Novel bifunctional alpha-L-arabinofuranosidase/xylobiohydrolase (ABF3) from Penicillium purpurogenum

Abstract

The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl alpha-L-arabinofuranoside and p-nitrophenyl beta-D-xylopyranoside with a K(m) of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both alpha-L-arabinofuranosidase and xylobiohydrolase functionalities.

FIG. 1.

SDS-polyacrylamide gel electrophoresis results of ABF3. Lane St, molecular mass standards; lane 1, ABF3. (A) Gel stained with Coomassie brilliant blue R 250. (B) Glycoprotein staining with periodic acid-Schiff reagent.

FIG. 2.

Thin-layer chromatography of the hydrolysis products of xylooligosaccharides by ABF3. Lane 1, pNPXyl; lane 2, pNPxyl plus ABF3; lane 3, xylose; lane 4, xylose plus ABF3; lane 5, xylobiose; lane 6, xylobiose plus ABF3; lane 7, xylotriose; lane 8, xylotriose plus ABF3; lane 9, xylotetraose; lane 10, xylotetraose plus ABF3; lane 11, xylopentaose; lane 12, xylopentaose plus ABF3; line 13, oligosaccharide standards. The incubation was for 21 h at 30°C.

FIG. 3.

Thin-layer chromatography of the hydrolysis products of arabinoxylan by ABF1 and ABF3. Lane 1, arabinoxylan plus buffer; lane 2, arabinoxylan plus ABF1; lane 3, arabinoxylan plus ABF3; lane 4, mixture of xylooligosaccharides (xylose, xylobiose, xylotriose, xylotetraose, and xylopentaose); lane 5, arabinose. Incubation conditions were those of the experiment presented in Table 4.

FIG. 4.

Synergistic effect of xylanase A (XYL) from P. purpurogenum on the liberation of arabinose from wheat arabinoxylan (AX) by the action of ABFs. Incubations were for 6 h at 28°C. Arabinose was quantified as described in Materials and Methods. The arabinose content indicated by the manufacturer was considered 100%. The asterisk indicates that the addition of XYL increases the liberation of arabinose significantly with 95% confidence (based on analysis of variance).