Dataset Information

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.

SUBMITTER: Ravanal MC

PROVIDER: S-EPMC2916492 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Ravanal María Cristina MC Callegari Eduardo E Eyzaguirre Jaime J

Applied and environmental microbiology 20100618 15

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-arabinofur ...[more]

PMID: 20562284

Similar Datasets

Project description:BackgroundXylan, the major constituent of hemicellulose, is composed of β-(1,4)-linked xylopyranosyl units that for the backbone, with side chains formed by other chemical moieties such as arabinose, galactose, mannose, ferulic acid and acetyl groups. Acetyl xylan esterases and α-L-arabinofuranosidases are two important accessory enzymes that remove side chain residues from xylan backbones and may act in synergy with other xylanolytic enzymes. Compared with enzymes possessing a single catalytic activity, multifunctional enzymes can achieve lignocellulosic biomass hydrolysis using a less complex mixture of enzymes.ResultsHere, we cloned an acetyl xylan esterase (PcAxe) from Penicillium chrysogenum P33 and expressed it in Pichia pastoris GS115. The optimal pH and temperature of the recombinant PcAxe (rPcAxe) for 4-nitrophenyl acetate were 7.0 and 40 °C, respectively. rPcAxe is stable across a broad pH range, retaining 100% enzyme activity om pH 6-9 after a 1 h incubation. The enzyme tolerates the presence of a wide range of metal ions. Sequence alignment revealed a GH62 domain exhibiting α-L-arabinofuranosidase activity with pH and temperature optima of pH 7.0 and 50 °C, in addition to the expected esterase domain. rPcAxe displayed significant synergy with a recombinant xylanase, with a degree of synergy of 1.35 for the hydrolysis of delignified corn stover. Release of glucose was increased by 51% from delignified corn stover when 2 mg of a commercial cellulase was replaced by an equivalent amount of rPcAxe, indicating superior hydrolytic efficiency.ConclusionsThe novel bifunctional enzyme PcAxe was identified in P. chrysogenum P33. rPcAxe includes a carbohydrate esterase domain and a glycosyl hydrolase family 62 domain. This is the first detailed report on a novel bifunctional enzyme possessing acetyl xylan esterase and α-L-arabinofuranosidase activities. These findings expand our current knowledge of glycoside hydrolases and pave the way for the discovery of similar novel enzymes.

Project description:BACKGROUND:Improving the hydrolytic performance of xylanolytic enzymes on arabinoxylan is of importance in the ethanol fermentation industry. Supplementation of debranching (arabinofuranosidase) and depolymerizing (xylanase) enzymes is a way to address the problem. In the present study, we identified a bifunctional ?-l-arabinofuranosidase/endo-xylanase (Ac-Abf51A) of glycoside hydrolase family 51 in Alicyclobacillus sp. strain A4. Its biochemical stability and great hydrolysis efficiency against complex biomass make it a potential candidate for the production of biofuels. RESULTS:The gene encoding Ac-Abf51A was cloned. The comparison of its sequence with reference proteins having resolved 3D-structures revealed nine key residues involved in catalysis and substrate-binding interaction. Recombinant Ac-Abf51A produced in Escherichia coli showed optimal activity at pH 6.0 and 60 °C with 4-nitrophenyl-?-l-arabinofuranoside as the substrate. The enzyme exhibited an exo-type mode of action on polyarabinosides by catalyzing the cleavage of ?-1,2- and ?-1,3-linked arabinofuranose side chains in sugar beet arabinan and water-soluble wheat arabinoxylan and ?-1,5-linked arabinofuranosidic bonds in debranched sugar beet arabinan. Surprisingly, it had capacity to release xylobiose and xylotriose from wheat arabinoxylan and was active on xylooligosaccharides (xylohexaose 1.2/mM/min, xylopentaose 6.9/mM/min, and xylotetraose 19.7/mM/min), however a lower level of activity. Moreover, Ac-Abf51A showed greater synergistic effect in combination with xylanase (2.92-fold) on wheat arabinoxylan degradation than other reported enzymes, for the amounts of arabinose, xylose, and xylobiose were all increased in comparison to that by the enzymes acting individually. CONCLUSIONS:This study for the first time reports a GH51 enzyme with both exo-?-l-arabinofuranosidase and endo-xylanase activities. It was stable over a broad pH range and at high temperature, and showed greater synergistic effect with xylanase on the degradation of wheat arabinoxylan than other counterparts. The distinguished synergy might be ascribed to its bifunctional ?-l-arabinofuranosidase/xylanase activity, which may represent a possible way to degrade biomass at lower enzyme loadings.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets