Rhamnogalacturonan Acetylesterase Research Articles

One-pot biocatalysis of potato rhamnogalacturonan and the role of its deacetylation in efficient inhibition of colon cancer cells and hydrogel mediated colon-targeted drug delivery

Deacetylation of potato rhamnogalacturonan (PRG) by rhamnogalacturonan acetyl esterase (CtPae12B) was explored for enhanced hydrolysis of PRG by rhamnogalacturonan lyase (CtRGLf) and the effects of deacetylated PRG were studied in enhancing inhibition of colon-cancer cells and formation of colon-targeting drug delivery material. Pre-treatment of PRG with CtPae12B resulted in increased relative activity of CtRGLf. CtPae12B removed acetyl groups from both O-2 and O-3 positions of D-galactopyranosyluronic acid residues of PRG, resulting in 98 % deacetylation. PRG displayed 21.9 % degree of acetylation and 7.7 % degree of methylation. TLC and ESI-MS analysis of CtRGLf hydrolysed PRG showed unsaturated RG di-saccharide as the smallest product, with m/z 322. Deacetylated PRG-oligosaccharides displayed higher, 50 % inhibition of colon-cancer HCT-116 cells (with shrunken and globular morphology) than 35 % inhibition by acetylated PRG-oligosaccharides. FESEM and BET analysis of CtPae12B-treated PRG showed porous structure and significantly higher total surface area and pore volume than non-enzyme treated PRG. Higher drug entrapment efficiency and lower drug release rate of CtPae12B-treated PRG hydrogel (0.0033 min−1 at pH 1.2 and 0.009 min−1 at pH 7.4), than non-enzyme treated PRG hydrogel, (0.0057 min−1 at pH 1.2 and 0.02 min−1 at pH 7.4), showed it to be a potential biomaterial for sustainable colon-targeted drug delivery.

A thermotolerant and pH stable rhamnogalacturonan acetylesterase (CtPae12B), a family 12 carbohydrate esterase from Clostridium thermocellum with broad substrate specificity

The gene encoding rhamnogalacturonan acetylesterase, CtPae12B from Clostridium thermocellum was cloned, expressed, purified and biochemically characterized. Purified CtPae12B was soluble and exhibited homogenous single band. Phylogenetically it was most closely related to an RGAE, YesT from B. subtilis. CtPae12B production was maximum with LB medium. CtPae12B showed optimal temperature, 65 °C and thermostability with half-life, 5.1 h at 80 °C. CtPae12B was alkaliphilic with optimal pH, 8.0, while it displayed stability at both acidic and alkaline pH ranges. Inhibition of CtPae12B activity by PMSF showed the importance of nucleophilic serine in the catalytic triad. The metal ions, chemical or chelating agents used, did not enhance CtPae12B activity, which was also corroborated by protein melting study. The enzymatic activity of CtPae12B remained unaffected by 5 M urea. CtPae12B showed broad substrate specificity as it displayed activity against a range of synthetic substrates showing highest Vmax, 770 U/mg and Km, 1.2 mM with β-D-gluco pentaacetate. CtPae12B could deacetylate both pectic and xylan substrates showing highest Vmax, 770 U/mg and Km, 13.4 mg/mL with potato rhamnogalacturonan and Vmax, 105 U/mg and Km, 7.1 mg/mL with acetylated birchwood xylan. The thermostability, pH stability and broad substrate specificity of CtPae12B makes it a versatile enzyme for industrial applications.

Coexpression of Fungal Cell Wall-Modifying Enzymes Reveals Their Additive Impact on Arabidopsis Resistance to the Fungal Pathogen, Botrytis cinerea.

Simple SummaryIn the present study, we created transgenic Arabidopsis plants overexpressing two fungal acetylesterases and a fungal feruloylesterase that acts on cell wall polysaccharides and studied their possible complementary additive effects on host defense reactions against the fungal pathogen, Botrytis cinerea. Our results showed that the Arabidopsis plants overexpressing two acetylesterases together contributed significantly higher resistance to B. cinerea in comparison with single protein expression. Conversely, coexpression of either of the acetyl esterases together with feruloylesterase compensates the latter’s negative impact on plant resistance. The results also provided evidence that combinatorial coexpression of some cell wall polysaccharide-modifying enzymes might exert an additive effect on plant immune response by constitutively priming plant defense pathways even before pathogen invasion. These findings have potential uses in protecting valuable crops against pathogens.The plant cell wall (CW) is an outer cell skeleton that plays an important role in plant growth and protection against both biotic and abiotic stresses. Signals and molecules produced during host–pathogen interactions have been proven to be involved in plant stress responses initiating signal pathways. Based on our previous research findings, the present study explored the possibility of additively or synergistically increasing plant stress resistance by stacking beneficial genes. In order to prove our hypothesis, we generated transgenic Arabidopsis plants constitutively overexpressing three different Aspergillus nidulans CW-modifying enzymes: a xylan acetylesterase, a rhamnogalacturonan acetylesterase and a feruloylesterase. The two acetylesterases were expressed either together or in combination with the feruloylesterase to study the effect of CW polysaccharide deacetylation and deferuloylation on Arabidopsis defense reactions against a fungal pathogen, Botrytis cinerea. The transgenic Arabidopsis plants expressing two acetylesterases together showed higher CW deacetylation and increased resistance to B. cinerea in comparison to wild-type (WT) Col-0 and plants expressing single acetylesterases. While the expression of feruloylesterase alone compromised plant resistance, coexpression of feruloylesterase together with either one of the two acetylesterases restored plant resistance to the pathogen. These CW modifications induced several defense-related genes in uninfected healthy plants, confirming their impact on plant resistance. These results demonstrated that coexpression of complementary CW-modifying enzymes in different combinations have an additive effect on plant stress response by constitutively priming the plant defense pathways. These findings might be useful for generating valuable crops with higher protections against biotic stresses.

Protein engineering of a cold-adapted rhamnogalacturonan acetylesterase: In vivo functional expression and cinnamyl acetate synthesis

The rhamnogalacturonan acetylesterase gene (named pp1113), encoding a novel cold-adapted rhamnogalacturonan acetylesterase, was cloned from Paenibacillus polymyxa. pp1113 was functionally expressed in Escherichia coli by inducing expression at 20 °C for 16 h. The pH and temperature optimums for the recombinant enzyme were 8.5 and 30 °C, respectively. As an SGNH-type acetylesterase, pp1113 demonstrated high activity against acetyl ester substrates and could maintain approximately 60 % activity at 0 °C.The specific activity of purified pp1113 was 65.95 U/mg (C2). The Km and Vmax values of purified pp1113 were 3.89 mM and 81.07 μmol/min, respectively. We obtained an R232L variant with improved thermal stability and catalytic activity through site-directed mutagenesis. T1/2 of R232L at 50 °C increased from less than 30 min to 6 h when compared to wild-type pp1113, and T1/2 extended to 8 h after immobilization. The R232L mutant improved the conversion rate of 0.1 M cinnamyl alcohol at 40 °C to 94 % in the reaction of synthesis of cinnamyl acetate. After purification, the R232L mutant was immobilized (pH 8.0 phosphate buffer 0.5 M, 20 h at 20 °C, protein loading reached 17 mg/g). This was the first report of the rhamnogalacturonan acetylesterase gene obtained from Paenibacillus polymyxa with detailed enzymatic properties.

Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration

Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus.

Genome Sequence of the Multiple-Protease-Producing Strain Geobacillus thermoleovorans N7 , a Thermophilic Bacterium Isolated from Paniphala Hot Spring, West Bengal, India

Here, we present the draft genome sequence of Geobacillus thermoleovorans strain N7 (MCC 3175), isolated from Paniphala Hot Spring, West Bengal, India, which contains genes that encode several industrially and medically important thermostable enzymes like neutral protease, xylose isomerase, rhamnogalacturonan acetylesterase, nitrate and nitrite reductase, l-asparaginase, glutaminase, and RNase P.

Sequence Analysis and Heterologous Expression of Two Rhamnogalacturonan Acetylesterase Genes (<i>Asrgae1</i> and <i>Asrgaea2</i>) from a <i>Shoyu Koji</i> Mold, <i>Aspergillus sojae</i> KBN1340

Two rhamnogalacturonan acetylesterase genes, designated as Asrgae1 and Asrgae2, were isolated from a shoyu koji mold, Aspergillus sojae KBN1340, and characterized. Asrgae1 comprised 793 bp, encoding 248 amino acids with a predicted molecular mass of 26,092 Da, interrupted by a single putative intron of 46 bp in length. The mature AsRgae1 of 232 amino acids had a calculated molecular mass of 24,415 Da. The coding region of Asrgae2 was determined to be 777 bp in length with no introns. The predicted protein of 258 amino acids had an estimated molecular mass of 26,902 Da. The mature AsRgae2 of 238 amino acids had a calculated molecular mass of 25,015 Da. Utilizing the A. oryzae taaG2 gene promoter and the A. oryzae taaG3 gene terminator, AsRgae1 and AsRgae2 were successfully expressed in A. oryzae and secreted into the culture medium. AsRgae1 and AsRgae2 had a molecular mass of 28.0 kDa and 33.0 kDa, respectively.

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 26.7kDa 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.

Gene expression profiling of candidate virulence factors in the laminated root rot pathogen Phellinus sulphurascens

BackgroundPhellinus sulphurascens is a fungal pathogen that causes laminar root rot in conifers, one of the most damaging root diseases in western North America. Despite its importance as a forest pathogen, this fungus is still poorly studied at the genomic level. An understanding of the molecular events involved in establishment of the disease should help to develop new methods for control of this disease.ResultsWe generated over 4600 expressed sequence tags from two cDNA libraries constructed using either mycelia grown on cellophane sheets and exposed to Douglas-fir roots or tissues from P. sulphurascens-infected Douglas-fir roots. A total of 890 unique genes were identified from the two libraries, and functional classification of 636 of these genes was possible using the Functional Catalogue (FunCat) annotation scheme. cDNAs were identified that encoded 79 potential virulence factors, including numerous genes implicated in virulence in a variety of phytopathogenic fungi. Many of these putative virulence factors were also among 82 genes identified as encoding putatively secreted proteins. The expression patterns of 86 selected fungal genes over 7 days of infection of Douglas-fir were examined using real-time PCR, and those significantly up-regulated included rhamnogalacturonan acetylesterase, 1,4-benzoquinone reductase, a cyclophilin, a glucoamylase, 3 hydrophobins, a lipase, a serine carboxypeptidase, a putative Ran-binding protein, and two unknown putatively secreted proteins called 1 J04 and 2 J12. Significantly down-regulated genes included a manganese-superoxide dismutase, two metalloproteases, and an unknown putatively secreted protein called Ps0058.ConclusionsThis first collection of Phellinus sulphurascens EST sequences and its annotation provide an important resource for future research aimed at understanding key virulence factors of this forest pathogen. We examined the expression patterns of numerous fungal genes with potential roles in virulence, and found a collection of functionally diverse genes that are significantly up- or down-regulated during infection of Douglas-fir seedling roots by P. sulphurascens.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-603) contains supplementary material, which is available to authorized users.

Assays for Determination of Acetylesterase Activity and Specificity Using pNP-acetyl and Acetylated Polysaccharides as Substrates

The acetylesterases are hydrolytic enzymes which in plants cleave acetyl groups from acetylated cell wall components, primarily polysaccharides. To estimate acetylesterase activity in plant apoplast, two assays can be used. First assay is a direct measurement of the acetylesterase activity in protein extract using synthetic substrate, pNP-acetyl. In this assay, amount of pNP released after hydrolysis of pNP-acetyl is determined by measuring the intensity of developed yellow color using spectrophotometer. The absorbance of reaction mixture is directly proportional to the activity of acetylesterases in the reaction mixture. Second assay is a determination of acetylesterase activity and its specificity towards natural polysaccharides and based on interaction between ferric perchlorate and acetyl residues resulting in ferric acetohydroxamic complex that can be quantified using spectrophotometer. In this assay, commercially available acetylated polysaccharides (xylan from Birchwood for acetylxylan esterase; pectin from citrus fruit for rhamnogalacturonan acetylesterase; or any other available polysaccharide of interest) incubated with apoplastic extract and amount of acetyl residues released from this polysaccharide is estimated using ferric perchlorate reagent (protocol was modified from McComb and McCready, 1957). The absorbance of produced colored complex is directly proportional to the amount of acetyls released from acetylated polysaccharide.

Involvement of TBL/DUF231 proteins into cell wall biology

Through map-based cloning we determined TRICHOME BIREFRINGENCE (TBR) to belong to a plant-specific, yet anonymous gene family with 46 members in Arabidopsis thaliana. These genes all encode the domain of unknown function 231 (DUF231). TBR and its homolog TRICHOME BIREFRINGENCE-LIKE3 (TBL3) are transcriptionally coordinated with CELLULOSE SYNTHASE (CESA) genes, and loss of TBR or TBL3 results in decreased levels of crystalline secondary wall cellulose in trichomes and stems, respectively. Loss of TBR or TBL3 further results in increased pectin methylesterase (PME) activity and reduced pectin esterification in etiolated Arabidopsis hypocotyls. Together, the results suggest that DUF231 proteins might function in the maintenance of pectin- and probably homogalacturonan esterification, and that this is a requirement for normal secondary wall cellulose synthesis, at least in some tissues and organs. Here we expand the discussion about the role of TBL/DUF231 proteins in cell wall biology based on sequence and structure analyses. Our analysis revealed structural similarities of TBR with a rhamnogalacturonan acetylesterase (RGAE) of Aspergillus aculeatus and the protein LUSTRIN A-LIKE (Oryza sativa). The implications of these findings in regard to TBL functions are discussed.

Short strong hydrogen bonds in proteins: a case study of rhamnogalacturonan acetylesterase

An extremely low-field signal (at approximately 18 p.p.m.) in the (1)H NMR spectrum of rhamnogalacturonan acetylesterase (RGAE) shows the presence of a short strong hydrogen bond in the structure. This signal was also present in the mutant RGAE D192N, in which Asp192, which is part of the catalytic triad, has been replaced with Asn. A careful analysis of wild-type RGAE and RGAE D192N was conducted with the purpose of identifying possible candidates for the short hydrogen bond with the 18 p.p.m. deshielded proton. Theoretical calculations of chemical shift values were used in the interpretation of the experimental (1)H NMR spectra. The crystal structure of RGAE D192N was determined to 1.33 A resolution and refined to an R value of 11.6% for all data. The structure is virtually identical to the high-resolution (1.12 A) structure of the wild-type enzyme except for the interactions involving the mutation and a disordered loop. Searches of the Cambridge Structural Database were conducted to obtain information on the donor-acceptor distances of different types of hydrogen bonds. The short hydrogen-bond interactions found in RGAE have equivalents in small-molecule structures. An examination of the short hydrogen bonds in RGAE, the calculated pK(a) values and solvent-accessibilities identified a buried carboxylic acid carboxylate hydrogen bond between Asp75 and Asp87 as the likely origin of the 18 p.p.m. signal. Similar hydrogen-bond interactions between two Asp or Glu carboxy groups were found in 16% of a homology-reduced set of high-quality structures extracted from the PDB. The shortest hydrogen bonds in RGAE are all located close to the active site and short interactions between Ser and Thr side-chain OH groups and backbone carbonyl O atoms seem to play an important role in the stability of the protein structure. These results illustrate the significance of short strong hydrogen bonds in proteins.

Pectin acetylesterases from Aspergillus are able to deacetylate homogalacturonan as well as rhamnogalacturonan

An acetylesterase was purified from Aspergillus aculeatus on the basis of its ability to deacetylate homogalacturonan. A series of well-characterized substrates was prepared and used to investigate its specificity, including pectin with various degrees of methylation and acetylation, pectic domains with various structures, and oligomers. It was then compared to a rhamnogalacturonan acetylesterase previously isolated from the same fungus. Both enzymes were active towards various acetylated pectins, and were able to release acetyl groups from acetylated homogalacturonan, oligogalacturonates as well as rhamnogalacturonan at different extent. It was thus concluded that while the two enzymes differed in their efficiency, they are both pectin acetylesterases, able to act in the “smooth” as well as in the “hairy” regions of pectin.

Characterization of a New Rhamnogalacturonan Acetyl Esterase from Bacillus halodurans C-125 with a New Putative Carbohydrate Binding Domain

BH1115 is a gene from Bacillus halodurans strain C-125 that hypothetically encodes a rhamnogalacturonan acetyl esterase (RGAE) of the CE-12 family. As confirmation, this gene was cloned, and the product was expressed in Escherichia coli strain Rosetta (DE3) cells and purified. The enzyme obtained was monomeric, with a molecular mass of 45 kDa, and exhibited alkaliphilic properties. A study of the inhibition of the activity by some modulators confirmed that the catalytic triad for the esterase activity was Ser-His-Asp. This enzyme also presents broad substrate specificity and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, RGAE from B. halodurans achieves a synergistic effect with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The homology between the folds of RGAE from Aspergillus aculeatus and the hypothetical YxiM precursor from Bacillus subtilis, which both belong to the SGNH family, illustrates the divergence of such proteins from a common ancestor. Furthermore, the enzyme possesses a putative substrate binding region at the N terminus of the protein which has never been described to date for any RGAE.

A molecular and in silico characterization of Hev b 4, a glycosylated latex allergen

Hev b 4 is a heavily glycosylated latex allergen with seven attached N-glycans, comprising of both oligomannose and complex type structures. Treatment with a mixture of N-glycosidase A and N-glycosidase F resulted in lowering Hev b 4 protein on SDS-gel from 53 to 55 kDa to circa 40 kDa, this being comparable to the 38.53 kDa mass predicted by its cDNA. In Western-immunoblots, the enzymatically deglycosylated Hev b 4 showed negligible binding to IgE from latex allergic patients; the results indicated that IgE essentially binds to Hev b 4 via its N-glycan moiety. Structural modelling of the Hev b 4 was carried out based on the template protein and carbohydrate crystal coordinates of rhamnogalacturonan acetylesterase (PDB ID 1DEO). We managed to link four N-glycan structures on to the Hev b 4 model; the glycans were scattered over the surface of the model. The structural and functional features of Hev b 4 could prove useful to elucidate its exposed epitopes which are important for IgE binding.

YesT: A new rhamnogalacturonan acetyl esterase from Bacillus subtilis

YesT, a putative protein from Bacillus subtilis ATCC 6633 that has been provisionally classified as a rhamnogalacturonan acetyl esterase (RGAE) in CE-12 family, was cloned, expressed in Escherichiacoli Rosetta (DE3), and purified. The enzyme is monomeric with a molecular mass of 37 kDa and presents thermophilic properties similar to RGAE from Aspergillus aculeatus, although YesT is more alkaliphilic. The study of inhibitors confirmed the importance of the His and the nucleophilic Ser for the esterase activity, apart from the Asp from the catalytic triad. This enzyme also presents broad substrate specificity, and is active toward 7-aminocephalosporanic acid, cephalosporin C, p-nitrophenyl acetate, beta-naphthyl acetate, glucose pentaacetate, and acetylated xylan. Moreover, YesT achieves a synergistic effect together with xylanase A toward acetylated xylan. As a member of the SGNH family, it does not adopt the common alpha/beta hydrolase fold. The primary sequence analysis and multiple sequence alignment revealed the lack of a two beta-stranded antiparallel sheet, which results in a clear change in the structure together with the disappearance of one of the three 3(10)-helices presented in RGAE structure. The similarities found in this article among the topological diagrams of RGAE, YesT, and Esterase A from Streptomyces scabies, Platelet-Activating Factor AcetylHydrolase, isoform Ib, alpha subunit [PAF-AH(Ib)alpha(1)], PAF-AH(Ib)alpha(2), the esterase domain from hemagglutinin esterase fusion glycoprotein (HEF1) from Influenza C virus, the thioesterase I (TAP) from E. coli, the hypothetical protein a1r1529 from Nostoc sp., and the hypothetical YxiM precursor that all belong to the SGNH family could indicate a possible divergence of such proteins from a common ancestor.

Crystal packing in two pH-dependent crystal forms of rhamnogalacturonan acetylesterase.

The glycoprotein rhamnogalacturonan acetylesterase from Aspergillus aculeatus has been crystallized in two crystal forms, an orthorhombic and a trigonal crystal form. In the orthorhombic crystal form, the covalently bound carbohydrate at one of the two N-glycosylation sites is involved in crystal contacts. The orthorhombic crystal form was obtained at pH 5.0 and the trigonal crystal form at pH 4.5. In one case, the two crystal forms were found in the same drop at pH 4.7. The differences in crystal packing in the two crystal forms can be explained by the pH-dependent variation in the protonation state of the glutamic acid residues on the protein surface.

Expression profiling of pectinolytic genes from Aspergillus niger

The expression of 26 pectinolytic genes from Aspergillus niger was studied in a wild type strain and a CreA derepressed strain, under 16 different growth conditions, to obtain an expression profile for each gene. These expression profiles were then submitted to cluster analysis to identify subsets of genes with similar expression profiles. With the exception of the feruloyl esterase encoding genes, all genes were expressed in the presence of D-galacturonic acid, polygalacturonate, and/or sugar beet pectin. Despite this general observation five distinct groups of genes were identified. The major group consisted of 12 genes of which the corresponding enzymes act on the pectin backbone and for which the expression, in general, is higher after 8 and 24 h of incubation, than after 2 or 4 h. Two other groups of genes encoding pectin main chain acting enzymes were detected. Two additional groups contained genes encoding L-arabinose and D-galactose releasing enzymes, and ferulic acid releasing enzymes, respectively. The genes encoding β-galactosidase and the L-arabinose releasing enzymes were not only expressed in the presence of D-galacturonic acid, but also in the presence of L-arabinose, suggesting that they are under the control of two regulatory systems. Similarly, the rhamnogalacturonan acetylesterase encoding gene was not only expressed in the presence of D-galacturonic acid, polygalacturonate and sugar beet pectin, but also in the presence of L-rhamnose. The data presented provides indications for a general pectinolytic regulatory system responding to D-galacturonic acid or a metabolite derived from it. In addition, subsets of pectinolytic genes are expressed in response to the presence of L-arabinose, L-rhamnose or ferulic acid.

A branched N-linked glycan at atomic resolution in the 1.12 A structure of rhamnogalacturonan acetylesterase.

The crystal structure of the glycoprotein rhamnogalacturonan acetylesterase from Aspergillus aculeatus has been refined to a resolution of 1.12 A using synchrotron data collected at 263 K. Both of the two putative N-glycosylation sites at Asn104 and Asn182 are glycosylated and, owing to crystal contacts, the glycan structure at Asn182 is exceptionally well defined in the electron-density maps, showing the six-carbohydrate structure Manalpha1-6(Manalpha1-3)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAcbeta-Asn182. Equivalent carbohydrate residues were restrained to have similar geometries, but were refined without target values. The refined bond lengths and angles were compared with the values obtained from small-molecule studies that form the basis for the dictionaries used for glycoprotein refinement.

Enzymatic modification of pectic polysaccharides obtained from sugar beet pulp

Rhamnogalacturonans and arabinans, purified from an autoclave extract of sugar beet pulp, as well as an acid extracted beet pectin (ABP) were treated with enzymes in order to modify their physico-chemical properties. The enzymes used were arabinofuranosidase B (AF), endo-arabinanase plus arabinofuranosidase (EA+AF), rhamnogalacturonase plus rhamnogalacturonan acetyl esterase (RGase+RGAE), and polygalacturonase plus pectin methyl esterase (PG+PE). During the enzyme treatments the intrinsic viscosity ([η]w), apparent molecular weight (Mw), and radius of gyration (Rgw) of the polysaccharides were monitored.We found in most cases that treatment with glycanases decreased the [η]w of the polysaccharides investigated. However, results showed that degradation of the rhamnogalacturonan backbone of a beet pectin obtained by acid extraction had little influence on the [η]w which led to the conclusion that the rhamnogalacturonans present in this type of pectin are mainly located at the extremities of the pectin molecules. Some evidence was found that the rhamnogalacturonans obtained by autoclaving are linked through the arabinose side-chains, probably by diferulic acid cross-links. Removal of the side-chains of the arabinans present in the rhamnogalacturonans of ABPs with enzymes also showed that the arabinan side-chains contribute relatively little to the [η]w.In our experiments we found that modification with glycanases can be used to change the structural characteristics of pectic polysaccharides without a significant loss of viscosity. In this way glycanases can be used for the removal of structural elements of pectic polysaccharides, which limit their physico-chemical properties.