Search

The fungus Aspergillus niger is an industrial producer of pectin-degrading enzymes. The recent solving of the genomic sequence of A. niger allowed an inventory of the entire genome of the fungus for potential carbohydrate-degrading enzymes. By applying bioinformatics tools, 12 new genes, putatively encoding family 28 glycoside hydrolases, were identified. Seven of the newly discovered genes form a new gene group, which we show to encode exoacting pectinolytic glycoside hydrolases. This group includes four exo-polygalacturonan hydrolases (PGAX, PGXA, PGXB and PGXC) and three putative exo-rhamnogalacturonan hydrolases (RGXA, RGXB and RGXC). Biochemical identification using polygalacturonic acid and xylogalacturonan as substrates demonstrated that indeed PGXB and PGXC act as exo-polygalacturonases, whereas PGXA acts as an exo-xylogalacturonan hydrolase. The expression levels of all 21 genes were assessed by microarray analysis. The results from the present study demonstrate that exo-acting glycoside hydrolases play a prominent role in pectin degradation.

XGH (xylogalacturonan hydrolase; GH 28) is an enzyme that is capable of degrading XGA (xylogalacturonan), which is a polymer of α-D-galacturonic acid, highly substituted with β-D-xylose. XGA is present in cell walls of various plants and exudates, such as gum tragacanth. XGA oligosaccharides were derived from an XGH digestion of gum tragacanth, then fractionated, and analysed for their sugar composition and structure by matrix-assisted laser-desorption ionization–time-of-flight MS and nanospray MS. Several oligosaccharides from XGA were identified with different galacturonic acid/xylose ratios including five oligosaccharide isomers. Although XGH can act as an endo-enzyme, product-progression profiling showed that the disaccharide GalAXyl was predominantly produced from XGA by XGH, which indicated also an exolytic action. The latter was further supported by degradation studies of purified oligosaccharide GalA4Xyl3. It was shown that XGH acted from the non-reducing end towards the reducing end of this oligosaccharide, and showed the processive character of XGH. The results from this study further show that although XGH prefers to act between two xylosidated GalA units, it tolerates unsubstituted GalA units in its −1 and +1 subsites.

It is claimed that bifidobacteria have several health-promoting effects. To increase the amount of bifidobacteria in the colon the concept of probiotics and/or prebiotics can be applied. Bifidobacterium adolescentis is one of the main species of bifidobacteria in the gastro-intestinal tract of human adults. B. adolescentis is able to degrade a wide range of oligosaccharides and a number of glycosyl hydrolases have been characterized in detail. The hydrolytic activity of the glycosyl hydrolases from B. adolescentis toward prebiotics like arabinoxylan-oligosaccharides, isomalto-oligosaccharides, arabinogalactan, and sucrose-based oligosaccharides (raffinose, stachyose, and fructo-oligosaccharides) is reviewed. Alternatively, some of these glycosyl hydrolases are able to catalyze transglycosylation, which allows them to elongate oligosaccharides and to prepare potentially prebiotic oligosaccharides. Such oligosaccharides might be used to influence the microbial composition in the more distal parts of the colon. In nature, not all enzyme-substrate encounters are transglycosylating. So, the hydrolytic activity of the enzyme makes the oligosaccharide elongation less efficient than desired. Site-directed mutagenesis was applied to improve the transglycosylation reaction of the ?-galactosidase from B. adolescentis. Les glycosyl hydrolases de Bifidobacterium adolescentis DSM20083. Il est affirm? que les bifidobact?ries ont des propri?t?s b?n?fiques sur la sant?. Pour augmenter la teneur en bifidobact?ries au niveau du colon, le concept de probiotiques et/ou pr?biotiques peut ?tre appliqu?. Bifidobacterium adolescentis est l?une des principales esp?ces de bifidobact?ries dans le tractus gastro-intestinal des adultes. Certaines des glycosyl hydrolases de B. adolescentis ont ?t? caract?ris?es en d?tail et elles semblent d?grader un large ?ventail d?oligosaccharides. L?activit? d?hydrolyse des glycosyl hydrolases de B. adolescentis envers des pr?biotiques tels que les arabino-xylanes, isomalto-oligosaccharides, arabino-galactanes et les oligosaccharides contenant du saccharose (raffinose, stachyose et fructo-oligosaccharides) est pass?e en revue. De plus, certaines de ces glycosyl hydrolases peuvent ?tre utilis?es pour pr?parer des oligosaccharides pr?biotiques potentiels par transglycosylation, ce qui permet d?allonger les oligosaccharides. De tels oligosaccharides peuvent ?tre utilis?s pour influencer la composition microbienne au niveau des parties plus distales du colon. Les produits de rencontre enzymes-substrates ne sont pas tous transglycosyl?s naturellement et l?activit? d?hydrolyse de l?enzyme entra?ne une ?longation de l?oligosaccharide moindre que celle d?sir?e. La mutagen?se dirig?e a ?t? appliqu?e pour am?liorer la r?action de transglycosylation de l??-galactosidase de B. adolescentis.

It is claimed that bifidobacteria have several health-promoting effects. To increase the amount of bifidobacteria in the colon the concept of probiotics and/or prebiotics can be applied. Bifidobacterium adolescentis is one of the main species of bifidobacteria in the gastro-intestinal tract of human adults. B. adolescentis is able to degrade a wide range of oligosaccharides and a number of glycosyl hydrolases have been characterized in detail. The hydrolytic activity of the glycosyl hydrolases from B. adolescentis toward prebiotics like arabinoxylan-oligosaccharides, isomalto-oligosaccharides, arabinogalactan, and sucrose-based oligosaccharides (raffinose, stachyose, and fructo-oligosaccharides) is reviewed. Alternatively, some of these glycosyl hydrolases are able to catalyze transglycosylation, which allows them to elongate oligosaccharides and to prepare potentially prebiotic oligosaccharides. Such oligosaccharides might be used to influence the microbial composition in the more distal parts of the colon. In nature, not all enzyme-substrate encounters are transglycosylating. So, the hydrolytic activity of the enzyme makes the oligosaccharide elongation less efficient than desired. Site-directed mutagenesis was applied to improve the transglycosylation reaction of the $\alpha$-galactosidase from B. adolescentis.

Investigations on the mode of action of Aspergillus niger pectin methylesterase (PME) towards differently C6- and C1-substituted oligogalacturonides (oligoGalpA) are described. De-esterification of methyl-esterified (un)saturated oligoGalpA proceeds via a specific pattern, depending on the degree of polymerization. Initially, a first methyl ester of the oligomer is hydrolysed, resulting in one free carboxyl group. Subsequently, this first product is preferred as a substrate and is de-esterified for a second time. This product is then accumulated and hereafter de-esterified further to the final product, i.e. oligoGalpA containing one methyl ester located at the non-reducing end residue for both saturated and unsaturated oligoGalpA, as found by post-source decay matrix-assisted laser-desorption/ionization–time-of-flight MS. The saturated hexamer is an exception to this: three methyl esters are removed very rapidly, instead of two methyl esters. When unsaturated oligoGalpA were used, the formation of the end product differed slightly, suggesting that the unsaturated bond at the non-reducing end influences the de-esterification process. In vivo, PME prefers methyl esters, but the enzyme appeared to be tolerant for other C6- and C1-substituents. Changing the type of ester (ethyl esterification) or addition of a methyl glycoside (C1) only reduced the activity or had no effect respectively. The specific product pattern was identical for all methyl- and ethyl-esterified oligoGalpA and methyl-glycosidated oligoGalpA, which strongly indicates that one or perhaps two non-esterified oligoGalpA are preferred in the active-site cleft.

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.

Abstract: The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of α-amylase, β-amylase, exo-(1→3, 1→4)-β-d-glucanase, and endo-(1→3)-β-d-glucanase were detected in these leaves. The endo-(1→3)-β-d-glucanase (EC3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI≥10.3. Its optimal activity occurred at pH 4.5 and 50°C. Kinetic analysis gave V max, k cat , and K m values of 659 U/mg, 395 s−1, and 0.42 mg/mL, respectively, for laminarin as substrate. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry and high-performance liquid chromatography revealed that the enzyme hydrolyzes not only soluble but also insoluble (1→3)-β-glucan chains in an endo fashion. This property is unusual for endo-acting (1→3)-β-d-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.

The use of post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the structural analysis of ((partly) methyl-esterified) oligogalacturonides (oligoGalA) is described. The fragmentation behavior of purified (un)saturated oligoGalA (degree of polymerization 3–6), methyl-esterified and methyl-glycosydated oligoGalA was studied. General fragmentation patterns are described and used for the elucidation of the positions of methyl esters on partly methyl-esterified oligoGalA. This technique now permits the determination of the position of methyl esters or other substituents on pectic fragments, helping in understanding the mode of action of pectinolytic enzymes. Copyright © 2000 John Wiley & Sons, Ltd.

Alterations in cell wall composition during low temperature acclimation have been investigated. Low temperature temporarily ceased the growth of winter wheat roots and reduced wall matrix polysaccharide content. The 14C-glucose incorporation into all polysaccharide fractions of cell wall was strongly inhibited by the 6th h of low temperature treatment. After 24 h of plant exposure to low temperature the rate of incorporation of a label into cellulose was still low; incorporation into pectins was restored to about initial level, whereas hemicellulose synthesis considerably increased compared with the initial level. Analyses of monosaccharide composition and leakages in different fractions of wall matrix polysaccharides showed that during the first hours of low temperature exposure a decrease in the proportion of glucans and branched glucoronoarabinoxylans took place, indicating the most dramatic modifications of hemicelluloses. At the same time an increase of some glycosidase activities was observed (β-fucosidase, β-glucosidase and β-cellobiosidase). The results obtained demonstrate involvement of cell wall metabolism (including anabolic and catabolic processes) in adaptation reactions of plants to low temperature, which probably is aimed at the alteration of physical properties of the wall.

A bacterium belonging to the Bacillus firmus/lentus-complex and capable of growth on native potato starch was isolated from sludge of a pilot plant unit for potato-starch production. Utilization of a crude enzyme preparation obtained from the culture fluid after growth of the microorganism on native starch, resulted in complete degradation of native starch granules from potato, maize and wheat at a temperature of 37°C. Glucose was found as a major product. Production of maltose, maltotriose and maltotetraose was also observed. Native-starch-degrading activity (NSDA) could be selectively adsorbed on potato-starch granules, whereas soluble-starch-degrading activity (SSDA) remained mainly in solution. The use of such a starch-adsorbed enzyme preparation on native starch resulted in a completely changed product pattern. An increase in oligosaccharides concomitant with less glucose formation was observed. An increased conversion of soluble starch to maltopentaose was possible with this starch-adsorbed enzyme preparation. It is concluded that NSDA comes from a-amylase(s) and SSDA from glucoamylase(s) and/or a-glucosidase(s). Cultivation of B. firmus/lentus on glucose, maltose, or soluble starch resulted in substantially smaller quantities of (native) starch-degrading activity.

The stereochemical course of hydrolysis catalysed by fourAspergillus aculeatusenzymes acting on α-l-rhamnosyl and α-d-galacturonosyl linkages in the hairy regions of pectins has been determined using1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e→a), shown by the initial release of β-d-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e→a) during hydrolysis of α-d-GalpA-(1→2)-α-l-Rhaplinkages in RG, initially releasing oligosaccharides with β-d-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. α-l-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a→e), initially releasing β-l-Rhapfromp-nitrophenyl α-l-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

The stereochemical course of hydrolysis catalyzed by various enzymes acting on arabinofuranosyl linkages has been determined. 1H-NMR analysis of the action of endo-(1 → 5)-α- l- arabinanasesfrom Aspergillus nigerand Aspergillus aculeatusshowed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. nigerenzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of α- l-arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatumand Bacillus subtiliswere investigated using both 1H-NMR and high performance anion exchange chromatography to follow glycosyl transfer reactions to methanol. In all cases these enzymes catalyzed the reaction with retention of configuration, and therefore probably operate via double displacement hydrolytic mechanisms. From the results with arabinofuranosidase A and B from A. nigerwe predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1 → 4)-β- d- arabinoxylanarabinohydrolases from A. awamori, Trichoderma reeseiand Bifidobacterium adolescentisonly enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.

The stereochemical course of hydrolysis catalyzed by various enzymes acting on arabinofuranosyl linkages has been determined. 1H‐NMR analysis of the action of endo‐(1 → 5)‐α‐l‐arabinanases from Aspergillus nigerand Aspergillus aculeatusshowed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. nigerenzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of α‐l‐arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatumand Bacillus subtiliswere investigated using both 1H‐NMR and high performance anion exchange chromatography to follow glycosyl transfer reactions to methanol. In all cases these enzymes catalyzed the reaction with retention of configuration, and therefore probably operate via double displacement hydrolytic mechanisms. From the results with arabinofuranosidase A and B from A. nigerwe predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1 → 4)‐β‐d‐arabinoxylan arabinohydrolases from A. awamori, Trichoderma reeseiand Bifidobacterium adolescentisonly enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.

The maceration of potato tuber (Solanum tuberosum cv Bintje) by technical enzyme preparations was examined with the aid of fluorescence microscopy using fluorescein diacetate as a dye. Treatment with Pectinex Ultra-SP-L from Aspergillus aculeatus resulted in a higher release of single viable cells and smaller clumps of cells than treatment with Rohament P from Aspergillus niger. Fractions with high pectin lyase and low polygalacturonase activity obtained by anion exchange chromatography of Pectinex Ultra SP-L showed the highest degree of maceration. When two different cloned polygalacturonases (PG1 and PG2) and a cloned pectin lyase (PL1) from A aculeatus were examined, the highest degree of maceration was observed with PL1. In conclusion, pectin lyase is probably the main enzyme in Pectinex Ultra-SP-L responsible for the maceration of potato tuber. © 1997 SCI.

Efficient enzymic degradation of cellulose in isolated cell wall material of apples requires prior removal of its xyloglucan coating. In this study, raw and blanched apple fruit tissues were treated with pectin lyase and various (combinations of purified) cellulases. These experiments confirmed that xyloglucanase activity is important for cellulose degradation in apple fruit tissue. Apart from this, it was observed that raw apple material disintegrated faster than blanched. Typically, the release of xyloglucan oligosaccharide XXXG from raw apple material was slower (relative to XXFG) when compared to that from blanched material. The endogenous enzyme, xyloglucan endotransglycosylase (XET), is probably responsible for these phenomena. It is hypothesised that XET activity accelerates disintegration of apple tissue once its depolymerising mode is triggered by xyloglucan oligosaccharides released by exogenous endoglucanases. © 1997 SCI.

In order to assess the role of endogenous enzymes during apple juice manufacturing by enzymic liquefaction, apples of different maturity were assayed for various enzyme activities. Xyloglucan endotransglycosylase (XET) activity increased dramatically from early September to mid October, and decreased slowly during cold storage. It was shown that XET is probably the most important, endo-acting activity in apple. The apples of different maturity were also treated by pectin lyase and various concentrations of cellulase. It appeared that less cellulase was needed for disintegration of apples with a high level of XET activity. This suggests that XET facilitates the liquefaction process. Under certain conditions, ‘high XET’ apples could be transformed into a cloudy apple juice. This cloud may be regarded as a dispersion of particles which is predominantly composed of cellulose and xyloglucan. © 1998 Society of Chemical Industry.