ABSTRACT
The gene encoding an α-L-arabinofuranosidase (BvAF) GH51 from Bacillus velezensis FZB42 was cloned and expressed in Escherichia coli. The corresponding open reading frame consists of 1,491 nucleotides which encode 496 amino acids with the molecular mass of 56.9 kDa. BvAF showed the highest activity against sugar beet (branched) arabinan in 50 mM sodium acetate buffer (pH 6.0) at 45°C. However, it could hardly hydrolyze debranched arabinan and arabinoxylans. The time-course hydrolyses of branched arabinan and arabinooligosaccharides (AOS) revealed that BvAF is a unique exo-hydrolase producing exclusively L-arabinose. BvAF could cleave α-(1,2)- and/or α-(1,3)-L-arabinofuranosidic linkages of the branched substrates to produce the debranched forms of arabinan and AOS. Although the excessive amount of BvAF could liberate L-arabinose from linear AOS, it was extremely lower than that on branched AOS. In conclusion, BvAF is the arabinan-specific exo-acting α-L-arabinofuranosidase possessing high debranching activity towards α-(1,2)- and/or α-(1,3)-linked branches of arabinan, which can facilitate the successive degradation of arabinan by endo-α-(1,5)-L-arabinanase.
Subject(s)
Bacillus/enzymology , Bacterial Proteins/metabolism , Glycoside Hydrolases/metabolism , Polysaccharides/metabolism , Amino Acid Sequence , Arabinose/metabolism , Bacillus/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Beta vulgaris/chemistry , Cloning, Molecular , Escherichia coli/enzymology , Escherichia coli/genetics , Gene Expression , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Glycoside Hydrolases/isolation & purification , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Molecular Weight , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , TemperatureABSTRACT
Two recombinant arabinosyl hydrolases, α-L-arabinofuranosidase from Geobacillus sp. KCTC 3012 (GAFase) and endo-(1,5)-α-L-arabinanase from Bacillus licheniformis DSM13 (BlABNase), were overexpressed in Escherichia coli, and their synergistic modes of action against sugar beet (branched) arabinan were investigated. Whereas GAFase hydrolyzed 35.9% of L-arabinose residues from sugar beet (branched) arabinan, endo-action of BlABNase released only 0.5% of L-arabinose owing to its extremely low accessibility towards branched arabinan. Interestingly, the simultaneous treatment of GAFase and BlABNase could liberate approximately 91.2% of L-arabinose from arabinan, which was significantly higher than any single exo-enzyme treatment (35.9%) or even stepwise exo- after endo-enzyme treatment (75.5%). Based on their unique modes of action, both exo- and endo-arabinosyl hydrolases can work in concert to catalyze the hydrolysis of arabinan to L-arabinose. At the early stage in arabinan degradation, exo-acting GAFase could remove the terminal arabinose branches to generate debranched arabinan, which could be successively hydrolyzed into arabinooligosaccharides via the endoaction of BlABNase. At the final stage, the simultaneous actions of exo- and endo-hydrolases could synergistically accelerate the L-arabinose production with high conversion yield.
