The thermostable ß-1,3-1,4-glucanase from Clostridium thermocellum improves the nutritive value of highly viscous barley-based diets for broilers.

1. Microbial ß-1,3-1,4-glucanases improve the nutritive value of barley-based diets for poultry by effectively decreasing the degree of polymerisation of the anti-nutritive soluble ß-glucans. Glycoside hydrolases (GHs) acting on recalcitrant polysaccharides display a modular architecture comprising a catalytic domain linked to one or more non-catalytic Carbohydrate-Binding Modules (CBMs). 2. GHs and CBMs have been classified in different families based on primary structure similarity (see CAZy webpage at http://www.cazy.org ). The role of CBMs is to anchor the appended GHs into their target substrates, therefore eliciting the efficient hydrolysis of structural carbohydrates. 3. Here we describe the biochemical properties of the family 16 GH from Clostridium thermocellum, termed CtGlc16A. CtGlc16A is a thermostable enzyme that specifically acts on ß-1,3-1,4-glucans with a remarkable catalytic activity (38000 U/mg protein). 4. CtGlc16A, individually or fused to the family 11 ß-glucan-binding domain of cellulase CtLic26A-Cel5E of C. thermocellum, was used to supplement a highly viscous barley-based diet for broilers. 5. The data showed that birds fed on diets supplemented with the recombinant enzymes displayed an improved performance when compared with birds given diets not supplemented with exogenous enzymes. However, inclusion of the non-catalytic CBMs had no influence on the capacity of CtGlc16A to reduce the anti-nutritive effects of soluble ß-1,3-1,4-glucans. 6. The data suggest that at elevated dosage rates, CBMs might be unable to potentiate the catalytic activity of appended catalytic domains; this effect may only be revealed when feed enzymes are incorporated at low levels.

Levels of endogenous ß-glucanase activity in barley affect the efficacy of exogenous enzymes used to supplement barley-based diets for poultry.

To improve the nutritive value of barley-based diet for broilers, 2 experiments using 2 different barley lots were performed to evaluate the capacity of a mesophilic cellulase when fused to a ß-glucan specific family 11 carbohydrate-binding module. The data revealed that the recombinant ß-glucanase derivatives were not appropriate for feed supplementation because of a lack of stability at acidic pH levels. However, under the same experimental conditions, a commercial enzyme mixture improved the nutritive value of 1 of the cereal lots used. Analysis of the nutritive value of the 2 barleys revealed intrinsic differences in the levels of endogenous ß-glucanase activity. These differences were extensively evident when the studies were expanded to a range of 64 barley lots. Thus, to clarify the effect of endogenous cellulases on the efficacy of exogenous ß-glucanases used to supplement barley-based diets for poultry, 2 barley lots presenting low and high levels of endogenous plant cell wall-degrading enzymes were selected. These lots were used to prepare 2 barley-based diets, which were supplemented with or without a commercial enzyme product and fed to broiler chicks. The data revealed that the exogenous enzymes were effective when the basal diet presented low levels of endogenous ß-glucanases but were unable to improve the nutritive value of the barley lot displaying higher ß-glucanase activity. Thus, these studies suggest that levels of endogenous ß-glucanases may affect the efficacy of exogenous enzymes used to improve the nutritive value of barley-based diets for broilers. The development of a quick ß-glucanase assay that could be applied for cereal-based feeds may help identify those barley-based diets that are more responsive to the action of feed enzymes.

Role of a family 11 carbohydrate-binding module in the function of a recombinant cellulase used to supplement a barley-based diet for broiler chickens.

1. Cellulases and xylanases display a modular architecture that comprises a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs). CBMs have been classified into 52 different families, based on primary structure similarity. These non-catalytic modules mediate a prolonged and intimate contact of the enzyme with the target substrate eliciting efficient hydrolysis of the target polysaccharides. 2. A study was undertaken to investigate the importance of a family 11 CBM, displaying high affinities for barley beta-glucans, in the function of recombinant derivatives of cellulase CtLic26A-Cel5E of Clostridium thermocellum used to supplement a barley-based diet for broiler chicken. 3. The results showed that birds fed on diets containing the recombinant CtLic26A-Cel5E modular derivatives or the commercial enzyme mixture Rovabio Excel AP displayed improved performance when compared with birds fed on diets not supplemented with exogenous enzymes. 4. It is suggested that the enzyme dosage used in this study (30 U/kg of basal diet), was probably too high for the efficacy of the family 11 CBM to be noticed. It remains to be established if the targeting effect resulting from the incorporation of CBMs in plant cell wall hydrolases may be effective at lower exogenous enzyme dosages.

Isoleucine and valine supplementation of a low-protein corn-wheat-soybean meal-based diet for piglets: growth performance and nitrogen balance.

The effects of Ile and Val supplementation of a low-CP, corn-wheat-soybean meal-based piglet diet on growth performance, incidence of diarrhea, and N balance were studied using 60 Landrace x Duroc male piglets in a 4-wk experiment. The 60 individually caged piglets were divided into 5 dietary treatments, each consisting of 12 piglets. Diet 1 was a positive control diet (20% CP); diet 2 was a low-CP negative control diet (17% CP); diets 3, 4, and 5 were low-CP diets to which Ile, Val, or the combination of Ile and Val were added, respectively. All diets were supplemented with Lys, Met, Thr, and Trp to provide the required concentrations of these AA according to the 1998 NRC. Average daily gain and ADFI were similar among pigs fed the positive control, Val-added, and the Val plus Ile-added diets. On wk-2 and wk-4, fecal score was greater (softer feces) in piglets fed the 20% CP level compared with the remaining treatments (P < 0.01). Nitrogen intake was decreased (P < 0.0001) in pigs fed diets containing low levels of CP compared with pigs fed the 20% CP diet. Fecal N excretion (g/d) was decreased (P < 0.05) in piglets fed low-CP diets at wk 1 and wk 4 of feeding, and in urine at wk 4 of feeding. Crude protein levels or AA supplementation had no effect on N retention efficiencies. These results indicate that the supplementation of Val alone, or in combination with Ile, to a low-CP piglet diet with adequate levels of Lys, Met, Thr, and Trp is necessary to achieve maximum performance in pigs consuming corn-wheat-soybean meal-based diets.

Effect of the source of dietary fat on postweaning lipogenesis in lean and fat pigs.

Twenty-four male piglets weaned after 21 days, 12 of the Large White lean breed (LW) and 12 of the Alentejano fat breed (AL), have been used to compare the effects of genotype and source of dietary fat on the activities of enzymes involved in lipogenesis and on the composition of selected fatty tissues. During 4 weeks the piglets were fed isoenergetic and isonitrogenous experimental diets, containing 5 % of either olive oil or tallow. In AL piglets the acetylcoenzyme A carboxylase activity was three- and ninefold higher, the malic enzyme activity six- and fivefold, and the glucose-6-phosphate dehydrogenase activity was four- and fivefold higher in the dorsal subcutaneous and in the perirenal fat, respectively, than in LW piglets. In general, fatty tissues of the AL piglets contained a higher proportion of saturated fatty acids. Olive oil induced a significant increase in the activities of malic enzyme and glucose-6-phosphate dehydrogenase in both tissues, but only slightly increased the acetylcoenzyme A carboxylase activity in perirenal fatty tissues (p < 0.05). The fatty acid profile of the subcutaneous and of the perirenal fat was strongly affected by the composition of dietary fat. These observations showed that the source of dietary fat influenced markedly lipid metabolism and body composition since a very early age.