Escherichia coli: A Comparative Study of Recombinant Arabinofuranosidase Originatingin Bacillus subtilis DB104 and Newly Isolated Bacillus licheniformis CW1

Abstract

is one of the most important enzymes involved in degradation of lignocelullose biomass. Two genes encoding α-L-Arabinofuranosidase (abfA), each from Bacillus subtilis DB104 (abfAa1) and an indigenous Indonesian B. licheniformis CW1 (abfAb3), were cloned by the PCR approach and expressed in Escherichia coli. Sequences analysis of abfAa1 and abfAb3 revealed that each consists of 1721 and 1739 base pairs long DNA, respectively. Each clone contains a hypothetical open reading frame of 1503 and 1509 bp that encode an Abfa protein of 500 and 502 amino acids for abfAa1 and abfAb3, respectively. The deduced amino acid sequence of AbfaB3 shares 75% identity to that of AbfaA1. The recombinant enzymes were expressed constitutively in E. coli. Partial characterization of those enzymes revealed that the AbfaA1 and AbfaB3 were optimally active at 50 ºC and 60 ºC at pH 6, respectively. Thermostability studies of the recombinant enzymes with p-nitrophenyl α-L-arabinofuranoside at their optimal conditions showed that up to 50% AbfaA1 activity was lost after 5 h incubation at 50 ºC, whereas the AbfaB3 retained its activity over 75% after 12 h pre-incubation o at 60 C. This thermostability study of recombinant AbfaB3 showed for the first time that the arabinofuranosidase from B. licheniformis is a thermostable enzyme. The recombinant enzyme showed a higher optimal reaction temperature (60 ºC) in comparison to the previously reported thermostable arabinofuranosidase. The thermostable AbfaB3 has a potential to be applied to the degradation of lignocellulose biomass synergistically with thermostable xylanases, for instance in the production of xylo-oligosaccharides.

Production and characterization of aroma compounds from apple pomace by solid-state fermentation with selected yeasts

“…b-xylosidase activity was carried out on agar plates containing 1.7 g/L yeast nitrogen base without amino acids and ammonium sulphate (Difco), 5 g/L ammonium sulphate, 5 g/L D-xylose and 20 g/ L agar (pH ¼ 5.5) (Manzanares et al, 1999). a-L-arabinofuranosidase activity was assayed according to the method described by Nurcholis et al (2012), based on the hydrolysis of p-nitrophenyl-a-L-arabinofuranoside (pNP-A, Aldrich, USA).…”

Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

“…Cysteine residues are known to form disulphide bridges with alternate cysteine residues within the catalytic domain and to increase thermostability by 10 to 20 °C [29, 47]. The contribution of cysteine residues to the thermostability has been previously shown through substitution of cysteine residues with alanine, resulting in a decreased thermostability of AFases from G. caldoxylolyticus , Geobacillus stearothermophilus , Thermobacillus xylanilyticus and B. subtilis [30, 48]. Further evidence of the contribution of subtle changes in the amino acid sequence to thermostability of the proteins has been shown through comparison of amino acid composition of thermophilic and mesophilic protein homologues.…”

DESIGN OF <i>CANDIDA ANTARCTICA</i> LIPASE B THERMOSTABILITY IMPROVEMENT BY INTRODUCING EXTRA DISULFIDE BOND INTO THE ENZYME

“…Although it is not already wide spread in industrial scale, enzyme stability studies have been conducted extensively in Indonesia (Susilowati et al, 2008;Nurcholis et al, 2012;Hanim et al, 2013). Eventually, the modeling study has been conducted as well (Hertadi et al, 2007).…”