Screening of a Novel Glycoside Hydrolase Family 51 α-L-Arabinofuranosidase from Paenibacillus polymyxa KF-1: Cloning, Expression, and Characterization

Paenibacillus polymyxa exhibits remarkable hemicellulolytic activity. In the present study, 13 hemicellulose-degrading enzymes were identified from the secreted proteome of P. polymyxa KF-1 by liquid chromatography-tandem mass spectrometry analysis. α-L-arabinofuranosidase is an important member of hemicellulose-degrading enzymes. A novel α-L-arabinofuranosidase (PpAbf51b), belonging to glycoside hydrolase family 51, was identified from P. polymyxa. Recombinant PpAbf51b was produced in Escherichia coli BL21 (DE3) and was found to be a tetramer using gel filtration chromatography. PpAbf51b hydrolyzed neutral arabinose-containing polysaccharides, including sugar beet arabinan, linear-1,5-α-L-arabinan, and wheat arabinoxylan, with L-arabinose as the main product. The products from hydrolysis indicate that PpAbf51b functions as an exo-α-L-arabinofuranosidase. Combining PpAbf51b and Trichoderma longibrachiatum endo-1,4-xylanase produced significant synergistic effects for the degradation of wheat arabinoxylan. The α-L-arabinofuranosidase identified from the secretome of P. polymyxa KF-1 is potentially suitable for application in biotechnological industries.

Pseudomonas cellulosa expresses a single membrane-bound glycoside hydrolase family 51 arabinofuranosidase

In the accompanying paper [Beylot, McKie, Voragen, Doeswijk-Voragen and Gilbert (2001) Biochem. J. 358, 607–614] the chromosome of Pseudomonas cellulosa was shown to contain two genes, abf51A and abf62A, that encode arabinofuranosidases belonging to glycoside hydrolase families 51 and 62, respectively. In this report we show that expression of Abf51A is induced by arabinose and arabinose-containing polysaccharides. Northern-blot analysis showed that abf51A was efficiently transcribed, whereas no transcript derived from abf62A was detected in the presence of arabinose-containing polysaccharides. Zymogram and Western-blot analyses revealed that Abf51A was located on the outer membrane of P. cellulosa. To investigate the importance of Abf51A in the release of arabinose from poly- and oligosaccharides, transposon mutagenesis was used to construct an abf51A-inactive mutant of P. cellulosa (Δabf51A). The mutant did not grow on linear arabinan or sugar beet arabinan, and utilized arabinoxylan much more slowly than the wild-type bacterium. Arabinofuranosidase activity in Δabf51A against aryl-α-arabinofuranosides, arabinan and α1,5-linked arabino-oligosaccharides was approx. 1% of the wild-type bacterium. The mutant bacterium did not exhibit arabinofuranosidase activity against arabinoxylan, supporting the view that abf62A is not expressed in P. cellulosa. These data indicate that P. cellulosa expresses a membrane-bound glycoside hydrolase family 51 arabinofuranosidase that plays a pivotal role in releasing arabinose from polysaccharides and arabino-oligosaccharides.

The Pseudomonas cellulosa glycoside hydrolase family 51 arabinofuranosidase exhibits wide substrate specificity

To investigate the mechanism by which Pseudomonas cellulosa releases arabinose from polysaccharides and oligosaccharides, a gene library of P. cellulosa genomic DNA was screened for 4-methylumbelliferyl-α-l-arabinofuranosidase (MUAase) activity. A single MUAase gene (abf51A) was isolated, which encoded a non-modular glycoside hydrolase family (GH) 51 arabinofuranosidase (Abf51A) of 57000Da. The substrate specificity of the Abf51A showed that it preferentially removed α1,2- and α1,3-linked arabinofuranose side chains from either arabinan or arabinoxylan, and hydrolysed α1,5-linked arabino-oligosaccharides, although at a much lower rate. The activity of Abf51A against arabinoxylan was similar to a GH62 arabinofuranosidase encoded by a P. cellulosa gene. Glu-194 and Glu-321 of Abf51A are conserved in GH51 enzymes, and it has been suggested that these amino acids comprise the key catalytic acid/base and nucleophile residues, respectively. To evaluate this hypothesis the biochemical properties of E194A and E321A mutants of Abf51A were evaluated. The data were consistent with the view that Glu-194 and Glu-321 comprise the key catalytic residues of Abf51A. These data, in conjunction with the results presented in the accompanying paper [Beylot, Emami, McKie, Gilbert and Pell (2001) Biochem. J. 358, 599–605], indicate that P. cellulosa expresses a membrane-bound GH51 arabinofuranosidase that plays a pivotal role in releasing arabinose from a range of polysaccharides and oligosaccharides.