Dataset Information

GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production.

ABSTRACT: BACKGROUND: The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZ?A vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data. RESULTS: High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified to homogeneity using one purification step. The apparent size on SDS-PAGE was around 64 kDa and was 46 kDa by mass spectrometry thus higher than the expected molecular mass of 41 kDa. The recombinant protein was N- and O-glycosylated, as demonstrated using glycoprotein staining and deglycosylation reactions, which explained the discrepancy in molecular mass. Enzyme-catalysed hydrolysis of low viscosity arabinoxylan (LVAX) was maximal at pH 5.0 with Km(app) and kcat/Km(app) of 3.7 ± 0.2 (mg.mL-1) and 132 (s-1mg-1.mL), respectively. The activity of XynD was optimal at 80°C and the recombinant enzyme has shown an interesting high thermal stability at 70°C for at least 180 min without loss of activity. The enzyme had an endo-mode of action on xylan forming mainly xylobiose and short-chain xylooligosaccharides (XOS). The initial rate data from the hydrolysis of short XOS indicated that the catalytic efficiency increased slightly with increasing their chain length with a small difference of the XynD catalytic efficiency against the different XOS. CONCLUSION: Because of its attractive properties XynD might be considered for biotechnological applications. Moreover, XOS hydrolysis suggested that XynD possess four catalytic subsites with a high energy of interaction with the substrate and a fifth subsite with a small energy of interaction, according to the GH10 xylanase literature data.

SUBMITTER: Lafond M

PROVIDER: S-EPMC3083334 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production.

Lafond Mickael M Tauzin Alexandra A Desseaux Véronique V Bonnin Estelle E Ajandouz El-Hassan el-H Giardina Thierry T

Microbial cell factories 20110405

<h4>Background</h4>The filamentous fungus Penicillium funiculosum produces a range of glycoside hydrolases (GH). The XynD gene, encoding the sole P. funiculosum GH10 xylanase described so far, was cloned into the pPICZαA vector and expressed in methylotrophe yeast Pichia pastoris, in order to compare the results obtained with the P. funiculosum GH11 xylanases data.<h4>Results</h4>High level expression of recombinant XynD was obtained with a secretion of around 60 mg.L-1. The protein was purified ...[more]

PMID: 21466666

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Project description:Xylanolytic enzymes have a broad range of applications in industrial biotechnology as biocatalytic components of various processes and products, such as food additives, bakery products, coffee extraction, agricultural silage and functional foods. An increasing market demand has driven the growing interest for the discovery of xylanases with specific industrially relevant characteristics, such as stability at elevated temperatures and in the presence of other denaturing factors, which will facilitate their incorporation into industrial processes. In this work, we report the discovery and biochemical characterization of a new thermostable GH10 xylanase, termed XynDZ5, exhibiting only 26% amino acid sequence identity to the closest characterized xylanolytic enzyme. This new enzyme was discovered in an Icelandic hot spring enrichment culture of a Thermoanaerobacterium species using a recently developed bioinformatic analysis platform. XynDZ5 was produced recombinantly in Escherichia coli, purified and characterized biochemically. This analysis revealed that it acts as an endo-1,4-β-xylanase that performs optimally at 65-75°C and pH 7.5. The enzyme is capable of retaining high levels of catalytic efficiency after several hours of incubation at high temperatures, as well as in the presence of significant concentrations of a range of metal ions and denaturing agents. Interestingly, the XynDZ5 biochemical profile was found to be atypical, as it also exhibits significant exo-activity. Computational modeling of its three-dimensional structure predicted a (β/α)8 TIM barrel fold, which is very frequently encountered among family GH10 enzymes. This modeled structure has provided clues about structural features that may explain aspects of its catalytic performance. Our results suggest that XynDZ5 represents a promising new candidate biocatalyst appropriate for several high-temperature biotechnological applications in the pulp, paper, baking, animal-feed and biofuel industries.

Project description:The functional screening of a Pseudacanthotermes militaris termite gut metagenomic library revealed an array of xylan-degrading enzymes, including P. militaris 25 (Pm25), a multimodular glycoside hydrolase family 10 (GH10). Sequence analysis showed details of the unusual domain organization of this enzyme. It consists of one catalytic domain, which is intercalated by two carbohydrate binding modules (CBMs) from family 4. The genes upstream of the genes encoding Pm25 are susC-susD-unk, suggesting Pm25 is a Xyn10C-like enzyme belonging to a polysaccharide utilization locus. The majority of Xyn10C-like enzymes shared the same interrupted domain architecture and were vastly distributed in different xylan utilization loci found in gut Bacteroidetes, indicating the importance of this enzyme in glycan acquisition for gut microbiota. To understand its unusual multimodularity and the possible role of the CBMs, a detailed characterization of the full-length Pm25 and truncated variants was performed. Results revealed that the GH10 catalytic module is specific toward the hydrolysis of xylan. Ligand binding results indicate that the GH10 module and the CBMs act independently, whereas the tandem CBM4s act synergistically with each other and improve enzymatic activity when assayed on insoluble polysaccharides. In addition, we show that the UNK protein upstream of Pm25 is able to bind arabinoxylan. Altogether, these findings contribute to a better understanding of the potential role of Xyn10C-like proteins in xylan utilization systems of gut bacteria.IMPORTANCE Xylan is the major hemicellulosic polysaccharide in cereals and contributes to the recalcitrance of the plant cell wall toward degradation. Members of the Bacteroidetes, one of the main phyla in rumen and human gut microbiota, have been shown to encode polysaccharide utilization loci dedicated to the degradation of xylan. Here, we present the biochemical characterization of a xylanase encoded by a Bacteroidetes strain isolated from the termite gut metagenome. This xylanase is a multimodular enzyme, the sequence of which is interrupted by the insertion of two CBMs from family 4. Our results show that this enzyme resembles homologues that were shown to be important for xylan degradation in rumen or human diet and show that the CBM insertion in the middle of the sequence seems to be a common feature in xylan utilization systems. This study shed light on our understanding of xylan degradation and plant cell wall deconstruction, which can be applied to several applications in food, feed, and bioeconomy.

Project description:BackgroundSugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig188, in response to SCB induction and examined their role in the saccharification of SCB.ResultsA systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0-45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 280 proteins were identified in the secretome of PfMig188, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-β-1,3(4)-glucanase (GH16), endo-α-1,3-glucanase (GH71), xylanase (GH30), β-xylosidase (GH5), β-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in β-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB.ConclusionOur findings provide an insight into the enzyme system of PfMig188 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse.

Dataset Information

GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production.

Publications

GH10 xylanase D from Penicillium funiculosum: biochemical studies and xylooligosaccharide production.

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