Thermostable xylanase inhibits and disassembles Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms are problematic and play a critical role in the persistence of chronic infections because of their ability to tolerate antimicrobial agents. In this study, various cell-wall degrading enzymes were investigated for their ability to inhibit biofilm formation of two P. aeruginosa strains, PAO1 and PA14. Xylanase markedly inhibited and detached P. aeruginosa biofilms without affecting planktonic growth. Xylanase treatment broke down extracellular polymeric substances and decreased the viscosity of P. aeruginosa strains. However, xylanase treatment did not change the production of pyochelin, pyocyanin, pyoverdine, the Pseudomonas quinolone signal, or rhamnolipid. In addition, the anti-biofilm activity of xylanase was thermally stable for > 100 days at 45°C. Also, xylanase showed anti-biofilm activity against one methicillin-resistance Staphylococcus aureus and two Escherichia coli strains.

Effect of fibrolytic enzymes added to a Andropogon gayanus grass silage-concentrate diet on rumen fermentation in batch cultures and the artificial rumen (Rusitec).

In vitro batch cultures were used to screen four fibrolytic enzyme mixtures at two dosages added to a 60 : 40 silage : concentrate diet containing the C(4) tropical grass Andropogon gayanus grass ensiled at two maturities - vegetative stage (VS) and flowering stage (FS). Based on these studies, one enzyme mixture was selected to treat the same diets and evaluate its impact on fermentation using an artificial rumen (Rusitec). In vitro batch cultures were conducted as a completely randomized design with two runs, four replicates per run and 12 treatments in a factorial arrangement (four enzyme mixtures×three doses). Enzyme additives (E1, E2, E3 and E4) were commercial products and contained a range of endoglucanase, exoglucanase and xylanase activities. Enzymes were added to the complete diet 2 h before incubation at 0, 2 and 4 µl/g of dry matter (DM). Gas production (GP) was measured after 3, 6, 12, 24 and 48 h of incubation. Disappearance of DM (DMD), NDF (NDFD) and ADF (ADFD) were determined after 24 and 48 h. For all four enzyme mixtures, a dosage effect (P>0.05) DM, N, NDF or ADF disappearance after 48 h of incubation nor daily ammonia-N, volatile fatty acids or CH(4) production. However, enzyme application increased (P<0.05) microbial N production in feed particle-associated (loosely-associated) and silage feed particle-bound (firmly associated) fractions. With A. gayanus silage diets, degradation may not be limited by microbial colonization, but rather by the ability of fibrolytic enzymes to degrade plant cell walls within this recalcitrant forage.

Effect of Mediterranean saltbush (Atriplex halimus) ensilaging with two developed enzyme cocktails on feed intake, nutrient digestibility and ruminal fermentation in sheep.

The aim of this study was to assess the effects of feeding Atriplex halimus (AH) silage treated with two developed enzyme cocktails to sheep on feed intake, nutrient digestibility and ruminal fermentation. The AH silage was treated without or with 2 L of ZAD1(®) or ZAD2(®) /1000 kg with 5% molasses and ensiled for 30 days. Barley grain (300 g/head/day) was fed as an energy supplement once daily at 10.00 hours and AH silage with or without enzyme treatment was offered ad libitum to animals twice daily at 09.00 and 16.00 hours. Sheep were fed on four experimental forage diets comprised of AH silage and barley (D1), AH silage treated with ZAD1(®) and barley (D2), AH silage treated with ZAD2(®) and barley (D3) and AH silage treated with a combination of ZAD1(®) and ZAD2(®) (1:1) and barley (D4). Ensiling AH with enzymes reduced its contents of neutral detergent fiber and acid detergent fiber. The dry matter intake of AH of D2, D3 and D4 decreased (P < 0.001) as compared to D1. However, enzyme-treated diets had greater total digestible nutrients intake (P < 0.001) as compared to D1. The nutrients digestibility for D2, D3 and D4 were higher than those for D1 (P < 0.001), and were higher for D3 as compared to both D2 and D4. Sheep fed on D3 had highest (P < 0.001) ruminal total volatile fatty acids concentration, ammonia nitrogen concentration and microbial protein yield. It could be concluded that AH silage treated with ZAD1(®) or ZAD2(®) improved digestibility and rumen fermentation in sheep.

Using a fibrolytic enzyme in barley-based diets containing wheat dried distillers grains with solubles: ruminal fermentation, digestibility, and growth performance of feedlot steers.

Two experiments were conducted to evaluate the effects of adding an exogenous fibrolytic enzyme (FE) on ruminal pH and fermentation, digestibility, and growth performance of feedlot beef cattle fed a finishing diet containing wheat dried distillers grains with solubles (DDGS). In Exp. 1, 4 ruminally cannulated Angus heifers (average BW of 807 ± 93.9 kg) were used in a replicated 4 × 4 Latin square design. Treatments were 1) control (CON; 10% barley silage and 90% barley grain-based concentrate), 2) CON diet substituting 30% wheat DDGS for barley grain (WDG), 3) WDG diet supplemented with low FE (WDGL), and 4) WDG diet supplemented with high FE (WDGH). Heifers fed WDG had less (P = 0.01) total tract DM digestibility than heifers fed CON. Increasing FE linearly (P < 0.05) increased starch digestibility without affecting digestibility of other nutrients. Addition of FE also reduced (P = 0.03) ruminal ammonia-N (NH3-N) concentration but did not affect VFA concentration. Moreover, application of FE to wheat DDGS linearly increased in situ ruminal DM (P < 0.01) and NDF (P = 0.02) disappearance after 48 h of incubation. In Exp. 2, 160 yearling steers (initial BW = 495 ± 37.9 kg) were fed the same diets as in Exp. 1. No differences in DMI, final BW, ADG, dietary NEg, or carcass characteristics were observed among diets. However, the steers fed WDG had less (P < 0.05) G:F and greater number of (P < 0.01) abscessed livers than steers fed CON. Increasing FE application in wheat DDGS diets did not affect DMI, final BW, or ADG but tended (P < 0.09) to linearly improve feed efficiency and decreased (P = 0.03) the incidence of abscessed livers. These results demonstrated adverse effects of including wheat DDGS in finishing diets on feed digestion, feed efficiency, and animal health. Application of FE in wheat DDGS-based diets potentially improved starch digestion, protein metabolism in the rumen, feed efficiency, and animal health.

Interactions between phytase and xylanase enzymes in male broiler chicks fed phosphorus-deficient diets from 1 to 18 days of age.

A total of 735 one-day-old male broiler chicks were used to evaluate the interactions between different levels of phytase and xylanase enzymes on performance and bone mineralization. Basal nonphytate P (nPP)-deficient diets (0.15%) were supplemented with different levels of phytase [0X, 1X, 2X, 3X, and 4X of recommended level (X = 500 phytase units per kg of feed)] alone or in combination with 3 levels of a xylanase preparation [0X, 1X, and 2X of recommended level (X = 0.1 g per kg of feed; a mixture with predominantly xylanase activity)]. A standard curve was developed using different levels of nPP (0.15 to 0.45%) to estimate the P equivalency of each enzyme combination. Treatments were replicated with 7 pens of 5 chicks each. The findings indicated that reducing dietary nPP level had a severely negative influence on bird performance and bone ash content. Results also showed that increasing levels of phytase significantly (P < 0.05) improved BW, feed intake, feed conversion ratio, mortality, and toe and tibia bone ash contents in a dose-dependent fashion. The P equivalency of phytase was also dose dependent, with estimates of 0.08, 0.11, 0.15, and 0.19 for 1X, 2X, 3X, and 4X supplementation levels of phytase, respectively. Xylanase preparation at 1X level failed to influence measured criterion; however, increasing the level of xylanase (2X) resulted in deteriorating BW and feed conversion ratio. The P equivalency of xylanase preparation at 1X and 2X was estimated as 0.010 and 0.014%. There were no interactions between phytase and xylanase preparation (P > 0.05). In conclusion, phytase was effective in improving bird performance and bone mineralization; however, xylanase supplementation failed to enhance phytase efficacy.

Interactive effects of phytase and xylanase supplementation with extractable salt-soluble protein content of corn in diets with adequate calcium and nonphytate phosphorus fed to broilers.

The objective was to determine the effects of extractable salt-soluble protein content of corn (PS) and exogenous enzyme supplementation on N, starch, and energy digestibility in broilers fed diets adequate in Ca and nonphytate P. Broilers were randomly distributed into floor pens (6 replicate pens per treatment) with 28 birds per pen at 1 d of age. Treatments consisting of 4 sources of corn varying in PS (A, 58.1; B, 54.2; C, 53.7; and D, 30.6 mg of BSA equivalent values) with or without phytase (0 and 1,000 phytase units/kg) and xylanase (0 and 16,000 units of xylanase activity/kg) were randomly assigned to each pen. Different sources of corn were provided from 1 to 9 and 24 to 29 d of age. However, enzyme treatments were provided throughout the experiment. From 1 to 9 d of age, no interactions were observed. Apparent ileal N digestibility (AIND) and apparent ileal digestible energy (IDE) of diets with the lowest PS (based on corn D) were lower (P ≤ 0.05) than those of diets with a higher PS. Phytase increased (P ≤ 0.01) AIND and IDE by 5 and 16%, respectively, and xylanase exerted the opposite effect (P ≤ 0.03). From 24 to 29 d of age, phytase and xylanase in combination resulted in reduced (P ≤ 0.05) AIND of diets with a low PS (based on corn D) compared with the basal diet in broilers. Broilers fed diets with the highest or lowest PS (based on corn A or D) had lower (3-way interaction; P ≤ 0.05) IDE when phytase and xylanase were supplemented in combination compared with either enzyme alone. In conclusion, responses to exogenous enzyme supplementation are not constant and are influenced by the source of ingredients as well as the age of broilers. The magnitudes of the responses to phytase on nutrient and energy digestibility were greater at 9 compared with 29 d of age.

The effect of protease and xylanase enzymes on growth performance and nutrient digestibility in finisher pigs.

Two 2 × 2 factorial experiments were conducted to investigate the interaction between xylanase (0 vs. 200 mg/kg) and protease (0 vs. 200 mg/kg) enzyme supplementation on growth performance (Exp. 1) and coefficient of ileal and total tract apparent digestibility in grower-finisher pigs (Exp. 2). One hundred and twenty-eight individual fed pigs (BW = 34.2 kg; n = 32) were assigned to 1 of 4 dietary treatments: basal diet (T1), T1 + xylanase enzyme (T2), T1 + protease enzyme (T3), or T1 + xylanase + protease enzymes (T4). The pigs offered diets containing protease enzymes had reduced daily gain (0.795 vs. 0.840 kg/d; P < 0.05) and final body weight (96.4 vs. 99.1 kg; P < 0.05) compared to pigs offered diets without protease enzymes. Pigs offered xylanase-supplemented diets had reduced daily gain (0.787 vs. 0.848 kg/d; P < 0.05) compared to pigs offered diets without xylanase enzymes. In Exp. 2, the nutrient digestibility experiment consisted of 24 intact male pigs (n = 6; BW = 78 kg), offered identical diets to that offered in Exp. 1. Following the fecal collections, the pigs were slaughtered and digesta samples were taken from the ileum in order to measure apparent ileal N and GE digestibilities. Pigs offered diets supplemented with protease had increased coefficients of ileal digestibility of N compared to pigs offered diets without protease supplementation (0.583 vs. 0.449; P < 0.05). There was a xylanase × protease interaction (P < 0.05) on the apparent ileal digestibility of GE. Pigs offered diets containing protease only had increased apparent ileal digestibility of GE compared to basal fed pigs; however, the ileal digestibility of GE decreased when protease was combined with xylanase. Neither xylanase nor protease enzymes had any effect on total tract digestibility of GE or N. In conclusion, xylanase and protease enzyme supplementation had no positive effects on grower-finisher pig performance.

Characterization of statistically produced xylanase for enrichment of fruit juice clarification process.

Critical factors for xylanase production of Bacillus stearothermophilus under batch fermentation and for clarification of citrus fruit juice using this xylanase were optimized through central composite design of response surface methodology. Statistical approach resulted in an increase of 1.19-fold in xylanase yield over conventional method. Model equation for juice clarification included independent variables viz. temperature, incubation time and enzyme dose to study the dependent variables such as yield, acidic neutrality and filterability etc. Coefficient of determination, R(2) for enzyme production model and for different juice properties were in accordance with the linearity of the model. On the basis of the contour plots the optimum enzyme dose was 12.5 IU/g of xylanase. Enzymatic treatment has resulted in the improvement of twofold in the release of reducing sugars and 52.97% in juice yield, whereas 35.34% reduction in turbidity was observed.

Effects of structure and xylanase treatment of brewers' spent grain on performance and nutrient availability in broiler chickens.

1. A factorial (2 x 3) feeding trial was set up to investigate the effects of coarse or finely ground brewers' spent grain (BSG) and xylanase treatment, either with no xylanase, top-dressed with xylanase or pre-treated with xylanase. 2. The experimental diets shared the same basal formulation and were fed to male broiler chickens (Ross 308) housed in individual cages from 12 to 29 d of age. 3. Xylanase pre-treatment reduced the dietary concentration of arabinoxylan by 15-30%. Pellet durability increased when BSG was ground. 4. Feed utilisation was significantly higher (6%) when the birds were given coarse BSG rather than ground BSG, whereas there was no significant effect of enzyme treatment. Apparent metabolisable energy was unaffected by the dietary treatments. 5. The overall starch digestibility was high (99%), with no dietary differences, whereas ileal protein digestibility was low (57%). Xylanase top-dressing tended to improve ileal protein digestibility but, in general, xylanase treatment had no major effect on overall performance in male broilers given diets with BSG.

Effect of fortifying concentrate supplement with fibrolytic enzymes on nutrient utilization, milk yield and composition in lactating goats.

Thirty lactating Beetle-sannen crossbred goats were randomly divided into 3 groups based on first fortnight milk yield, and then adjusted to body weights to study the effect of fibrolytic enzyme supplementation on digestibility and milk production. Goats in the control group (C) were fed 500 g of concentrate supplement (CS) without exogenous fibrolytic enzymes (control; CG) or CS fortified with cellulase and xylanases @ of 4000 and 12500 (EG(1)) or 8000 and 18 750 IU/kg (EG(2)), respectively. CS was offered in two equal proportions just after milking at 7 and 17 h. After feeding CS, goats were fed 1200 g of wheat straw as roughage in two equal parts at 08.00 and 18.00 hours. Milk yield was studied for 3 1/2 months. Milking was done by hand. Changes in body weight were also recorded. After a preliminary period of 60 days of feeding, a 7-day digestibility trial was conducted in two intervals with 5 animals from each group at a time. The increase in either wheat straw or NDF intake due to the enzyme mixture was only 3%. There was significant improvement (P < 0.05) in the diet digestibility of DM, OM, CP, NDF, ADF and total carbohydrates (TC) between the control and EG(1) and EG(2) goats. Improvements were greater (P < 0.05) in the latter group. Microbial protein (MBP) was estimated based on purine derivatives and creatinine excreted in spot urine samples. MBP increased about 10 and 15% in EG(1) and EG(2), but these values were statistically invalid. Phenomenally milk yield, fat and SNF were significantly (P < 0.001) changed with progression of lactation. On a temporal basis, fortnightly changes in FCM yield of this study could be grouped as 1 month each of first and last and 1 1/2 of middle phase of lactation. Repeated measure analysis showed 180 to 260 mL/day of higher FCM and 16 to 20 g/day of higher SNF yield (P < 0.001) in EG(2) than control or even EG(1) during 5 to 7th fortnight. However, no significant impact (P > 0.05) was noticed on TF. Feed intake, g/kg FCM yield was also 7% less in EG(2) than either CG or EG(1). The study concluded that apart from increased digestibility, fortification of CS with cellulase and xylanases enzymes improved FCM yield in the last quarter of lactation in goats, and improved body weights by 5% (P > 0.05). While the digestibility of the diet was higher in EG(1) and EG(2), the impact on FCM and SNF yield was noticed only on EG(2). Our study indicated that CS of lactating goats may be fortified with 8000 and 18 500 IU/kg CS in the last quarter of lactation.

Influence of wheat-based diets supplemented with xylanase, bile acid and antibiotics on performance, digestive tract measurements and gut morphology of broilers compared with a maize-based diet.

1. The aim of the present study was to compare wheat-based diets supplemented with arabinoxylanase, bile acid and virginiamycin with a maize-based diet in their effects on the performance of broilers. 2. The diets were fed to 5 groups of broilers. Group A received a maize-based diet (control), and groups B, C, D and E were fed on wheat-based diets. Diets C, D and E were supplemented with arabinoxylanase (150 mg/kg), bile acid (500 mg/kg) and virginiamycin (200 mg/kg), respectively. 3. A total of 450 1-d-old Ross 385 chicks were randomly divided into 5 groups, each with three pens as replicates (30 birds per pen). The experimental diets were given for 18 d. On d 18, three chickens from each group were slaughtered by cervical dislocation and the liver weight, and duodenal, jejunal and ileal lengths were measured for length and width of the villi and the height of crypts. 4. Arabinoxylanase, bile acid and virginiamycin supplementation significantly increased feed intake and daily gain but had no significant effect on feed conversion ratio. The liver weight was significantly greater in virginiamycin supplemented chicks as compared with those on the diet containing bile acid. 5. Virginiamycin inclusion increased full and empty caeca weight significantly compared with groups A and D. Group A had the lightest caeca. Significant effects of diets were observed on duodenal, ileal and intestinal length, but jejunal length was not affected by diet. The heights of duodenal and ileal, but not jejunal, villi were significantly affected by the treatments. The lengths of villi in groups B and E were significantly higher than other groups. The mean height of ileal villi in group A was significantly higher than in groups receiving the wheat-based diets. The mean width of duodenal and ileal villi was significantly affected by the ration. The height of crypts in group B was significantly higher than in group E. 6. It could be concluded that supplementation of the wheat-based broiler ration with virginiamycin had significant positive effects on gastrointestinal tract characteristics for absorption. Supplementation with bile acid reduced the weight of liver and caeca and the length of duodenum, jejunum, ileum and in small intestine and the height and width of villi.

Age-related influence of a cocktail of xylanase, amylase, and protease or phytase individually or in combination in broilers.

This 21-d experiment was conducted to determine if the response of chicks to a cocktail of xylanase, amylase, and protease (XAP) or Escherichia coli-derived phytase individually or in combination when fed a nutritionally marginal corn-soybean meal diet is age-dependent. Six hundred 1-d-old chicks were allocated to 5 dietary treatments in a randomized complete block design. The treatments were as follows: 1) positive control with supplemental inorganic P; 2) negative control (NC) marginal in P and ME; 3) NC plus XAP to provide (per kg of diet) 650, 1,650, and 4,000 U of xylanase, amylase, and protease, respectively; 4) NC plus phytase added to provide 1,000 phytase units/kg; and 5) NC plus a combination of XAP and phytase. Low ME and P in the NC diet depressed weight gain and gain:feed (P < 0.001). A cocktail of XAP alone did not improve performance, but phytase supplementation improved (P < 0.001) weight gain. The enzymes were additive in their effects on growth performance. The enzymes had no effect on ileal digestible energy. Ileal N digestibility was higher (P < 0.05) in diet with XAP or phytase individually compared with NC. Both phytase and XAP individually and in combination improved (P < 0.01) ileal P digestibility compared with NC. Total tract nutrient retention and ME increased (P < 0.01) as the birds grew older. There were age x diet interactions (P < 0.001) on total tract retention of P and Ca; improvement in P retention due to phytase use decreased by 50% as the chicks matured. The current study shows that a combination of XAP and phytase improved performance, but the enhancement in performance appears to be mainly from phytase. Both XAP and phytase were effective in improving P digestibility and retention of chicks receiving nutritionally marginal corn-soybean meal. The data also shows that the chicks benefited more from the enzyme addition at a younger age and that the contribution of the enzymes to nutrient retention decreased with age in chickens.

The effect of conditioning temperature and exogenous xylanase addition on the viscosity of wheat-based diets and the performance of broiler chickens.

1. A total of 1920 Ross broiler chicks were used in a 6 treatment by 8 replicate growth study to investigate the effect of conditioning temperature and exogenous xylanase addition on diet characteristics and bird performance. 2. Increasing conditioning temperature from 80 to 90 degrees C increased the viscosity of the diets by approximately 6 mPa.s and reduced body weight gain of the broiler chickens by 7%. 3. Furthermore, there was a significant interaction between temperature and the addition of exogenous xylanase, with a proportionately greater positive response to added xylanase in the diets that were pelleted at higher temperatures. 4. Addition of exogenous xylanase also increased the proportion of soluble arabinoxylan in the feed and reduced dietary viscosity, which is likely to be related to the degree of polymerisation of the soluble carbohydrate. 5. These results suggest that conditioning temperature should be considered as a factor when attempting to predict the response of broilers to added xylanase, especially when high-viscosity or 'low-quality' wheat is being used. 6. It can be concluded that pelleting wheat-based diets above 80 degrees C can compromise bird performance and that, if higher temperatures are to be employed, the use of exogenous xylanase is critical to maintain productivity.

Usage of enzymes in a novel baking process.

In this study, the effects of different enzymes (alpha-amylase, xylanase, lipase, protease) on quality of breads baked in different ovens (microwave, halogen lamp-microwave combination and conventional oven) were investigated. It was also aimed to reduce the quality problems of breads baked in microwave ovens with the usage of enzymes. As a control, bread dough containing no enzyme was used. Specific volume, firmness and color of the breads were measured as quality parameters. All of the enzymes were found to be effective in reducing the initial firmness and increasing the specific volume of breads baked in microwave and halogen lamp-microwave combination ovens. However, in conventional baking, the effects of enzymes on crumb firmness were seen mostly during storage. The color of protease enzyme added breads were found to be significantly different from that of the no enzyme and the other enzyme added breads in the case of all type of ovens.

The receptor for the fungal elicitor ethylene-inducing xylanase is a member of a resistance-like gene family in tomato.

An ethylene-inducing xylanase (EIX) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Lycopersicon esculentum). The LeEix locus in tomatoes was characterized by map-based cloning, which led to the identification of a novel gene cluster from which two members (LeEix1 and LeEix2) were isolated. Similar to the tomato Ve resistance genes in tomato plants, the deduced amino acid sequences encoded by LeEix1 and LeEix2 contain a Leu zipper, an extracellular Leu-rich repeat domain with glycosylation signals, a transmembrane domain, and a C-terminal domain with a mammalian endocytosis signal. Silencing expression of the LeEix genes prevented the binding of EIX to cells of an EIX-responsive plant and thus inhibited the hypersensitive response. Overexpression of either LeEix1 or LeEix2 genes in EIX-nonresponsive tobacco plants enabled the binding of EIX, although only LeEix2 could transmit the signal that induced the hypersensitive response. Overexpressing LeEix2 in mammalian COS-7 cells enables binding of EIX, indicating physical interaction between the EIX elicitor and LeEix2 gene product. Structural analysis of the LeEix proteins suggests that they belong to a class of cell-surface glycoproteins with a signal for receptor-mediated endocytosis. Mutating the endocytosis signal in LeEix2 (Tyr 993 to Ala) abolished its ability to induce the hypersensitive response, suggesting that endocytosis plays a key role in the signal transduction pathway.

Isolation and characterization of solventogenic, cellulase-free xylanolytic Clostridia from cow rumen.

Hemicellulolytic obligate anaerobes capable of converting a range of agricultural substrates and monomeric sugars of hemicellulose to solvents and acids were isolated from cow rumen fluid. The strains were identified as 5 strains of C. beijerinckii (3 different genotypes), 3 strains of C. acetobutylicum (2 different genotypes), 1 strain of C. butyricum and 3 strains of C. bifermentans (2 different genotypes). All of them produced cellulase-free xylanase activity. C. acetobutylicum LU1 produced solvent, a high of 5.1 gl(-1) and C.bifermentans LU1 produced xylanase activity a high of 4.2 IU ml(-1).

Bleaching of bagasse pulp with enzyme pre-treatment.

The effluent from pulp bleaching processes containing chlorinated lignin and degraded polyphenolic intermediates remains as a major source of water pollution from the pulp and paper industries. Alternative elemental chlorine free bleaching methods based on the usage of chlorine dioxide, ozone and hydrogen peroxide are potential substitutes. Bio-bleaching methods, which involve pre-treatment of pulp with microbial enzymes such as xylanases, have emerged as viable options. Investigations reported in this paper aim at exploring the suitability of commercial bacterial xylanase enzyme preparations for bio-bleaching of bagasse pulps in conjunction with specific chemical bleach sequences employing hydrogen peroxide (P), alkali extraction (E), ozonation (Z), hypochlorite (H) and chelation (Q) stages. The effluent profiles and pulp qualities obtained for each of the bleach combinations (involving bio-bleaching and chemical bleaching sequences) were monitored. Analysis of the results clearly indicates that the inclusion of enzyme pre-treatment with the TCF (total chlorine free) and ECF (elemental chlorine free) sequences has a significant effect on the effluent (COD, lignin and colour) and pulp quality (kappa number, brightness) parameters. In conclusion, the findings of this investigation indicate the potential promise of enzyme pre-treatment in combination with chemical bleaching to enhance the quality of pulps and combined effluents.

Nod factor and elicitors activate different phospholipid signaling pathways in suspension-cultured alfalfa cells.

Lipo-chitooligosaccharides (Nod factors) are produced by symbiotic Rhizobium sp. bacteria to elicit Nod responses on their legume hosts. One of the earliest responses is the formation of phosphatidic acid (PA), a novel second messenger in plant cells. Remarkably, pathogens have also been reported to trigger the formation of PA in nonlegume plants. To investigate how host plants can distinguish between symbionts and pathogens, the effects of Nod factor and elicitors (chitotetraose and xylanase) on the formation of PA were investigated in suspension-cultured alfalfa (Medicago sativa) cells. Theoretically, PA can be synthesized via two signaling pathways, i.e. via phospholipase D (PLD) and via phospholipase C in combination with diacylglycerol (DAG) kinase. Therefore, a strategy involving differential radiolabeling with [(32)P]orthophosphate was used to determine the contribution of each pathway to PA formation. In support, PLD activity was specifically measured by using the ability of the enzyme to transfer the phosphatidyl group of its substrate to a primary alcohol. In practice, Nod factor, chitotetraose, and xylanase induced the formation of PA and its phosphorylated product DAG pyrophosphate within 2 min of treatment. However, whereas phospholipase C and DAG kinase were activated during treatment with all three different compounds, PLD was only activated by Nod factor. No evidence was obtained for the activation of phospholipase A(2).

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.

Xylan-hydrolyzing enzyme system from Bacillus pumilus CBMAI 0008 and its effects on Eucalyptus grandis kraft pulp for pulp bleaching improvement.

The extracellular productions of beta-xylanase, beta-xylosidase, beta-glucosidase, beta-mannanase, arabinosidase, alpha-glucuronidase, alpha-galactosidase and Fpase from Bacillus pumilus CBMAI 0008 were investigated with three different xylan sources as substrate. The enzymatic profiles on birchwood, Eucalyptus grandis and oat were studied at alkaline and acidic pH conditions. B. pumilus CBMAI 0008 grown on the three carbon sources produced mainly beta-xylanase. At pH 10, the levels of xylanase were 328, 160 and 136 U/ml, for birch, oat and E. grandis, respectively. beta-Mannanase production was induced on E. grandis (5 U/ml) and arabinofuranosidase on oat (5 U/ml). Although small quantities of alpha-glucuronidase had been produced at pH 10, activity at pH 4.8 was 1.5 U/ml, higher than observed for Aspergillus sp. in literature reports. Preliminary assays carried out on E. grandis kraft pulp from an industrial paper mill (RIPASA S.A. Celulose e Papel, Limeira, SP, Brazil) showed a reduction of 0.3% of chlorine use in the pulp treated with the enzymes, resulting in increased brightness, compared to conventional bleaching. The enzymes were more efficient if applied before the initial bleaching sequence, in a non-pre-oxygenated pulp.