Citric acid production from xylan and xylan hydrolysate by semi-solid culture of Aspergillus niger.
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Citric acid production from xylan and xylan hydrolysate was done by Aspergillus niger Yang no. 2 cultivated in a semi-solid culture using bagasse as a carrier. Yang no. 2 produced 72.4 g/l and 52.6 g/l of citric acid in 5 d from 140 g/l of xylose and arabinose, respectively. Yang no. 2 produced 51.6 g/l of citric acid in 3 d from a concentrated xylan hydrolysate prepared by cellulase treatment, containing 100 g/l of reducing sugars. Moreover, Yang no. 2 directly produced 39.6 g/l of citric acid maximally in 3 d from 140 g/l of xylan. (+info)
Purification of xyloglucan endotransglycosylases (XETs): a generally applicable and simple method based on reversible formation of an enzyme-substrate complex.
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We describe a novel and general, mechanism-based, method for purification of xyloglucan endotransglycosylases (XETs) from crude plant extracts. Putative isoforms, obtained by step-wise precipitation with (NH4)2SO4, were incubated with tamarind xyloglucan (approximately 1 MDa) to form stable xyloglucan-XET complexes with apparent molecular masses >500 kDa on gel-permeation chromatography (GPC). Subsequent addition of xyloglucan-derived oligosaccharides (a mixture of XET acceptor substrates) caused a shift in the GPC elution volume of the activity back to that expected of a approximately 32 kDa protein, presumably by completing the transglycosylation reaction and so freeing the enzyme from the xyloglucan (donor substrate). This simple two-step method enabled the isolation of each XET activity attempted [various (NH4)2SO4 cuts from extracts of cauliflower florets and mung bean seedlings], in pure form as judged by SDS/PAGE. (+info)
Xyloglucan fucosyltransferase, an enzyme involved in plant cell wall biosynthesis.
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Cell walls are crucial for development, signal transduction, and disease resistance in plants. Cell walls are made of cellulose, hemicelluloses, and pectins. Xyloglucan (XG), the principal load-bearing hemicellulose of dicotyledonous plants, has a terminal fucosyl residue. A 60-kilodalton fucosyltransferase (FTase) that adds this residue was purified from pea epicotyls. Peptide sequence information from the pea FTase allowed the cloning of a homologous gene, AtFT1, from Arabidopsis. Antibodies raised against recombinant AtFTase immunoprecipitate FTase enzyme activity from solubilized Arabidopsis membrane proteins, and AtFT1 expressed in mammalian COS cells results in the presence of XG FTase activity in these cells. (+info)
The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production.
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In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate, C. cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition. (+info)
The predominant protein on the surface of maize pollen is an endoxylanase synthesized by a tapetum mRNA with a long 5' leader.
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In plants, the pollen coat covers the exine wall of the pollen and is the outermost layer that makes the initial contact with the stigma surface during sexual reproduction. Little is known about the constituents of the pollen coat, especially in wind-pollinated species. The pollen coat was extracted with diethyl ether from the pollen of maize (Zea mays L.), and a predominant protein of 35 kDa was identified. On the basis of the N-terminal sequence of this protein, a cDNA clone of the Xyl gene was obtained by reverse transcriptase-polymerase chain reaction. The deduced amino acid sequence of the 35-kDa protein shared similarities with the sequences of many microbial xylanases and a barley aleurone-layer xylanase. The 35-kDa protein in the pollen-coat extract was purified to homogeneity by fast protein liquid chromatography and determined to be an acidic endoxylanase that was most active on oat spelt xylan. Northern and in situ hybridization showed that Xyl was specifically expressed in the tapetum of the anther after the tetrad microspores had become individual microspores. Southern hybridization and gene-copy reconstruction studies showed only one copy of the Xyl gene per haploid genome. Analyses of the genomic DNA sequence of Xyl and RNase protection studies with the transcript revealed many regulatory motifs at the promoter region and an intron at the 5' leader region of the transcript. The Xyl transcript had a 562-nucleotide (nt) 5' leader, a 54-nt sequence encoding a putative signal peptide, a 933-nt coding sequence, and a 420-nt 3'-untranslated sequence. The unusually long 5' leader had an open reading frame encoding a putative 175-residue protein whose sequence was most similar to that of a microbial arabinosidase. The maize xylanase is the first enzyme documented to be present in the pollen coat. Its possible role in the hydrolysis of the maize type II primary cell wall (having xylose, glucose, and arabinose as the major moieties) of the tapetum cells and the stigma surface is discussed. (+info)
Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes.
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Clostridium thermocellum produces a consortium of plant-cell-wall hydrolases that form a cell-bound multi-enzyme complex called the cellulosome. In the present study two similar xylanase genes, xynU and xynV, were cloned from C. thermocellum strain YS and sequenced. The deduced primary structures of both xylanases, xylanase U (XylU) and xylanase V (XylV), were homologous with the previously characterized xylanases from C. thermocellum strain F1. Truncated derivatives of XylV were produced and their biochemical properties were characterized. The xylanases were shown to be remarkably thermostable and resistant to proteolytic inactivation. The catalytic domains hydrolysed xylan by a typical endo-mode of action. The type VI cellulose-binding domain (CBD) homologue of XylV bound xylan and, to a smaller extent, Avicel and acid-swollen cellulose. Deletion of the CBD from XylV abolished the capacity of the enzymes to bind polysaccharides. The polysaccharide-binding domain was shown to have a key role in the hydrolysis of insoluble substrates by XylV. The C-terminal domain of XylV, which is absent from XylU, removed acetyl groups from acetylated xylan and acted in synergy with the glycosyl hydrolase catalytic domain of the enzyme to elicit the hydrolysis of acetylated xylan. (+info)
Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains.
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Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28 degree C in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ss-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20. 45 cp. The efficiency of delignification was 33%. (+info)
The type II and X cellulose-binding domains of Pseudomonas xylanase A potentiate catalytic activity against complex substrates by a common mechanism.
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Xylanase A (Pf Xyn10A), in common with several other Pseudomonas fluorescens subsp. cellulosa polysaccharidases, consists of a Type II cellulose-binding domain (CBD), a catalytic domain (Pf Xyn10A(CD)) and an internal domain that exhibits homology to Type X CBDs. The Type X CBD of Pf Xyn10A, expressed as a discrete entity (CBD(X)) or fused to the catalytic domain (Pf Xyn10A'), bound to amorphous and bacterial microcrystalline cellulose with a K(a) of 2.5 x 10(5) M(-1). CBD(X) exhibited no affinity for soluble forms of cellulose or cello-oligosaccharides, suggesting that the domain interacts with multiple cellulose chains in the insoluble forms of the polysaccharide. Pf Xyn10A' was 2-3 times more active against cellulose-hemicellulose complexes than Pf Xyn10A(CD); however, Pf Xyn10A' and Pf Xyn10A(CD) exhibited the same activity against soluble substrates. CBD(X) did not disrupt the structure of plant-cell-wall material or bacterial microcrystalline cellulose, and did not potentiate Pf Xyn10A(CD) when not covalently linked to the enzyme. There was no substantial difference in the affinity of full-length Pf Xyn10A and the enzyme's Type II CBD for cellulose. The activity of Pf Xyn10A against cellulose-hemicellulose complexes was similar to that of Pf Xyn10A', and a derivative of Pf Xyn10A in which the Type II CBD is linked to the Pf Xyn10A(CD) via a serine-rich linker sequence [Bolam, Cireula, McQueen-Mason, Simpson, Williamson, Rixon, Boraston, Hazlewood and Gilbert (1998) Biochem J. 331, 775-781]. These data indicate that CBD(X) is functional in Pf Xyn10A and that no synergy, either in ligand binding or in the potentiation of catalysis, is evident between the Type II and X CBDs of the xylanase. (+info)