Means and methods for producing deacetylated xanthan

he present invention relates to a method for producing xanthan with an altered acetylation pattern, wherein an enzyme site-specifically cleaving acetyl residues off mannose moieties of acetylated xanthan is contacted with acetylated xanthan. Furthermore, the present invention refers to the xanthan with the characteristics obtainable from such method and to the use thereof. Moreover, the present invention also refers to the use of enzymes site-specifically cleaving off the acetyl residues of mannose moieties of acetylated xanthan

Формоутворення великопрогонових світлопрозорих металевих конструкцій покриття

Dandelion (Taraxacum officinale) possesses an unusually high degree of disease resistance. As this plant exhibits high polyphenol oxidase (PPO) activity and PPO have been implicated in resistance against pests and pathogens, we analyzed the potential involvement of five PPO isoenzymes in the resistance of dandelion against Botrytis cinerea and Pseudomonas syringae pv. tomato. Only one PPO (ppo-2) was induced during infection, and ppo-2 promoter and beta-gincuronidase marker gene fusions revealed strong induction of the gene surrounding lesions induced by B. cinerea. Specific RNAi silencing reduced ppo-2 expression only, and concomitantly increased plant susceptibility to P. syringae pv. tomato. At 4 days postinoculation, P syringae pv. tomato populations were strongly increased in the ppo-2 RNAi lines compared with wild-type plants. When the dandelion ppo-2 gene was expressed in Arabidopsis thaliana, a plant having no PPO gene, active protein was formed and protein extracts of the transgenic plants exhibited substrate-dependent antimicrobial activity against P. syringae pv. tomato. These results clearly indicate a strong contribution of a specific, single PPO isoform to disease resistance. Therefore, we propose that specific PPO isoenzymes be included in a new family of pathogenesis-related (PR) proteins

Mode of action of Bacillus licheniformis pectin methylesterase on highly methylesterified and acetylated pectins

A gene encoding a putative pectinesterase from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli. The resulting recombinant enzyme (BliPME) was purified and characterized as a pectin methylesterase. The enzyme showed maximum activity at pH 8.0 and 50 °C. BliPME is able to release up to 100% of the methylesters from lime pectin (DM 34–76 ¿ DM 0) and up to 73% of all methylesters from SBPs (DM 30–73 ¿ DM 14). BliPME efficiently de-methylesterifies lemon pectins and SBPs in a blockwise manner and is quite tolerant towards the acetyl groups present within the SBPs. Detailed analysis of the BliPME-modified pectins using HILIC–MSn and the classical calcium reactivity measurement showed that the enzyme generates pectins with low methylesterification (lime and SBP) and high acetyl content (SBP) while creating blocks of nonmethylesterified galacturonic acid residues. The high activity of BliPME towards highly methylesterified and acetylated pectins makes this novel esterase more efficient in removing methylesters from highly esterified beet pectin compared to other PMEs, e.g. Aspergillus niger PME

Characterization of an acetyl esterase from Myceliophthorathermophila C1 able to deacetylate xanthan

Screening of eight carbohydrate acetyl esterases for their activity towards xanthan resulted in the recogni-tion of one active esterase. AXE3, a CAZy family CE1 acetyl xylan esterase originating from Myceliophthorathermophila C1, removed 31% of all acetyl groups present in xanthan after a 48 h incubation. AXE3 activ-ity towards xanthan was only observed when xanthan molecules were in the disordered conformation.Optimal performance towards xanthan was observed at 53¿C in the complete absence of salt, a condi-tion favouring the disordered conformation. AXE3-deacetylated xanthan was hydrolyzed using cellulasesand analyzed for its repeating units using UPLC–HILIC–ELSD/ESI–MS. This showed that AXE3 specificallyremoves the acetyl groups positioned on the inner mannose and that acetyl groups positioned on theouter mannose are not removed at all. After a prolonged incubation at optimal conditions, 57% of allacetyl groups, representing 70% of all acetyl groups on the inner mannose units, were hydrolyzed

A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50 °C. It is stable in the range pH 5.0–7.0 and below 50 °C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, 1H NMR of polymer and LC-HILIC-MSn after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties

A Bacillus licheniformis pectin acetylesterase is specific for homogalacturonans acetylated at O-3

A recombinant acetylesterase from Bacillus licheniformis DSM13, belonging to carbohydrate esterase family 12, was purified and biochemically characterized. The purified enzyme, termed BliPAE, was capable of deacetylating acetylated pectins, e.g. sugar beet pectin (SBP). Contrary to its provisional annotation as rhamnogalacturonan acetylesterase, the enzyme specifically removed acetyl groups from the homogalacturonan region classifying it as a PAE. The recombinant enzyme has a molecular mass of 26.7 kDa and shows optimal activity at pH 8.0 and 50 °C. It is stable in the range pH 5.0–7.0 and below 50 °C. Methylesterification of the galacturonic acid (GalA) moieties reduces the deacetylation efficacy of BliPAE. The enzyme efficiently removes acetyl groups from SBPs with low degree of methylesterification (DM) 9-30, releasing about 75% of the acetyl groups present in the homogalacturonan. Furthermore, 1H NMR of polymer and LC-HILIC-MSn after endo-PGII and PL degradation were used to structurally characterize the BliPAE-modified pectins. The results show that BliPAE removes acetyl groups specifically when substituted at the O-3 position of GalA moieties

Combined HILIC-ELSD/ESI-MSn enables the separation, identification and quantification of sugar beet pectin derived oligomers

The combined action of endo-polygalacturonase (endo-PGII), pectin lyase (PL), pectin methyl esterase (fungal PME) and RG-I degrading enzymes enabled the extended degradation of methylesterified and acetylated sugar beet pectins (SBPs). The released oligomers were separated, identified and quantified using hydrophilic interaction liquid chromatography (HILIC) with online electrospray ionization ion trap mass spectrometry (ESI-IT-MSn) and evaporative light scattering detection (ELSD). By MSn, the structures of galacturonic acid (GalA) oligomers having an acetyl group in the O-2 and/or O-3 positions eluting from the HILIC column were elucidated. The presence of methylesterified and/or acetylated galacturonic acid units within an oligomer reduced the interaction with the HILIC column significantly compared to the unsubstituted GalA oligomers. The HILIC column enables a good separation of most oligomers present in the digest. The use of ELSD to quantify oligogalacturonides was validated using pure GalA standards and the signal was found to be independent of the chemical structure of the oligomer being detected. The combination of chromatographic and enzymatic strategies enables to distinguish SBPs having different methylesters and acetyl group distribution

A broad pH range and processive chitinase from a metagenome library

Chitinases are hydrolases that degrade chitin, a polymer of N-acetylglucosamine linked β(1-4) present in the exoskeleton of crustaceans, insects, nematodes and fungal cell walls. A metagenome fosmid library from a wastewater-contaminated soil was functionally screened for chitinase activity leading to the isolation and identification of a chitinase gene named metachi18A. The metachi18A gene was subcloned and overexpressed in Escherichia coli BL21 and the MetaChi18A chitinase was purified by affinity chromatography as a 6xHis-tagged fusion protein. The MetaChi18A enzyme is a 92-kDa protein with a conserved active site domain of glycosyl hydrolases family 18. It hydrolyses colloidal chitin with an optimum pH of 5 and temperature of 50°C. Moreover, the enzyme retained at least 80% of its activity in the pH range from 4 to 9 and 98% at 600 mM NaCl. Thin layer chromatography analyses identified chitobiose as the main product of MetaChi18A on chitin polymers as substrate. Kinetic analysis showed inhibition of MetaChi18A activity at high concentrations of colloidal chitin and 4-methylumbelliferyl N,N′-diacetylchitobiose and sigmoid kinetics at low concentrations of colloidal chitin, indicating a possible conformational change to lead the chitin chain from the chitin-binding to the catalytic domain. The observed stability and activity of MetaChi18A over a wide range of conditions suggest that this chitinase, now characterized, may be suitable for application in the industrial processing of chitin

A seven-membered cell wall related transglycosylase gene family in Aspergillus niger is relevant for cell wall integrity in cell wall mutants with reduced α-glucan or galactomannan

Plant science