Abstract
α-Linked xylose is a major component of xyloglucans in the cell walls of higher plants. An α-xylosidase (AxlA) was purified from a commercial enzyme preparation from Aspergillus niger, and the encoding gene was identified. The protein is a member of glycosyl hydrolase family 31. It was active on p-nitrophenyl-α-d-xyloside, isoprimeverose, xyloglucan heptasaccharide (XXXG), and tamarind xyloglucan. When expressed in Pichia pastoris, AxlA had activity comparable to the native enzyme on pNPαX and IP despite apparent hyperglycosylation. The pH optimum of AxlA was between 3.0 and 4.0. AxlA together with β-glucosidase depolymerized xyloglucan heptasaccharide. A combination of AxlA, β-glucosidase, xyloglucanase, and β-galactosidase in the optimal proportions of 51:5:19:25 or 59:5:11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. To the best of our knowledge, this is the first characterization of a secreted microbial α-xylosidase. Secreted α-xylosidases appear to be rare in nature, being absent from other tested commercial enzyme mixtures and from the genomes of most filamentous fungi.
Highlights
Hemicelluloses that can constitute a major fraction of plant cell walls include xyloglucan, glucuronoarabinoxylan, mannan, galactan, arabinan, mixed-linked glucan, and glucuronoarabinoxylan [6]
We report the identification and characterization of a secreted AX from A. niger
T. reesei has only two poor (E-value Ͼ e-10 and Ͻ25% amino acid identity) BLASTP hits to An ␣-xylosidase of Aspergillus niger (AxlA) (Trire2͉121351 and Trir2͉69944), and neither of these has a predicted signal peptide
Summary
A combination of AxlA, -glucosidase, xyloglucanase, and -galactosidase in the optimal proportions of 51:5:19:25 or 59:5: 11:25 could completely depolymerize tamarind XG to free Glc or Xyl, respectively. Commercial enzyme mixtures contain 25–150 proteins [3, 4] These preparations are not necessarily ideal in their range of catalytic activities or in their relative proportions, especially for diverse pretreatment/feedstock combinations and there is considerable scope for their improvement. Hemicelluloses that can constitute a major fraction of plant cell walls include xyloglucan, glucuronoarabinoxylan, mannan, galactan, arabinan, mixed-linked glucan, and glucuronoarabinoxylan [6]. These hemicelluloses contain a number of fermentable monosaccharides, including Glc, Xyl, Gal, Ara, Man, Fuc, rhamnose, and uronic acids. Considering that a significant fraction of the metabolizable sugars in plant cell walls are
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