Identification and characterization of GH62 bacterial α-l-arabinofuranosidase from thermotolerant Streptomyces sp. SWU10 that preferentially degrades branched l-arabinofuranoses in wheat arabinoxylan.

Abstract

We previously described thermotolerant Streptomyces sp. SWU10, which produced four endo-xylanases and one xylosidase able to digest xylan backbones. To achieve arabinoxylan degradation, the swu62A gene was cloned and overexpressed in Escherichia coli, and the recombinant enzyme, termed SWUAbf62A, was characterized. The 438 amino acids of SWUAbf62A revealed Glyco_hydro_62 and closely related with putative α-l-arabinofuranosidases belonging to glycoside hydrolase family 62. SWUAbf62A was purified in two steps, Ni-affinity and size-exclusion column chromatographies, and its molecular mass without signal peptide was determined to be 49 kDa. SWUAbf62A showed optimum activity at pH 5.0 and 50 °C, and more than 70% of its initial enzymatic activity remained after incubation at pH 4.1-10.5, while SWUAbf62A lost all activity after 1 h at 60 °C. SWUAbf62A activity was stimulated by Ba2+, Ca2+, and Mn2+ and decreased by Ag+, Cu2+, Fe2+, and EDTA. SWUAbf62A had no activity towards p-nitrophenyl-α-l-arabinofuranoside or p-nitrophenyl-β-d-xylopyranoside synthetic substrates. On the other hand, SWUAbf62A had the highest activity against wheat arabinoxylan, with a specific activity of 1.29 U/mg, and was also active against sugar beet arabinan, with a specific activity of 0.14 U/mg; these results indicated that SWUAbf62A is an arabinoxylan arabinofuranohydrolase. Using 1H-NMR analysis, SWUAbf62A was found to release l-arabinofuranoses singly linked to O-3 of wheat arabinoxylan. In addition, SWUAbf62A acted synergistically with endo-xylanase (XynSW3) and α-l-arabinofuranosidase, which releases arabinose linked to O-3 of double-substituted xylose residues on arabinoxylan, to digest the wheat arabinoxylan. SWUAbf62A is an important debranching enzyme for hydrolysis of hemicelluloses to monosaccharides and can be applied in various industrial biotechnologies.