[The effect of a short-term group stabilisation training in patients with complex posttraumatic stress disorder]. / Het effect van een kortdurende groepsgewijze stabilisatietraining bij patiënten met een complexe posttraumatische stressstoornis.

BACKGROUND: The international guideline for complex posttraumatic stress disorder (ptsd) from the International Society for Traumatic Stress Studies (istss) recommends treatment in phases, starting with stabilisation treatment. Different forms of stabilisation training have been developed the past few years, one being short-term group stabilisation training.
AIM: To map out the effects of the short-term group training.
METHOD: The research implemented a pre-post design. The training consisted of five group sessions. Questionnaires (bsi, OQ 45 and svl-15) were completed both prior to and after the training. Four domains were assessed: psychosymptomatology in general, depressive symptoms, problems with interpersonal functioning and ptsd-related symptoms. The effect of the training was calculated by paired t-tests.
RESULTS: The questionnaires of the 47 participants who had completed the training were analysed. No significant decrease was observed during the stabilisation training concerning the symptoms of the four evaluated domains.
CONCLUSION: Contrary our expectations, a short-term group-based stabilisation training does not seem to have added value when treating patients with complex ptsd. The results correspond with a recent trend in which the effectiveness of other stabilising methods is questioned. Alternative treatment options are discussed.

Effects of supplementing concentrates differing in carbohydrate composition in veal calf diets: I. Animal performance and rumen fermentation characteristics.

The aim of this experiment was to examine the effects of concentrates in feed, differing in carbohydrate source, on the growth performance and rumen fermentation characteristics of veal calves. For this purpose, 160 Holstein Friesian x Dutch Friesian crossbred male calves were used in a complete randomized block design with a 5 x 2 factorial arrangement. Dietary treatments consisted of 1) milk replacer control, 2) pectin-based concentrate, 3) neutral detergent fiber-based concentrate, 4) starch-based concentrate, and 5) mixed concentrate (equal amounts of concentrates of treatments 2, 3, and 4). Concentrate diets were provided as pellets in addition to a commercial milk replacer. Calves were euthanized either at the end of 8 or 12 wk of age. The overall dry matter intake of the concentrate diets varied between 0.37 and 0.52 kg/d. Among the concentrate diets, the dry matter intake was lower in the starch diet (0.37 kg/d of dry matter) and differed between the NDF and pectin diets. The average daily gain for all the dietary treatments varied between 0.70 and 0.78 kg/d. The mixed- and NDF-fed calves had an increased average daily gain (0.78 and 0.77 kg/d, respectively) compared with the starch- and pectin-fed calves (0.70 and 0.71 kg/d, respectively). Rumen fermentation in the calves fed concentrates was characterized by a low pH (4.9 to 5.2), volatile fatty acid concentrations between 100 and 121 mmol/L, and high concentrations of reducing sugars (33 to 66 g/kg of dry matter). The volatile fatty acid concentrations of calves fed concentrates were higher than those of the control calves. All concentrate treatments showed a low acetate-to-propionate ratio in rumen fluid (between 1.3 and 1.9). Among the concentrates, the NDF diet had the highest (55.5%) and starch the lowest (45.5%) molar proportions of acetate. Calves fed the mixed, pectin, and starch diets had significantly higher molar proportions of butyrate (13.1 to 15.8%) than the NDF- and control-fed groups (9.9 and 9.6%, respectively). Calves fed the control diet had a higher lactate concentration (21 mmol/L) than the concentrate-fed calves (between 5 and 11 mmol/L). With the exception of the NDF diet, polysaccharide-degrading enzyme activities in the rumen contents generally showed an adaptation of the microorganisms to the carbohydrate source in the diet. The mixed diet exhibited the least variation in rumen polysaccharide-degrading enzyme activities among the enzymes systems tested. Results indicated that the carbohydrate source can influence intake, growth rate, and rumen fermentation in young veal calves.

Physico-chemical and transglucosylation properties of recombinant sucrose phosphorylase from Bifidobacterium adolescentis DSM20083.

Clones of a genomic library of Bifidobacterium adolescentis were grown in minimal medium with sucrose as sole carbon source. An enzymatic fructose dehydrogenase assay was used to identify sucrose-degrading enzymes. Plasmids were isolated from the positive colonies and sequence analysis revealed that two types of insert were present, which only differed with respect to their orientation in the plasmid. An open reading frame of 1,515 nucleotides with high homology for sucrose phosphorylases was detected on these inserts. The gene was designated SucP and encoded a protein of 56,189 Da. SucP was heterologously expressed in Escherichia coli, purified, and characterized. The molecular mass of SucP was 58 kDa, as estimated by SDS-PAGE, while 129 kDa was found with gel permeation, suggesting that the native enzyme was a dimer. The enzyme showed high activity towards sucrose and a lower extent towards alpha-glucose-1-phosphate. The transglucosylation properties were investigated using a broad range of monomeric sugars as acceptor substrate for the recombinant enzyme, while alpha-glucose-1-phosphate served as donor. D- and L-arabinose, D- and L-arabitol, and xylitol showed the highest production of transglucosylation products. The investigated disaccharides and trisaccharides were not suitable as acceptors. The structure of the transglucosylation product obtained with D-arabinose as acceptor was elucidated by NMR. The structure of the synthesized non-reducing dimer was alpha-Glcp(1-->1)beta-Araf.

Cloning and characterization of two alpha-glucosidases from Bifidobacterium adolescentis DSM20083.

Two alpha-glucosidase encoding genes (aglA and aglB) from Bifidobacterium adolescentis DSM 20083 were isolated and characterized. Both alpha-glucosidases belong to family 13 of the glycosyl hydrolases. Recombinant AglA (EC 3.2.1.10) and AglB (EC 3.2.1.20), expressed in Escherichia coli, showed high hydrolytic activity towards isomaltose and pnp-alpha-glucoside. The K(m) for pnp-alpha-glucoside was 1.05 and 0.47 mM and the V(max) was 228 and 113 U mg(-1) for AglA and AglB, respectively. Using pnp-alpha-glucoside as substrate, the pH optimum for AglA was 6.6 and the temperature optimum was 37 degrees C. For AglB, values of pH 6.8 and 47 degrees C were found. AglA also showed high hydrolytic activity towards isomaltotriose and, to a lesser extent, towards trehalose. AglB has a high preference for maltose and less activity towards sucrose; minor activity was observed towards melizitose, low molecular weight dextrin, maltitol, and maltotriose. The recombinant alpha-glucosidases were tested for their transglucosylation activity. AglA was able to synthesize oligosaccharides from trehalose and sucrose. AglB formed oligosaccharides from sucrose, maltose, and melizitose.

Polysaccharide hydrolases from leaves of Boscia senegalensis: properties of endo-(1-->3)-beta-D-glucanase.

The leaves of Boscia senegalensis are traditionally used in West Africa in cereal protection against pathogens, pharmacologic applications, and food processing. Activities of alpha-amylase, beta-amylase, exo-(1-->3, 1-->4)-beta-D-glucanase, and endo-(1-->3)-beta-D-glucanase were detected in these leaves. The endo-(1-->3)-beta-D-glucanase (EC 3.2.1.39) was purified 203-fold with 57% yield. The purified enzyme is a nonglycosylated monomeric protein with a molecular mass of 36 kDa and pI > or = 10.3. Its optimal activity occurred at pH 4.5 and 50 degrees C. Kinetic analysis gave Vmax, kcat, and Km 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)-beta-glucan chains in an endo fashion. This property is unusual for endo-acting (1-->3)-beta-D-glucanase from plants. The involvement of the enzyme in plant defense against pathogenic microorganisms such as fungi is discussed.

Characterization of arabinose and ferulic acid rich pectic polysaccharides and hemicelluloses from sugar beet pulp.

Pectic polysaccharides were extracted from sugar beet pulp to yield fractions representing homogalacturonans, rhamnogalacturonans, arabinans and relatively small amounts of glucomannans and xyloglucans. The homogalacturonans had an apparent molecular weight of 21 kDa and contained relatively high amounts of methyl esters and relatively low amounts of acetyl groups as compared with the ramified 'hairy' regions. Three populations which originated from the ramified 'hairy' regions of pectin were distinguished. Two of these were rhamnogalacturonans with high apparent molecular weights of 1300 and 120 kDa, respectively. These populations had a high Ara and ferulic acid content. Despite the high neutral sugar content, these rhamnogalacturonans strongly bound to a DEAE column. The third population which originated from the ramified 'hairy' regions was a neutral population, which did not interact with the DEAE column and had a low apparent molecular weight and a high Ara and ferulic acid content. The arabinan side-chains of the rhamnogalacturonans were heavily branched in all populations. Enzymatic degradation of the xyloglucans showed similarities with apple xyloglucans with respect to the substitution with Fuc and Gal.

Oxidative cross-linking of pectic polysaccharides from sugar beet pulp.

Oxidative cross-linking of three beet pectin extracts with hydrogen peroxide/peroxidase resulted in an increase in viscosity at low concentrations and in the formation of a gel at higher concentrations. Gels were formed using concentrations of 1.5% for an autoclave preparation and one obtained by an acid extraction and of 3% for a second autoclaved extract. It was shown that in the autoclave extracts only rhamnogalacturonans and possibly the arabinans participated in the cross-linking reaction. Cross-linking of the autoclave extracts with ammonium persulfate resulted in a decrease in reduced viscosity and molecular weight, although ferulic acid dehydrodimers were formed. Treatment of the acid extracted pectin with ammonium persulfate gave a slow increase in viscosity and the formation of a high-molecular-weight population was observed. For both oxidative systems, the 8-5 dehydrodimer was predominant after cross-linking.

Structural analysis of (methyl-esterified) oligogalacturonides using post-source decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

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.

Characterization of a novel beta-galactosidase from Bifidobacterium adolescentis DSM 20083 active towards transgalactooligosaccharides.

This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel beta-galactosidase (beta-Gal II). In cells grown on TOS, in addition to the lactose-degrading beta-Gal (beta-Gal I), another beta-Gal (beta-Gal II) was detected and it showed activity towards TOS but not towards lactose. beta-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. Beta-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. Beta-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35 degrees C. The enzyme showed highest V(max) values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. Beta-Gal II was active towards beta-galactosyl residues that were 1-->4, 1-->6, 1-->3, and 1 <--> 1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B39.

Lactococcus lactis subsp. cremoris B39 grown on whey permeate produced an exopolysaccharide containing L-Rha, D-Gal and D-Glc in a molar ratio of 2:3:2. The polysaccharide was modified using an enzyme preparation from Aspergillus aculeatus, resulting in the release of Gal and a polymer with approximately the same hydrodynamic volume as the native polysaccharide. Linkage analysis and 1H NMR studies of both the native and modified exopolysaccharides elucidated that terminally linked Gal was released during modification and that the chemical structure of the branches within the repeating units is: beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. 2D NMR experiments (both 1H-1H and 1H-13C) revealed that exopolysaccharide B39 consists of a branched heptasaccharide repeating unit with the following structure: [structure: see text].

A universal assay for screening expression libraries for carbohydrases.

Although many assays are available for the screening of expression libraries for carbohydrases, some enzymes cannot be detected because their substrates are incompatible with the existing assays. One thing that all carbohydrases have in common is that they increase the number of reducing ends when degrading their substrates. In this paper we explore the possibility of detecting this increase with the highly sensitive bicinchoninic acid (BCA) reducing value assay. This assay can be used for the detection of all carbohydrases degrading any polysaccharide; enzymes with either an exo- or an endo-type of mechanism can be detected at the same time. A cDNA library of Aspergillus tubigensis expressed in Kluyveromyces lactis clones, was screened with this assay for the presence of xylogalacturonan degrading enzyme(s). High background absorbances caused by culture medium, by proteins produced by the clones and by substrate could be dealt with by using the precautions described in this note. Three xylogalacturonase producing clones were found using this procedure.

Transglycosidase activity of Bifidobacterium adolescentis DSM 20083 alpha-galactosidase.

Bifidobacterium adolescentis, a gram-positive saccharolytic bacterium found in the human colon, can, alongside other bacteria, utilise stachyose in vitro thanks to the production of an alpha-galactosidase. The enzyme was purified from the cell-free extract of Bi. adolescentis DSM 20083T. It was found to act with retention of configuration (alpha-->alpha), releasing alpha-galactose from p-nitrophenyl galactoside. This hydrolysis probably operates with a double-displacement mechanism, and is consistent with the observed glycosyltransferase activity. As alpha-galactosides are interesting substrates for bifidobacteria, we focused on the production of new types of alpha-galactosides using the transgalactosylation activity of Bi. adolescentis alpha-galactosides. Starting from melibiose, raffinose and stachyose oligosaccharides could be formed. The transferase activity was highest at pH 7 and 40 degrees C. Starting from 300 mM melibiose a maximum yield of 33% oligosaccharides was obtained. The oligosaccharides formed from melibiose were purified by size-exclusion chromatography and their structure was elucidated by NMR spectroscopy in combination with enzymatic degradation and sugar linkage analysis. The trisaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp-(1-->6)-D-Glcp and tetrasaccharide alpha-D-Galp-(1-->6)-alpha-D-Galp- (1-->6)-alpha-D-Galp-(1-->6)-D-Glcp were identified, and this indicates that the transgalactosylation to melibiose occurred selectively at the C-6 hydroxyl group of the galactosyl residue. The trisaccaride alpha-D-Galp-(1-->6)-alpha- D-Galp-(1-->6)-D-Glcp formed could be utilised by various intestinal bacteria, including various bifidobacteria, and might be an interesting pre- and synbiotic substrate.

Endoglucanase V and a phosphatase from Trichoderma viride are able to act on modified exopolysaccharide from Lactococcus lactis subsp. cremoris B40.

EPS B40 from Lactococcus lactis subsp. cremoris consists of a repeating unit of-->4)-beta-D-Glcp-(1-->4)-[alpha-L-Rhap-(1 -->2)][alpha-D-Galp-1-PO4-3]-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->. A phosphatase from Trichoderma viride was able to release phosphate, but only after removal of rhamnosyl and galactosyl residues by mild CF3CO2H treatment. Purified endoV from T. viride was able to act on the backbone of the polymer, but only if rhamnosyl substituents and phosphate had been removed. After complete removal of phosphate and partial removal of rhamnosyl residues by HF treatment, incubation with endoV resulted in a homologous series of oligomers. Purification of these oligomers and subsequent characterisation by NMR demonstrated that endoV was able to cleave the beta-(1-->4) linkage between two glucopyranosyl residues when the galactopyranosyl residue towards the nonreducing end is unsubstituted. The mode of action of endoV on HF-treated EPS B40 is discussed on the basis of the subsite model described for endoV [J.-P. Vincken, G. Beldman, A.G.J. Voragen, Carbohydr. Res., 298 (1997) 299-310].

Mode of action of RG-hydrolase and RG-lyase toward rhamnogalacturonan oligomers. Characterization of degradation products using RG-rhamnohydrolase and RG-galacturonohydrolase.

The mode of action of RG-hydrolase and RG-lyase toward purified linear rhamnogalacturonan (RG) oligomers has been studied. Major tools in the characterization of the degradation products were the exo-acting RG-rhamnohydrolase and RG-galacturonohydrolase. They were used to prepare a series of standards of RG oligomers for HPAEC. 1H NMR spectroscopy confirmed the structure assignment made using HPAEC for a selection of isolated degradation products. Identification of degradation products from purified RG oligomers was then performed by comparing retention times of HPAEC peaks with those of standards. RG-hydrolase was able to cleave RG oligomers which contained five Rha units or more, i.e. DP 9 with a Rha unit at both nonreducing and reducing end. Its preferential cleavage site was at four units from the first nonreducing Rha. RG-lyase was active toward oligomers that contained at least six GalA units, i.e. DP 12 with a GalA at the nonreducing and a Rha at the reducing end. The preferential cleavage site was for the smaller oligomers four residues, and for the largest oligomer six residues from the reducing Rha. From the observed cleavage patterns it can be speculated that in hairy regions, the RG stretches have to be at least 13 residues long for RG-hydrolase and 16 residues long for RG-lyase in order to produce one tetramer.

Enzymic degradation of sorghum glucuronoarabinoxylans leading to tentative structures.

Three glucuronoarabinoxylan (GAX) populations, obtained from water-unextractable cell wall material from sorghum by different alkali extractants, were digested by combinations of endo-xylanases (Xyl I, Xyl III and GXH), arabinofuranosidases (AXH and AraB) and an alpha-glucuronidase (GlcAase). All three GAX populations were shown to be rather poorly degradable, due to the very high degree of substitution, as well as the substitution pattern. The barium hydroxide-extracted GAX showed a maximum degree of degradation of almost 12%, using Xyl I combined with GXH and AXH. The GAX population extracted by 4 M KOH was hardly degraded by any of the tested combinations. In all cases, Xyl III showed lowest activity upon the three extracts. Synergistic effects were observed between Xyl I and AXH. Both neutral and acidic arabinoxylan oligomers were formed. The GlcAase acted only upon oligomeric material released by Xyl I. No synergistic effects were observed between the GXH and AXH. Combining the patterns of degradation with the modes of action of the enzymes, structures were proposed for the different populations of sorghum GAX. Evidence was obtained that the xylan backbone of especially the GAX extracted by 4 M KOH, is substituted by arabinose and glucuronic acid according to a strict pattern, which hinders the enzymes to act.

Structures of enzymically derived oligosaccharides from sorghum glucuronoarabinoxylan.

Oligosaccharides derived from alkali-extracted sorghum glucuronoarabinoxylan by digestion with a combination of (1-->4)-beta-D-arabinoxylan arabinofuranohydrolase (AXH) and endo-(1-->4)-beta-D-xylanase (Xyl I), both from Aspergillus awamori, were purified by size-exclusion chromatography followed by preparative high-performance anion-exchange chromatography. Structural studies including monosaccharide analysis, methylation analysis, 1H NMR spectroscopy, and mass spectrometry were carried out, resulting in the characterisation of four novel oligosaccharides, namely, alpha-D-GlcpA-(1-->2)-beta-D-Xyl p-(1-->4)-beta-D-Xyl p-(1-->4) -beta-D-Xyl p, alpha-D-GlcpA-(1-->2)-beta-D-Xyl p-(1-->4) [alpha-L-Araf-(1-->3)]-beta-D-Xyl p-(1-->4)-beta-D-Xyl p, alpha-D-GlcpA-(1-->2)-beta-D- Xyl p-(1-->4)-beta-D-Xyl p-(1-->4)[alpha-L-Araf-(1-->2)-alpha)-alpha- L-Araf-(1-->3)]-beta-D-Xyl p-(1-->4)-beta-D-Xyl p, and alpha-D-GlcpA-(1-->2) -beta-D-Xyl p-(1-->4)[alpha-L-Araf-(1-->3] beta-D-Xyl p-(1-->4)[alpha-L-Araf-(1-->2)-alpha-L-Araf-(1-->3)] -beta-D-Xyl p-(1-->4)-beta-D-Xyl p. The various oligosaccharides identified provide additional insight into the structure of sorghum glucuronoarabinoxylan. Furthermore, novel data were generated with respect to the substrate specificity of AXH and Xyl I towards glucuronoarabinoxylans in general.

Characterization of recombinant rhamnogalacturonan alpha-L-rhamnopyranosyl-(1,4)-alpha-D-galactopyranosyluronide lyase from Aspergillus aculeatus. An enzyme that fragments rhamnogalacturonan I regions of pectin.

The four major oligomeric reaction products from saponified modified hairy regions (MHR-S) from apple, produced by recombinant rhamnogalacturonan (RG) alpha-L-rhamnopyranosyl-(1, 4)-alpha-D-galactopyranosyluronide lyase (rRG-lyase) from Aspergillus aculeatus, were isolated and characterized by 1H-nuclear magnetic resonance spectroscopy. They contain an alternating RG backbone with a degree of polymerization of 4, 6, 8, and 10 and with an alpha-Delta-(4,5)-unsaturated D-galactopyranosyluronic acid at the nonreducing end and an L-rhamnopyranose at the reducing end. L-Rhamnopyranose units are substituted at C-4 with beta-galactose. The maximum reaction rate of rRG-lyase toward MHR-S at pH 6.0 and 31 degreesC was 28 units mg-1. rRG-lyase and RG-hydrolase cleave the same alternating RG I subunit in MHR. Both of these enzymes fragment MHR by a multiple attack mechanism. The catalytic efficiency of rRG-lyase for MHR increases with decreasing degree of acetylation. Removal of arabinose side chains improves the action of rRG-lyase toward MHR-S. In contrast, removal of galactose side chains decreased the catalytic efficiency of rRG-lyase. Native RG-lyase was purified from A. aculeatus, characterized, and found to be similar to the rRG-lyase expressed in Aspergillus oryzae.

Rhamnogalacturonan alpha-d-galactopyranosyluronohydrolase. An enzyme that specifically removes the terminal nonreducing galacturonosyl residue in rhamnogalacturonan regions of pectin

A new enzyme, rhamnogalacturonan (RG) alpha-d-galactopyranosyluronohydrolase (RG-galacturonohydrolase), able to release a galacturonic acid residue from the nonreducing end of RG chains but not from homogalacturonan, was purified from an Aspergillus aculeatus enzyme preparation. RG-galacturonohydrolase acted with inversion of anomeric configuration, initially releasing beta-d-galactopyranosyluronic acid. The enzyme cleaved smaller RG substrates with the highest catalytic efficiency. A Michaelis constant of 85 &mgr;m and a maximum reaction rate of 160 units mg-1 was found toward a linear RG fragment with a degree of polymerization of 6. RG-galacturonohydrolase had a molecular mass of 66 kD, an isoelectric point of 5.12, a pH optimum of 4.0, and a temperature optimum of 50 degreesC. The enzyme was most stable between pH 3.0 and 6.0 (for 24 h at 40 degreesC) and up to 60 degreesC (for 3 h).

Stereochemical course of hydrolysis catalysed by alpha-L-rhamnosyl and alpha-D-galacturonosyl hydrolases from Aspergillus aculeatus.

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-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 alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-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. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

Action patterns and mapping of the substrate-binding regions of endo-(1-->5)-alpha-L-arabinanases from Aspergillus niger and Aspergillus aculeatus.

The substrate binding sites of endo-(1-->5)-alpha-L-arabinanases (EC 3.2.1.99) from Aspergillus niger and Aspergillus aculeatus were investigated using reduced and regular (1-->5)-alpha-L-arabino-oligosaccharides and high performance anion exchange chromatographic analysis. Calculation of bond cleavage frequencies and kcat/K(m) parameters for these substrates enabled the determination of the number of arabinofuranosyl binding subsites and the estimation of the binding affinities of each subsite. The A. aculeatus endo-arabinanase has six subsites arranged symmetrically around the catalytic site, while the A. niger endo-arabinanase has five subsites; two from the catalytic site towards the non-reducing end of the bound substrate and three toward the reducing end. The two subsites directly adjacent to the catalytic sites in both the A. niger and A. aculeatus endo-arabinanase have near-zero net free energy of binding. These results are unlike most glycopyranosyl endo-hydrolases studied which have net negative (unfavourable) energies of interaction at these two subsites, and may be related to the greater conformational flexibility of arabinofuranosyl residues than glycopyranosyl residues. The complete subsite maps are also rationalized with regard to the observed action patterns of these enzymes on linear (1-->5)-alpha-L-arabinan.