The exoproteome of the fungus Fusarium graminearum grown on glucose and on hop (Humulus lupulus, L.) cell wall has been investigated. The culture medium was found to contain a higher quantity of proteins and the proteins are more diverse when the fungus is grown on cell wall. Using both 1D and 2D electrophoresis followed by mass spectrometry analysis and protein identification based on similarity searches, 84 unique proteins were identified in the cell wall-grown fungal exoproteome. Many are putatively implicated in carbohydrate metabolism, mainly in cell wall polysaccharide degradation. The predicted carbohydrate-active enzymes fell into 24 different enzymes classes, and up to eight different proteins within a same class are secreted. This indicates that fungal metabolism becomes oriented towards synthesis and secretion of a whole arsenal of enzymes able to digest almost the complete plant cell wall. Cellobiohydrolase is one of the only four proteins found both after growth on glucose and on plant cell wall and we propose that this enzyme could act as a sensor of the extracellular environment. Extensive knowledge of this very diverse F. graminearum exoproteome is an important step towards the full understanding of Fusarium/plants interactions.
Polyurethanes (PU) are a family of versatile synthetic polymers intended for diverse applications. Biological degradation of PU is a blooming research domain as it contributes to the design of ecofriendly materials sensitive to biodegradation phenomena and the development of green recycling processes. In this field, an increasing number of studies deal with the discovery and characterization of enzymes and microorganisms able to degrade PU chains. The synthesis of short lifespan PU material sensitive to biological degradation is also of growing interest. Measurement of PU degradation can be performed by a wide range of analytical tools depending on the architecture of the materials and the biological entities. Recent developments of these analytical techniques allowed for a better understanding of the mechanisms involved in PU biodegradation. Here, we reviewed the evaluation of biological PU degradation, including the required analytics. Advantages, drawbacks, specific uses, and results of these analytics are largely discussed to provide a critical overview and support future studies.
Bacteria usually use two-component systems for signal transduction, while eukaryotic organisms employ Ser/Thr and Tyr kinases and phosphatases for the same purpose. Many prokaryotes turn out to harbor Ser/Thr and Tyr kinases, Ser/Thr and Tyr phosphatases, and their accessory components as well. The sequence determination of the genome of the cyanobacterium Synechocystis sp. strain PCC 6803 offers the possibility to survey the extent of such molecules in a prokaryotic organism. This cyanobacterium possesses seven Ser/Thr kinases, seven Ser/Thr and Tyr phosphatases, one protein kinase interacting protein, one protein kinase regulatory subunit and several WD40-repeat-containing proteins. The majority of the protein phosphatases presented in this study were previously reported as hypothetical proteins. We analyze here the structure and genetic organization of these ORFs in the hope of providing a guidance for their functional analysis. Unlike their eukaryotic counterparts, many of these genes are clustered on the chromosome, and this genetic organization offers the opportunity to study their possible interaction. In several cases, genes of two-component transducers are found within the same cluster as those encoding a Ser/Thr kinase or a Ser/Thr phosphatase; the implication for signal transduction mechanism will be discussed.
Abstractor-Acetolactate decarboxylase from Lacrococcus lactis subsp. Zactis NCDO 2118 was expressed at low levels in cell extracts and was also unstable. The purification was carried out from E. coli in which the enzyme was expressed 36-fold higher. The specific activity was 24-fold enhanced after purification. The main characteristics of a-acetolactate decarboxylase were: (i) activation by the three branched chain amino acids leucine, valine and isoleucine; (ii) allosteric properties displayed in absence and Michaelis kinetics in the presence of leucine. The enzyme is composed of six identical subunits of 26,500 Da. Kev words: Lactococcus:a-Acetolactate decarboxvlase: Branched-chain amino acids
Itaconic acid (IA) is a dicarboxylic acid included in the US Department of Energy's (DOE) 2004 list of the most promising chemical platforms derived from sugars. IA is produced industrially using liquid-state fermentation (LSF) by Aspergillus terreus with glucose as the carbon source. To utilize IA production in renewable resource-based biorefinery, the present study investigated the use of lignocellulosic biomass as a carbon source for LSF. We also investigated the production of fumaric acid (FA), which is also on the DOE's list. FA is a primary metabolite, whereas IA is a secondary metabolite and requires the enzyme cis-aconitate decarboxylase for its production. Two lignocellulosic biomasses (wheat bran and corn cobs) were tested for fungal fermentation. Liquid hydrolysates obtained after acid or enzymatic treatment were used in LSF. We show that each treatment resulted in different concentrations of sugars, metals, or inhibitors. Furthermore, different acid yields (IA and FA) were obtained depending on which of the four Aspergillus strains tested were employed. The maximum FA yield was obtained when A. terreus was used for LSF of corn cob hydrolysate (1.9% total glucose); whereas an IA yield of 0.14% was obtained by LSF of corn cob hydrolysates by A. oryzae.
The phytopathogenic fungus Fusarium graminearum secretes a very diverse pool of glycoside hydrolases (GHs) aimed at degrading plant cell walls. ␣-L-Arabinanases are essential GHs participating in the complete hydrolysis of hemicellulose, a natural resource for various industrial processes, such as bioethanol or pharmaceuticals production. Arb93A, the exo-1,5-␣-L-arabinanase of F. graminearum encoded by the gene fg03054.1, belongs to the GH93 family, for which no structural data exists. The enzyme is highly active (1065 units/mg) and displays a strict substrate specificity for linear ␣-1,5-L-arabinan. Biochemical assays and NMR experiments demonstrated that the enzyme releases ␣-1,5-L-arabinobiose from the nonreducing end of the polysaccharide. We determined the crystal structure of the native enzyme and its complex with ␣-1,5-L-arabinobiose, a degradation product of ␣-Me-1,5-L-arabinotetraose, at 1.85 and 2.05 Å resolution, respectively. Arb93A is a monomeric enzyme, which presents the six-bladed -propeller fold characteristic of sialidases of clan GHE. The configuration of the bound arabinobiose is consistent with the retaining mechanism proposed for the GH93 family. Catalytic residues were proposed from the structural analysis, and site-directed mutagenesis was used to validate their role. They are significantly different from those observed for GHE sialidases.The plant cell wall consists mainly of a complex aggregation of polysaccharides, such as cellulose, hemicellulose, and pectin. Hemicellulose is one of the most abundant renewable biopolymers on earth and constitutes an important source of energy for the biofuel industry. It represents 20 -40% of plant biomass and is principally composed of pentoses, such as xylose and arabinose (1). Due to the high complexity and structural variability of this polysaccharide, many enzymes are necessary for its complete degradation (2). A number of microorganisms are able to break down hemicellulose, through the action of various glycoside hydrolases (GHs).2 The latter catalyze the cleavage of glycosidic bonds between sugars with either inversion or retention of the anomeric configuration (3). GHs have been classified into more than 114 different families based on their amino acid sequence similarity (CAZY (Carbohydrate Active Enzymes) server, available on the World Wide Web) (4, 5).␣-L-Arabinanases (EC 3.2.1.-) are accessory hemicellulases that hydrolyze ␣-L-arabinofuranosic linkages and act synergistically with other GHs to break down hemicellulose fully (6). These enzymes have become of interest in recent years because of their potential rate-limiting role in the degradation of lignocelluloses and their practical application in various industrial processes, such as the production of important medicinal compounds, the improvement of wine flavors, pulp treatment, juice clarification, the production of bioethanol, and the synthesis of oligosaccharides (7). According to the CAZY classification, ␣-Larabinanases are present in six GH families (3,43,51,54, 62, and 93) whose ...
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