[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.

The Effect of Xyloglucans on the Degradation of Cell-Wall-Embedded Cellulose by the Combined Action of Cellobiohydrolase and Endoglucanases from Trichoderma viride.

Two endoglucanases of Trichoderma viride, endoI and endoIV, were assayed for their activity toward alkali-extracted apple xyloglucans. EndoIV was shown to have a 60-fold higher activity toward xyloglucan than endoI, whereas carboxymethyl cellulose and crystalline cellulose were better substrates for the latter. The enzymic degradation of cellulose embedded in the complex cell-wall matrix of apple fruit tissue has been studied using cellobiohydrolase (CBH) and these two different endoglucanases. A high-performance liquid chromatographic method (Aminex HPX-22H) was used to monitor the release of cellobiose and oligomeric xyloglucan fragments. Synergistic action between CBH and endoglucanases on cell-wall-embedded cellulose was, with respect to their optimal ratio, slightly different from that reported for crystalline cellulose. The combination of endoIV and CBH solubilized twice as much cellobiose compared to a combination of endoI and CBH. Apparently, the concomitant removal of the xyloglucan coating from cellulose microfibrils by endoIV is essential for an efficient degradation of cellulose in a complex matrix. Cellulose degradation slightly enhanced the solubilization of xyloglucans. These results indicate optimal degradation of cell-wall-embedded cellulose by a three-enzyme system consisting of an endoglucanase with high affinity toward cellulose (endoI), a xyloglucanase (endoIV), and CBH.

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).

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis.

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.

Cloning and expression of the branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli.

Using the glgB gene from Escherichia coli as a hybridization probe, the gene encoding the branching enzyme of the cyanobacterium Synechococcus sp. PCC7942 has been identified on a 3.9-kb PstI fragment which was cloned into plasmid pUC9. Two types of plasmids have been isolated. Plasmid pKVN1 was expressing the Synechococcus sp. gene as was shown by complementation of the glgB mutation of E. coli KV832. Plasmid pKVN2, which carried the same insert in the opposite orientation was unable to complement E. coli KV832, indicating that the promoter of the cloned gene was either absent or was not recognized in E. coli. Determination of branching activity in extracts of Synechococcus sp. and E. coli KV832[pKVN1] showed that the enzyme was optimally active at approximately 35 degrees C. No significant activity was present at temperatures higher than 55 degrees C, reflecting the mesophilic nature of the cloned enzyme. In a cell-free coupled transcription-translation system the cloned gene specified two proteins of 84 kDa and 72 kDa, respectively, which are probably translated independently from the same gene by initiation at two different start codons.

Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases.

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)-alpha-L-arabinanases from Aspergillus niger and Aspergillus aculeatus showed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. niger enzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of alpha-L-arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatum and Bacillus subtilis were 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. niger we predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1-->4)-beta-D-arabinoxylan arabinohydrolases from A. awamori, Trichoderma reesei and Bifidobacterium adolescentis only enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.

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.

Substrate specificity of endoglucanases: what determines xyloglucanase activity?

Endoglucanases from Trichoderma viride differ in their activity and mode of action towards xyloglucans. In order to explain the basis for their different behavior, the number of substrate-binding sites of three endoglucanases (endoI, endoIV, and endoV) were determined using bond cleavage frequencies of both normal and reduced cellodextrins and Ko/K(m). EndoIV differed from other endoglucanases described so far, in having at least nine putative binding sites. The specificities of the three endoglucanases towards various xyloglucans derived from apple fruit and potato were determined. Also, the release of oligosaccharides from these substrates in time was monitored. It was concluded that the endoglucanases prefer to bind unbranched glucosyl residues. Because most xyloglucans are composed of XXXG-type of building units, distant subsites are needed to bind xyloglucan. Having at least nine substrate-binding sites, endoIV seems to be well equipped to degrade xyloglucans which was confirmed by its high xyloglucanase activity.

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.

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.

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.

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.

Potato xyloglucan is built from XXGG-type subunits.

Extraction of potato cell-wall material with solutions of increasing strength of alkali yielded a xyloglucan-rich fraction which was further purified by anion-exchange chromatography and treatment with alpha-amylase and endogalactanase. Methylation analysis indicated that the purified xyloglucan contained a high percentage of unsubstituted glucosyl residues compared to, for instance, apple xyloglucan, and equal amounts of Xyl-(1-->6)-, Gal-(1-->2)-Xyl(1-->6)-, and Ara-(1-->2)-Xyl-(1-->6)-sidechains. This xyloglucan was degraded with endoglucanase (endoV), purified from Trichoderma viride. The resulting digest was fractionated by BioGel P-2 chromatography, followed by preparative high-performance anion-exchange chromatography of the pentamer to nonamer fractions. The purified oligosaccharides were characterized by monosaccharide analysis, mass spectrometry, and degradation with an exoglucanase. Degradation of potato xyloglucan by another endoglucanase (endoIV) of Trichoderma viride yielded a different set of products. EndoIV released predominantly oligosaccharides with two unbranched glucosyl residues at the reducing terminus, whereas endoV also released products containing unbranched glucosyl residues on both ends of the molecule. A difference in the mode of action of endoglucanases with xyloglucan-degrading activity is demonstrated.

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].

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.

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].

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.

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.

The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme.

We have cloned the structural gene for the Bacillus caldolyticus glycogen branching enzyme (glgB) in Escherichia coli. The glgB gene consisted of a 1998 bp open reading frame (ORF) encoding a 78,087 Da protein, which was highly similar to the Bacillus stearothermophilus branching enzyme. The 5' end of a second gene that encoded a protein with extensive similarity to E. coli ADP-glucose pyrophosphorylase (ADPGP) partly overlapped the 3' end of the glgB gene. A putative promoter recognized by Bacillus subtilis RNA polymerase containing the sigma factor H (E-sigma H) preceded the genes. These data suggest that in contrast to the situation observed in B. stearothermophilus, the genes involved in glycogen synthesis in B. caldolyticus are clustered on the chromosome, and are presumably coordinately expressed during the early stages of sporulation. An incomplete third gene started upstream of B. caldolyticus glgB. This gene was highly similar to a gene found directly upstream of B. stearothermophilus glgB, which encodes a putative membrane protein with unknown function. The B. caldolyticus glgB gene was expressed in E. coli and B. subtilis. Surprisingly, the branching enzyme appeared to be thermolabile, the temperature of optimal activity being only 39 degrees C.