Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans.

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.

Structure, chemical composition, and xylanase degradation of external layers isolated from developing wheat grain.

The external layers of wheat grain were investigated during maturation with respect to chemical and structural features and xylanase degradability. Cytochemical changes were observed in the isolated peripheral tissues of the wheat grain at four defined stages following anthesis. Marked chemical changes were highlighted at 11 days after anthesis, for which protein and lipid contents varied weakly. The profile of esterified ferulic acid showed large variation in the maturing peripheral layers of grain in contrast to the deposition of ferulate dimers, p-coumaric and sinapic acids. Lignin was monitored at the latest stages of ripening, which corresponds to the cessation of reserve accumulation in the grain. Arabinoxylans (AX) reached a maximum at 20 days and did not display any significant change in arabinosyl substitution proportion until ripeness. When submitted to xylanase, all outer layers were similarly altered in the proportion of soluble AX except for the peripheral tissues of the 11-day-aged wheat grain that had very little AX. Aleurone and nucellar layers were mostly degraded, whereas pericarp stayed intact at all stages of maturation. This degradation pattern was connected with the preferential immunolocalization of xylanase in aleurone and nucellar layers irrespective of the developmental stages. Further chemical examination of the enzyme-digested peripheral tissues of the grain supports the facts that ferulic ester is not a limiting factor in enzyme efficiency. Arabinose branching, ferulic dimers, and ether-linked monomers that are deposited early in the external layers would have more relevance to the in situ degradability of AX.

Enzymatic cocktails produced by Fusarium graminearum under submerged fermentation using different lignocellulosic biomasses.

Fusarium graminearum was grown on four lignocellulosic substrates (corn cobs, wheat bran, hop cell walls, and birchwood) and glucose as the sole carbon source. Proteomic studies performed on the resulting enzymatic cocktails highlighted a great diversity in the number and type of proteins secreted. The cell wall-degrading enzymes (CWDE) proportion varied greatly from 20% to 69%. Only one of the 57 CWDEs detected in this study was common to the five proteomes. In contrast, 35 CWDEs were specific to one proteome only. The polysaccharide-degradation activities were different depending on the cocktail and the polysaccharide used. F. graminearum strongly modifies the enzymatic cocktail it secretes as a function of the biomass used for growth.

Product patterns of a feruloyl esterase from Aspergillus nidulans on large feruloyl-arabino-xylo-oligosaccharides from wheat bran.

A purified feruloyl esterase (EC 3.1.1.73) from Aspergillus nidulans produced in Pichia pastoris was used to study the de-esterification of large feruloyl oligosaccharides consisting of 4 to 20 pentose residues and (xylose plus arabinose) and one ferulic acid residue. The feruloyl oligosaccharides were prepared from total oligosaccharidic hydrolysates from wheat bran treated with a purified endoxylanase from Thermobacillus xylanilyticus. The feruloyl esterase showed similar specific activity but an affinity about 3.5-fold higher towards feruloyl oligosaccharides than towards methyl ferulate. Mass spectrometry analysis of the products after long-term enzymatic hydrolyses showed that the esterase was able to hydrolyze the largest feruloyl oligosaccharides and therefore could act alone on feruloyled xylans. Consequently, the feruloyl esterase from A. nidulans could be useful for the enzymatic deconstruction of xylans in plant cell walls.

Impact of epiphytic and endogenous enzyme activities of senescent maize leaves and roots on the soil biodegradation process.

This study was focused on investigating the role of the initial residue community, i.e. microorganisms and enzymes from the epiphytic and endophytic compartments, in soil decomposition processes. Aerial and underground parts (leaves and roots) of maize (Zea mays L.) plants were γ-irradiated, surface-sterilized with sodium hypochlorite (NaOCl)/ethanol or non-sterilized (controls), while the outer surface morphology of maize leaves and roots was examined by scanning electron microscopy (SEM). Non-sterilized and sterilized maize leaves and roots were incubated in soil to study carbon (C) mineralization kinetics and enzyme dynamics (L-leucine aminopeptidase, CBH-1, xylanase, cellulase and laccase). SEM results showed that initial microbial colonization was more pronounced on non-sterilized leaf and root surfaces than on sterilized samples. The hypochlorite treatment removed a part of the soluble components of leaves by washing and no specific effect of any type of colonizing microorganisms was observed on C mineralization. In contrast, γ irradiation and hypochlorite treatments did not affect root chemical characteristics and the quantitative effect of initial residue-colonizing microorganisms on C mineralization was demonstrated. The variations in C mineralization and enzyme dynamics between non-sterilized and sterilized roots suggested that activities of epiphytic and endogenic microorganisms were of the same order of magnitude.

Probing the cell wall heterogeneity of micro-dissected wheat caryopsis using both active and inactive forms of a GH11 xylanase.

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (K(d)) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.

Probing the catalytically essential residues of the alpha-L-arabinofuranosidase from Thermobacillus xylanilyticus.

The alpha-L-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of alpha-L-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu(28), Glu(176) and Glu(298) are important for catalytic activity. Kinetic data obtained for the mutant Glu(176)-->Gln, combined with the results of chemical rescue using the mutant Glu(176)-->Ala, have shown that Glu(176) is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu(176)-->Ala, indicated that alpha-L-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu(298) might be the catalytic nucleophile and that Glu(28) is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that alpha-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.