Philippe Debeire

Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides

Hydrolysis of wheat bran and wheat straw by a 20.7 kDa thermostable endoxylanase released 35 and 18% of the cell-wall xylan content, respectively. Separation of the cinnamoyl-oligosaccharides (accounting for 6%) from the bulk of total oligosaccharides was achieved by specific anion-exchange chromatography. The cinnamoyl-oligosaccharides were further purified by preparative paper chromatography (PPC) and their molecular weight was determined by MALDI-TOF mass spectrometry. The partially purified hydrolysis end-products contained from 4 to 16 and from 4 to 12 pentose residues for wheat bran and straw, respectively, and only one cinnamic acid per molecule. The primary structure of the new feruloyl arabinoxylopentasaccharide from wheat bran hydrolysis, which has been determined using 2D NMR spectroscopy, is O-beta-D-xylopyranosyl-(1-->4)-O-[5-O- (feruloyl)-alpha-L-arabinofuranosyl-(1-->3)]-O-beta-D-xylopyranosy l-(1-->4) -O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose.

Hydrolysis of wheat straw by a thermostable endoxylanase : Adsorption and kinetic studies

The adsorption of a purified 20.7 kDa thermostable endo-1-4-β-xylanase (EC 3.2.1.8) from a Bacillus sp. on wheat straw at 4°C was studied. Adsorption data fitted the Langmuir-type adsorption isotherm with the maximum amount of adsorbed xylanase being 521 μg protein g−1 straw. Adsorption of the xylanase on straw, lignin, and insoluble xylans was irreversible at 4°C. The extent of hydrolysis was quantified by the measurement of total neutral sugars liberated from wheat straw-xylanase complexes at 60°C. Maximum hydrolysis was observed using 350 μg enzyme g−1 straw and reached 11% of the xylans in the straw after 5 h of reaction. No proportionality could be found between the level of xylanase adsorption on straw and the extent of hydrolysis at 60°C. Adsorption and hydrolysis experiments indicated that all the bound xylanase was not hydrolytically active. This suggested that nonspecific adsorption occurred on lignin. Analysis of the end products of the reaction indicated that xylose and neutral and uronic acid-containing xylo-oligosaccharides were the major compounds.

Genetic and Biochemical Characterization of a Highly Thermostable α-l-Arabinofuranosidase from Thermobacillus xylanilyticus

ABSTRACT The gene encoding an α-l-arabinofuranosidase fromThermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90°C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56,071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.

Enzymic production of oligosaccharides from corncob xylan

Abstract An endo-1,4-β-xylanase (E.C.3.2.1.8) fraction of high specific activity was isolated from a 180-l culture supernatant of Clostridium thermolacticum and partially purified by a one-step ion-exchange chromatography. This xylanase fraction, which contained 290 U mg −1 of protein, was used for the large-scale deploymerization of a xylan extracted from corncob meal. The oligosaccharides produced were absorbed on charcoal and eluted stepwise with ethanol to yield mainly xylobiose, xylotriose, and arabino-xylo-oligosaccharides .

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 (Kd) 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.

A single domain thermophilic xylanase can bind insoluble xylan: evidence for surface aromatic clusters

A clone expressing xylanase activity in Escherichia coli has been selected from a genomic plasmid library of the thermophilic Bacillus strain D3. Subcloning from the 9-kb insert located the xylanase activity to a 2.7-kb HindII/BamHI fragment. The DNA sequence of this clone revealed an ORF of 367 codons encoding a single domain type-F or family 10 enzyme, which was designated as XynA. Purification of the enzyme following over-expression in E. coli produced an enzyme of 42 kDa with a temperature optimum of 75 degrees C which can efficiently bind and hydrolyse insoluble xylan. The pH optimum of the enzyme is 6.5, but it is active over a broad pH range. A homology model of the xylanase has been constructed which reveals a series of surface aromatic residues which form hydrophobic clusters. This unusual structural feature is strikingly similar to the situation observed in the structure determined for the type-G xylanase from the Bacillus D3 strain and may constitute a common evolutionary mechanism imposed on different structural frameworks by which these xylanases may bind potential substrates and exhibit thermostability.

Purification and properties of the catalytic domain of the thermostable pullulanase type II from Thermococcus hydrothermalis

A pullulanase type II was produced in Escherichia coli using the relevant gene from Thermococcus hydrothermalis. This protein was purified and its pullulanolytic and amylolytic activities were characterised. The optimum temperature and Ca2+ concentration for each activity were identical (105 °C and 0.09 mM), whereas the optimum pH (pHpullulan 5.75, pHamylose 5) and the influence of Ca2+ ions on the kinetic parameters were different. Further analyses revealed that this enzyme exhibits an endo-processive-like action and specifically cleaves α-1,6 bonds in pullulan.

XYLANASE-MEDIATED HYDROLYSIS OF WHEAT BRAN: EVIDENCE FOR SUBCELLULAR HETEROGENEITY OF CELL WALLS

Previous work has shown that (1→4)‐β‐D‐endoxylanase‐mediated hydrolysis of wheat bran leads to solubilization of 50% of arabinoxylans. However, xylanase efficiency on the individual bran tissues is unequal because of histological and chemical heterogeneity. We describe here the results of an immunocytochemical study that is aimed at an understanding of in situ enzyme action on bran xylans at different hydrolysis kinetic stages. Two polyclonal antibodies were used, one against xylanase and the other against (1→4)‐β‐D‐unsubstituted xylopyranosyl residues, to target poorly substituted arabinoxylans. These antibodies were used on optical microscopy or transmission electron microscopy sections of xylanase‐treated and water‐treated wheat bran. After 30 min of xylanase treatment, xylanase distribution was found to be confined to the aleurone cell walls close to the endosperm, and arabinoxylan labeling had been lost. After 75 min, xylanase had progressed throughout the aleurone and had begun to attack the nucellar layer. After 24 h, the aleurone was completely lost, while some remnants of the nucellar layer were still observable. In contrast, the morphology of the pericarp and the testa was unaltered, and no xylanase labeling in these tissues was detected. Xylanase localization was correlated to the level of arabinoxylan substitution. That way, we showed that xylanase is preferentially localized and degrades poorly substituted arabinoxylans, as shown by visible subcellular heterogeneity of aleurone and nucellus walls.

Regulation and optimization of xylanase production inClostridiumthermolacticum

SummaryIn substrate-limited continuous or fed-batch cultures,Clostridium thermolacticum excreted high yield of xylanases even when readily metabolizable compounds such as glucose were used as substrate. These results demonstrated that theC. thermolacticum xylanases were constitutive and were catabolite repressed. Optimization of culture conditions showed that the highest yields were obtained in fed-batch culture.