The effect of different dietary levels of hybrid rye and xylanase addition on the performance and egg quality in laying hens.

1. In this study, 240 ISA Brown hens were fed diets containing different levels of hybrid rye, and the influence of xylanase addition on laying performance and egg quality was evaluated. 2. Birds were allocated to 10 treatment groups with 12 replicates (cages) of two hens and were fed, from week 26 to 50, isocaloric and isonitrogenous experimental diets. A 5 × 2 experimental arrangement was applied, using diets with increasing level of rye (0%, 10%, 15%, 20% or 25%) with or without xylanase supplementation (200 mg/kg of feed; Ronozyme WX (CT) with minimum xylanase activity of 1,000 FXU/g). 3. Increasing dietary level of rye did not affect daily mass of eggs, mean egg weight or feed conversion ratio (P > 0.05). Laying rate decreased in all groups fed with rye. Egg and eggshell quality indices were unaffected by dietary rye grain (P > 0.05); however, rye inclusion significantly decreased yolk colour on the DSM scale (P < 0.05). In comparison with the control group, high dietary levels of rye (25%) significantly increased viscosity of small intestine content (P < 0.05). Diet supplementation with xylanase had no significant effect on egg production indices and egg quality (except for yolk colour) but decreased the viscosity of intestinal content in laying hens fed high levels of rye (P < 0.05). 4. The results of this experiment suggest that rye may be incorporated to a level of 25% in the diet of laying hens without any strong negative effect on egg performance, while xylanase added to high-rye grain reduced the viscosity of intestinal content; however, it did not positively affect the laying performance or egg quality.

Effect of exogenous fibrolytic enzymes on ruminal fermentation and nutrient digestion in dairy cows.

The aim of the present study was to examine the effects of an exogenous fibrolytic enzyme product applied to a total mixed ration (TMR) prior to feeding on ruminal fermentation, microbial protein synthesis, nutrient digestion, and milk yield and composition. Six multiparous lactating Holstein cows (598 +/- 29 kg initial live weight and 98 +/- 30 days in milk) fitted with rumen and duodenal cannulae were allocated to two treatments in a crossover design over three consecutive 28-d periods. The TMR containing 50% concentrates, 30% corn silage and 20% grass silage on dry matter (DM) basis, was mixed once daily and fed twice a day. Treatments were TMR alone (Control) or TMR with an enzyme product containing primarily cellulase and xylanase activities (9000 U endo-1,4-beta glucanase, 24000 U endo-1,3(4)-beta glucanase and 40000 U 1,4-beta xylanase per ml). The enzyme product was applied at a rate of 6.2 ml/kg TMR (DM basis). It was diluted at a rate of 1:5 with water and applied daily to the TMR. During the control period the cows received a TMR supplemented with 36 ml water/kg TMR on DM basis. Duodenal digesta flow was measured using Cr2O3 as flow marker and microbial protein in the duodenal digesta was estimated by near-infrared spectroscopy (NIRS). There were no significant differences in ruminal pH-values, NH3-N concentrations, total SCFA concentrations and molar proportions of SCFA. No treatment effects on microbial N flow to the duodenum and efficiency of microbial protein synthesis were observed. The apparent ruminal digestibilities of DM, organic matter, NDF and ADF, milk yield and composition were also not affected by the enzyme supplementation. In this study the application of exogenous fibrolytic enzymes fed to dairy cows did not show a significant effect on any parameter tested.

Enzyme complex containing carbohydrases and phytase improves growth performance and bone mineralization of broilers fed reduced nutrient corn-soybean-based diets.

One experiment was conducted to investigate the benefits of a multi-enzyme complex, containing carbohydrases (from Penicillium funiculosum) and phytase (bacterial 6-phytase) activities, on the performance and bone mineralization of broiler chickens fed corn-soybean meal diets. A total of 2,268 male broilers were allocated to 9 treatments, replicated 6 times, in a randomized complete block design from 1 to 43 d. A positive control (PC) diet formulated to be adequate in nutrients and 4 reduced nutrient diets (NC1 to NC4), with gradual decrease on AME, CP, and digestible amino acids (CP-dAA) and available P (avP) and Ca contents, with or without enzyme supplementation, were tested. The nutrient reductions applied were NC1 (-65 kcal/kg, -1.5% CP-dAA) and NC2 (-85 kcal/kg, -3.0% CP-dAA) both with -0.15 percent point avP and -0.12 percent point Ca and NC3 (-65 kcal/kg, -1.5% CP-dAA) and NC4 (-85 kcal/kg, -3.0% CP-dAA) both with -0.20 percent point avP and -0.16 percent point Ca. Supplementation of the NC diets with the enzyme complex increased ADFI (P<0.001), ADG (P<0.001), and reduced feed:gain (P<0.01). The magnitude of the enzyme effect in increasing feed intake and weight gain was greater for the diets with greatest reductions in avP and Ca. Enzyme supplementation increased (P<0.001) feed intake of birds fed on NC diets close to the level of feed consumption of the PC. Enzyme supplementation to NC diets resulted in all cases in lower (P<0.05) feed:gain than the PC. Enzyme supplementation to NC1 and NC3 diets restored bone mineralization to that of the PC, whereas ash and Ca with NC2 and NC4 diets and P with NC4 diet remained lower (P<0.05). These results suggest that the dietary supplementation with a multi-enzyme complex containing nonstarch polysaccharide enzymes and phytase is efficient in reducing the P, energy, protein, and amino acid specifications of corn-soybean meal diets.

Influence of enzyme supplementation and heat processing of barley on digestive traits and productive performance of broilers.

We studied the influence of enzyme supplementation (ES) of the diet and heat processing (HP) of barley on digestive traits and productive performance of broilers from 1 to 42 d of age. There were 6 diets arranged factorially with 2 doses (0 and 500 ppm) of a fungal enzyme complex with beta-glucanase and xylanase activity and 3 HP of barley (raw, micronized, and expanded). In addition, a control diet based on raw corn without ES was also included from 1 to 21 d of age. Enzymes reduced intestinal viscosity (IV) at all ages (P < or = 0.001) and water intake at 21 d of age (P < or = 0.01) and increased DM of the ileal contents at 28 d (P < or = 0.001). Also, ES increased total tract apparent retention of nutrients and BW gain and feed conversion ratio from 1 to 42 d of age (P < or = 0.001). Heat processing of barley increased IV at 7 and at 28 d of age, and DM of ileal contents (P < or = 0.05) at 28 d of age. In addition, HP improved feed intake (P < or = 0.01) and BW gain (P < or = 0.001) from 1 to 7 d of age, but the effects disappeared after 21 d of age. From 1 to 7 d of age, chicks fed micronized barley had higher IV, gained less weight, and had poorer feed conversion ratio than chicks fed expanded barley (P < or = 0.05). It is concluded that barley with enzymes can substitute for all of the corn in diets fed to broilers from 1 to 21 d of age. Enzymes improved digestive traits, retention of nutrients, and broiler performance from 1 to 42 d of age, and HP of barley improved performance from 1 to 7 d of age. The effects of HP of barley on broiler performance were more evident with expansion than with micronization.

Molecular identification of wheat endoxylanase inhibitor TAXI-II and the determinants of its inhibition specificity.

Wheat grains contain Triticum aestivum xylanase inhibitor (TAXI) proteins which inhibit microbial xylanases, some of which are used in cereal based food industries. These inhibitors may play a role in plant defence. Among the TAXI isoforms described so far, TAXI-II displays a deviating inhibition specificity pattern. Here, we report on the molecular identity of TAXI-II and the basis of its inhibition specificity. Three candidate TAXI-II encoding sequences were isolated and recombinantly expressed in Pichia pastoris. To identify TAXI-II, the resulting proteins were tested against glycoside hydrolase family (GHF) 11 xylanases of Aspergillus niger (ANX) and Bacillus subtilis (BSX). One of these proteins (rTAXI-IB) inhibited both enzymes, like natural TAXI-I. The other candidates (rTAXI-IIA and rTAXI-IIB) showed an inhibition pattern typical for natural TAXI-II, only clearly inhibiting BSX. Comparative analysis of these highly similar sequences with distinct inhibition activity patterns, combined with information on the structural basis for ANX inhibition by TAXI-I [S. Sansen, C.J. De Ranter, K. Gebruers, K. Brijs, C.M. Courtin, J.A. Delcour, A. Rabijns, Structural basis for inhibition of Aspergillus niger xylanase by Triticum aestivum xylanase inhibitor-I, J. Biol. Chem. 279 (2004) 36022-36028], indicated a crucial role for Pro294 of TAXI-IIA and Gln376 of TAXI-IIB in determining the reduced inhibition activity towards ANX. Consequently, single point mutants rTAXI-IIA[P294L] and rTAXI-IIB[Q376H], both displaying the Leu/His combination corresponding to TAXI-I, were able to inhibit ANX. These results show that TAXI-II inhibition specificity bears on the identity of two key residues at positions 294 and 376, which are involved in the interaction at the -2 glycon subsite and the active site of GHF 11, respectively.

Amino acid supplementation, controlled oxygen limitation and sequential double induction improves heterologous xylanase production by Pichia stipitis.

Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-density culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter.

Heat processing of barley and enzyme supplementation of diets for broilers.

The influence of heat processing (HP) of barley and enzyme supplementation (ES) of the diet on digestive and performance traits of broilers to 21 d was studied. There were four treatments arranged factorially with two barley-processing treatments (raw or heated), two levels of ES (0 or 500 ppm), and five replicates per treatment. Chicks fed HP barley grew faster than broilers fed raw barley until 8 d of age, but the effect disappeared thereafter. In general, ES improved broiler performance at all ages. Intestinal viscosity was increased by HP of barley (P < or = 0.05) and reduced by ES (P < or = 0.001), and the decrease in viscosity caused by ES was greater for HP than for raw barley diets (HP x ES; P < or = 0.05). Heat processing of barley and ES of the diet improved apparent retention of nutrients (P < or = 0.001). Age affected apparent retention of nutrients differently. For starch and neutral detergent fiber, retention increased linearly with age (P < or = 0.01), but for the remaining nutrients the retention decreased from d 4 to 8 and then increased until d 21 (P < or = 0.001). Also, the beneficial effects of HP on retention of nutrients were more pronounced at younger ages (HP x age; P < or = 0.05). Both HP (P < or = 0.001) and ES (P < or = 0.01) increased liver weight, and enzymes reduced the weights of pancreas (P < or = 0.05) and small intestine (P < or = 0.001). Villus height was improved by HP (P < or = 0.001) and ES (P < or = 0.01), but villus surface area was only improved by enzymes (P < or = 0.01). It was concluded that broiler performance is improved by HP of barley at early ages and by ES of the diet throughout the trial. Also, HP and ES increased apparent retention of nutrients, AMEn of the diet, and villus height.

Effects of dietary fat type and non-starch-polysaccharide-hydrolysing enzyme addition to rye-based diets on muscle protein turnover in broilers.

1. Muscle protein turnover was measured in broilers fed on rye-based diets containing either beef tallow (T) or soybean oil (S) at an inclusion rate of 100 g/kg. Each of these diet types was tested either in the absence (S[-], T[-]) or presence (S[+], T[+]) of a xylanase-containing enzyme preparation. Protein turnover was measured in gastrocnemius muscle (GM) and pectoralis major muscle (PM). 2. Fractional rate of protein synthesis (FSR) was measured by the large dose technique using [15N]-labelled phenylalanine whereas fractional protein growth rate (FGR) was estimated by regressing tissue protein content over time. Fractional breakdown rates (FBR) were calculated by the difference between FSR and FGR. 3. In PM, FSR (%/d) was 22.1, 23.4, 21.5 and 24.4 in groups S[-], S[+], T[-] and T[+], respectively, and FBR (%/d) was 8.3, 9.8, 4.5 and 10.8 with the xylanase effect being significant. The FGR of 17.0%/d calculated for the broilers fed on the T[-] diet tended to be higher than for the other groups (13.6 to 13.8%/d). No significant effects were detected for these parameters in the GM. 4. The absolute amounts of protein which were synthesised daily and accreted in both muscles were significantly higher with xylanase supplementation in both fat type diets but at a significantly higher level when soybean oil was the dietary fat type. 5. The observed effects on protein turnover have to be seen in the context of an overall adverse effect of dietary soluble pentosans from rye in combination with tallow on physico-chemical chyme conditions, digestion and absorption of energy and nutrients and tissue-specific metabolic changes.

Effect of enzyme preparation containing xylanase and beta-glucanase on performance of laying hens fed wheat/barley- or maize/soybean meal-based diets.

1. A commercial enzyme preparation (Quatrazyme HP) containing xylanase and beta-glucanase was examined in two laying hen experiments with wheat/barley- or maize-based diets. The activities of other enzymes were measured also. Starch, cell wall contents and effects of Quatrazyme HP on in vitro viscosity of wheat, barley, maize and soybean meal were determined. 2. In the first experiment, 90 ISA Brown laying hens at 28 weeks of age were given a wheat/barley basal diet with or without 20mg of Quatrazyme HP, which provided 560 and 2,800 IU of xylanase and beta-glucanase/kg diet. In the second experiment, 66 ISA Brown laying hens at 45 weeks of age were given a maize/soybean meal basal diet with or without 20 mg of Quatrazyme HP/kg diet for 9 weeks. Egg production, egg weight, egg mass, feed conversion ratio and change in body weight were recorded as response criteria. 3. There was a significant improvement in feed conversion ratio with enzyme supplementation. Birds given an enzyme-supplemented diet gained 86 g while those fed on the unsupplemented diet lost 103 g of their body weight by the end of the experiment. 4. The enzyme preparation did not affect either egg production, egg weight or egg mass of birds fed on the maize/soybean meal diet. However, a significant improvement in feed conversion ratio was detected. Birds on either the supplemented or unsupplemented diet exhibited an increase in their body weight at the end of the experiment. 5. Addition of xylanase and beta-glucanase decreased in vitro viscosity of wheat, barley, maize and soybean meal. This effect was greater for wheat and barley than for maize and soybean meal. 6. It was concluded that the beneficial effect of using an enzyme preparation containing xylanase and beta-glucanase is not limited to wheat/barley-based diets but also occurs with maize/soybean meal-based diets.

Effect of feeding supplemental fibrolytic enzymes or soluble sugars with malic acid on milk production.

Two trials were conducted to evaluate effects of feeding supplemental fibrolytic enzymes or soluble sugars and malic acid on milk production. In trial 1, 257 cows at four sites were fed a basal diet consisting of no more than 60% of forage DM as corn silage and less than 40% as alfalfa hay. Cows were assigned randomly within site, parity, and two stages of lactation to: 1) control; 2) enzyme A; 3) enzyme B; and 4) soluble sugars and malic acid. There was a 14-d pretreatment and an 84-d treatment period. Enzyme solutions were sprayed on either the forage component or the TMR each day while mixing feed. Trial 2 was similar, except 122 cows at one site in the United Kingdom were fed diets containing forage that was 75% corn silage and 25% grass silage, and all cows began the study between 25 to 31 DIM. Mean milk productions for 233 cows that completed trial 1 were 32.9, 32.5, 32.4, and 32.9 kg/d for control, enzyme A, enzyme B, and soluble sugars and malic acid, respectively. Mean milk productions for 116 cows that completed trial 2 were 28.2, 27.9, 28.8, and 28.4 kg/d, respectively. In vitro analyses of the activities of enzyme solutions indicated that all major cellulose and hemicellulose degrading activities were present; however, the pH optima (approximate pH = 4 to 5) were more acidic, and the temperature optimum (approximately 50 degrees C) was greater than normal pH and temperature in the rumen. If fibrolytic activity in the rumen is a major mechanism of action of supplemental fibrolytic enzymes, it appears that considerable activity of these preparations was lost due to conditions in the rumen. In conclusion, feeding supplemental fibrolytic enzymes or malic acid with soluble sugars had no effect on milk production under the conditions used in this study.

Influence of enzymes on performance and digestive parameters of broilers fed rye-based diets.

An experiment was conducted to study the influence of enzyme supplementation (ES) to rye-based diets on rate of food passage through the digestive tract, viscosity of jejunum content, volatile fatty acid concentration in ceca, and performance of broilers. There were seven treatments; six diets arranged factorially with three varieties of rye (Petkus, Prima, and Saratov V) and two levels of ES (0 or 500 ppm of an enzyme complex containing 858 IU of beta-glucanase and 864 IU of xylanase/g) and an additional control diet based on corn. Each treatment was replicated seven times (12 chicks caged together), and the trial lasted 25 d. Rye feeding increased intestinal viscosity and impaired bird performance at 25 d (P < 0.001). Among rye diets the greatest feed intake and weight gain were obtained with Petkus variety, which also produced the lowest intestinal viscosity. Enzyme addition reduced the time needed to recover 1% (0.78 vs. 0.98 h; P < 0.05) and 50% (4.2 vs. 6.5 h; P < 0.01) of the marker in feces and reduced the mean retention time of marker in the gastrointestinal tract (17.1 vs. 18.8 h; P < 0.05). Also, ES reduced intestinal viscosity (P < 0.001) and improved feed intake, daily gain, and feed conversion of birds from 4 to 25 d (P < 0.01) but did not modify volatile fatty acid concentration in ceca. We concluded that ES added to rye diets decreased intestinal viscosity and accelerated digestive transit, improving productive performance of broilers.

Effects of xylanase treatments on gelling and water-uptaking properties of psyllium.

The effects of a commercial food-grade xylanase on the physicochemical properties of psyllium were evaluated. The enzymatic reactions were conducted in solid state at ambient temperature. The enzyme-treated psyllium preparations were analyzed and compared with the starting psyllium for their water-uptake properties, gelling capacities, soluble and insoluble fiber contents, and surface structures. The solid-state xylanase treatment significantly reduced both water-uptake and gelling capacities of psyllium (p < 0.01), with a slight decrease of soluble fiber content, whereas no effect on insoluble fiber content was observed. The xylanase treatment also resulted in a smoother surface structure of psyllium particles. In addition, no special equipment and operation were required to conduct the enzymatic reaction, which generated no waste. These data indicated a potential to improve the physicochemical properties of psyllium by use of the solid-state xylanase reactions to promote the utilization of psyllium fiber in functional foods for promoting human health.

Xylanase and beta-glucanase supplementation improve conjugated bile acid fraction in intestinal contents and increase villus size of small intestine wall in broiler chickens fed a rye-based diet.

This study was performed with growing chickens (4 to 22 d of age) to evaluate the effects of feeding a rye-based diet supplemented with commercial enzyme preparation containing xylanase and beta-glucanase (Quatrazyme HP, Nutri-Tomen, France) on small intestine wall morphology, bile acid composition, nutrient digestibility, and bird performance compared with unsupplemented rye- or corn-based diets. The rye-based diet decreased (P < or = 0.05) weight gain, feed intake, and feed efficiency and increased water intake compared with the corn-based diet. Moreover, rye consumption reduced crude fat and protein digestibility as well as apparent metabolizable energy (P < or = 0.05). The small intestine wall showed that villus length, width, and surface were decreased in broiler chickens fed the rye-based diet compared with those fed the corn-based diet. However, crypt morphometry parameters were not affected by diet type. The concentration of conjugated bile acids in the small intestine contents of broiler chickens fed the rye-based diet was decreased (P < or = 0.05) compared with those fed the corn-based diet. These findings suggest that feeding a rye-based diet reduces villus capacity for nutrient absorption and bile acid capacity for fat solubilization and emulsification, resulting in decreased bird performance. The addition of xylanase and beta-glucanase to the rye-based diet improved (P < or = 0.05) weight gain, feed intake, and feed efficiency, and decreased water intake. The digestibility of nutrients and apparent metabolizable energy were also increased (P < or = 0.05). Addition of xylanase and beta-glucanase increased (P < or = 0.05) villus size and the villus height-to-crypt depth ratio, as well as the concentration of conjugated bile acids (P < or = 0.05) in the small intestine contents. Exogenous enzymes improved nutrient digestibility and broiler chicken performance, probably by improving the absorption capacity ofthe small intestine through increased villus surface and intestinal concentration of conjugated bile acids.

Bacterial responses to different dietary cereal types and xylanase supplementation in the intestine of broiler chicken.

Several studies were carried out to investigate the influence of dietary cereals differing in soluble non starch polysaccharides (NSP) content and a xylanase preparation on selected bacterial parameters in the small intestine of broiler chicken. Compared to a maize diet colony forming units (CFU) of mucosa associated bacteria were higher in a wheat/rye diet, most notably for enterobacteria and enterococci. Xylanase supplementation to the wheat/rye diet generally led to lower CFU, especially in the first week of life. However, xylanase supplementation also displayed higher in vitro growth potentials for enterobacteria and enterococci. Bacterial growth of luminal samples in minimal media supplemented with selected NSP showed that the wheat/rye diet enhanced bacterial capacities to utilize NSP only in ileal samples. The xylanase application generally shifted respective maximum growth to the proximal part of the small intestine. The presence of soluble NSP from wheat or rye in the diet per se did not enhance bacterial NSP hydrolyzing enzyme activities in the small intestine, but xylanase supplementation resulted in higher 1,3-1,4-beta-glucanase activity. Compared to a maize diet the activity of bacterial bile salt hydrolases in samples of the small intestine was not increased due to inclusion of wheat/rye or triticale to the diet. However, xylanase supplementation led to a reduction with a corresponding increase of lipase activity. It was concluded that dietary cereals producing high intestinal viscosities lead to increased overall bacterial activity in the small intestine. The supplementation of a xylanase to cereal based diets producing high intestinal viscosity, changes composition and metabolic potential of bacterial populations and may specifically influence fat absorption in young animals.

Maize tapetum xylanase is synthesized as a precursor, processed and activated by a serine protease, and deposited on the pollen.

Pollen coat contains ingredients that interact with the stigma surface during sexual reproduction. In maize (Zea mays L.) pollen coat, the predominant protein is a 35-kDa endoxylanase, whose mRNA is located in the tapetum cells enclosing the maturing pollen in the anthers. This 2.0-kb mRNA was found to have an open reading frame of 1,635 nucleotides encoding a 60-kDa pre-xylanase. In developing anthers, the pre-xylanase protein appeared prior to the 35-kDa xylanase protein and enzyme activity and then peaked and declined, whereas the 35-kDa xylanase protein and activity continued to increase until anther maturation. An acid protease in the anther extract converted the inactive pre-xylanase to the active 35-kDa xylanase in vitro. The protease activity was inhibited by inhibitors of serine proteases but unaffected by inhibitors of cysteine, aspartic, or metallic proteases. Sequence analysis revealed that the 60-kDa pre-xylanase was converted to the 35-kDa xylanase with the removal of 198 and 48 residues from the N and C termini, respectively. During in vitro and in vivo conversions, no intermediates of 60-35 kDa were observed, and the 35-kDa xylanase was highly stable. The pre-xylanase was localized in the tapetum-containing anther wall, whereas the 35-kDa xylanase was found in the pollen coat. The significance of having a large non-active pre-xylanase and the mode of transfer of the xylanase to the pollen coat are discussed. A gene encoding the barley (Hordeum vulgare L.) tapetum xylanase was cloned; this gene and the gene encoding the seed aleurone-layer xylanase had strict tissue-specific expressions.

Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99.

An alkalophilic Aspergillus nidulans KK-99 produced an alkaline, thermostable xylanase (40 IU/ml) in a basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15% N) pH 10.0 and 37 degrees C. The partially purified xylanase was optimally active at pH 8.0 and 55 degrees C. The xylanase was stable in a broad pH range of 4.0-9.5 for 1 h at 55 degrees C, retaining more than 80% of its activity. The enzyme exhibited greater binding affinity for xylan from hardwood than from softwood. The xylanase activity was stimulated (+25%) by Na+ and Fe2+ and was strongly inhibited (maximum by 70%) by Tween-20, 40, 60, SDS, acetic anhydride, phenylmethane sulphonyl fluoride, Triton-X-100. The xylanase dose of 1.0 IU/g dry weight pulp gave optimum bleach boosting of Kraft pulp at pH 8.0 and temperature 55 degrees C for 3 h reaction time.

A xylanase from roots of sanchi ginseng (Panax notoginseng) with inhibitory effects on human immunodeficiency virus-1 reverse transcriptase.

A xylanase with a molecular weight of 15 kDa, which is lower than those of previously reported xylanases, was isolated from the roots of the medicinal herb Panax notoginseng. The xylanase exhibits a temperature optimum of 50 degrees C and a pH optimum between 5 and 6. It inhibits HIV-1 reverse transcriptase with an IC(50) of 10 microM, but does not affect translation in a cell-free rabbit reticulocyte system when tested up to 70 microM. The enzyme is adsorbed on CM-cellulose, Affi-gel blue gel and Mono S. Previously xylanases have been isolated from seeds and not from roots, and have not been demonstrated to inhibit HIV-1 reverse transcriptase.

Monitoring on-line desalted lignocellulosic hydrolysates by microdialysis sampling micro-high performance anion exchange chromatography with integrated pulsed electrochemical detection/mass spectrometry.

An on-line system based on microdialysis sampling (MD), micro-high performance anion exchange chromatography (micro-HPAEC), integrated pulsed electrochemical detection (IPED), and electrospray ionization mass spectrometry (MS) for the monitoring of on-line desalted enzymatic hydrolysates is presented. Continuous monitoring of the enzymatic degradation of dissolving pulp from Eucalyptus grandis as well as degradation of sugar cane bagasse in a 5-mL reaction vessel was achieved up to 24 h without any additional sample handling steps. Combining MD with micro-HPAEC-IPED/MS and on-line desalting of hydrolysates enabled injection (5 microL) of at least 23 samples in a study of the sequential action of hydrolytic enzymes in an unmodified environment where the enzymes and substrate were not depleted due to the perm-selectivity of the MD membrane (30 kDa cut-off). Xylanase, phenolic acid esterase and a combination of endoglucanase (EG II) with cellobiohydrolase (CBH I) resulted in the production of DP 1 after the addition of esterase, DP 2 and DP 3 after the addition of EG II and CBH I, from the dissolving pulp substrate. Similar sequential enzyme addition to sugar cane bagasse resulted in DP 1 production after the addition of esterase and DP 1, DP 2 and DP 3 production after the addition of the EG II and CBH I mixture. Combining MS on-line with micro-HPAEC-IPED proved to be a versatile and necessary tool for such a study compared to conventional methods. The mass selectivity of MS revealed complementary information, including the co-elution of saccharides as well as the presence of more than one type of DP 2 in the case of dissolving pulp and several types of DP 2 and DP 3 for sugar cane bagasse. This study demonstrates the limitation of the use of retention time alone for confirmation of the identity of saccharides especially when dealing with complex enzymatic hydrolysates. In situ sampling and sample clean-up combined with on-line desalting of the chromatographic effluent, provides a generic approach to achieve real time monitoring of enzymatic hydrolysates when they are detected by a combination of IPED and MS.

Changes in the cell wall network during the thermal dehydration of alfalfa stems.

The effects of heat treatments used to dry alfalfa stems were investigated. Heating at 70 or 100 degrees C caused no major change in the cell wall composition, but xylanase had lower activity on the cell wall of heated material and the amount of xylose released varied with the temperature used. Chemical fractionation of cell wall carbohydrates showed that the main changes occurring during stem dehydration concerned pectic polymers and probably hemicelluloses. There was less material soluble in ammonium oxalate from alfalfa heated at 100 degrees C than from fresh alfalfa. The results suggest that heat processing causes some changes in the cell wall network. Environmental scanning electron microscopy was used to examine fully hydrated tissues at high resolution. There was cell distortion without disruption of cell walls as water was lost.

Soybean (Glycine max) cell wall composition and availability to feed enzymes.

Defatted untoasted soybean cotyledons and hulls were fractionated as water solutes (WSc and WSh) and water unextractable (WUc and WUh). Further fractionation of WUc through deproteinization yielded the isolation of a water unextractable solid (WUS) fraction that was mainly composed (molar percent) of galactose (28.1%), glucose (27.8%), arabinose (13.3%), and uronic acids (17.6%), which accounted for 76% of the water insoluble polysaccharides in soybean cotyledons (WUc). The cell wall (WUS) was sequentially fractionated with chelating agents (chelating agent soluble solids, ChSS) and a gradient of agents (dilute alkali, DASS; 1 M alkali, 1MASS; and 4M alkali, 4MASS), which gave a final cellulosic residue. The ChSS and DASS extracts were characterized as pectin-rich fractions, whereas 1MASS and 4MASS were hemicellulose- and cellulose-rich fractions. Incubation in vitro of the WUc fraction with pectinase, cellulase, and xylanase resulted in the release of low amounts (not more than 5% bound basis) of monosaccharides, mostly uronic acids, xylose, and arabinose. Protein extraction hardly increased this release after enzymatic incubation (<7%). However, progressive fractionation of the cell wall matrix markedly increased the release of monosaccharides from pectin- (ChSS and DASS) and hemicellulose-rich fractions (1MASS and 4MASS). Significant degradation of cellulose (up to 20%) was achieved only after complete protein, pectin, and hemicellulose extraction.