Effects of the inclusion of a Bacillus direct-fed microbial on performance parameters, bone quality, recovered gut microflora, and intestinal morphology in broilers consuming a grower diet containing corn distillers dried grains with solubles.

Distillers dried grains with solubles (DDGS) have increasingly been used in poultry diets as a consequence of rising grain costs. Some, but not all, sources of DDGS have a variable compositional value, and a high inclusion of this by-product could be considered a risk factor for presentation of enteric diseases. Presently, 2 experiments were conducted using a starter corn-soybean diet (zero to 7 d) and a corn-DDGS-soybean grower diet (8 to 28 d) with or without inclusion of a Bacillus-direct-fed microbial (DFM). In both experiments, day-of-hatch chicks were randomly assigned to 2 different groups: control group without DFM or Bacillus-DFM group, containing 106 spores/g of feed. In each experiment, 8 pens of 20 chicks (n = 160/group) were used. Performance parameters of BW, BW gain (BWG), feed intake (FI), and feed conversion (FCR) were evaluated in each growth phase. Additionally, in experiment 2, intestinal samples were collected to determine duodenal and ileal morphology (n = 8/group), as well as the microbiota population of total lactic acid bacteria (TLAB), total Gram-negative bacteria (TGNB), and total anaerobic bacteria (TAB) on d 28 (n = 16/group). Furthermore, both tibias were evaluated for bone strength and bone composition (n = 16/group). In both experiments BW, BWG, and FCR were improved by the DFM when compared to the control group (P < 0.05). In experiment 2, chickens supplemented with the DFM had less TGNB in the foregut intestinal segment and higher TLAB counts in both foregut and hindgut sections (P < 0.05). In addition significant increases in tibia breaking strength and bone mineralization were observed in the DFM group when compared with the control. In the case of intestinal morphology, DFM dietary inclusion increased villus height (VH), villus width, villus area, muscular thickness, and the VH to crypt depth ratio (VH:CD) in both duodenum and ileum sections. Results of the present study suggest that consumption of a selected Bacillus-DFM producing a variable set of enzymes could contribute to enhanced performance, intestinal microbial balance, and bone quality in broiler chickens consuming a grower diet that contains corn-DDGS.

Evaluation of a Bacillus direct-fed microbial candidate on digesta viscosity, bacterial translocation, microbiota composition and bone mineralisation in broiler chickens fed on a rye-based diet.

1. The effects of the dietary inclusion of a Bacillus-based direct-fed microbial (DFM) candidate on digesta viscosity, bacterial translocation, microbiota composition and bone mineralisation were evaluated in broilers consuming rye-based diets. 2. In the present study, control mash rye-based diets (CON) or Bacillus-DFM supplemented diets (TRT) were administered ad libitum to male broilers in three independent experiments. 3. In Experiments 1 and 2 (n = 25/group), liver samples were taken to evaluate bacterial translocation, digesta samples were used for viscosity measurements and the intestinal microbial flora was evaluated from different intestinal sections to enumerate total recovered gram-negative bacteria (TGB), lactic acid bacteria (LAB) and anaerobic bacteria (TAB). Additionally, both tibias were removed for assessment of bone quality. 4. In Experiment 3, each experimental group had 8 replicates of 20 chickens (n = 160/group). Weekly, body weight (BW), feed intake (FI) and feed conversion ratio (FCR) were evaluated. At d 28-of-age, samples were taken to determine bacterial translocation, digesta viscosity and bone quality characteristics. 5. In all experiments, consumption of Bacillus-DFM reduced bacterial translocation to the liver and digesta viscosity. Additionally, DFM supplementation improved BW, bone quality measurements and FCR. Moreover, chickens fed on the Bacillus-DFM diet in Experiments 1 and 2 showed a significant reduction in the number of gram-negative and anaerobic bacteria in the duodenal content compared to control. 6. In summary, chickens fed on a rye-based diet without DFM inclusion showed an increase in bacterial translocation and digesta viscosity, accompanied by reduced performance and bone quality variables relative to the Bacillus-DFM candidate group. Hence, incorporation into the feed of a selected DFM ameliorated the adverse anti-nutritional effects related to utilisation of rye-based diets in broilers chickens.

Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.

Complementary truncations of a hydrogen bond to ribose involved in transition-state stabilization by cytidine deaminase.

The crystal structure of the complex formed between Escherichia coli cytidine deaminase and the transition-state analogue inhibitor 3,4-dihydrouridine [Betts, L., Xiang, S., Short, S. A., Wolfenden, R., & Carter, C. W. (1994) J. Mol. Biol. 235, 635] shows the presence of an H-bond between Glu-91 and the 3'-OH group of substituent ribose, a part of the substrate that is not directly involved in its chemical transformation. To test the contribution of this interaction to transition-state stabilization, Glu-91 was converted to alanine. The mutant enzyme is very much less active than the wild-type enzyme, with a 500-fold increase in Km and a 32-fold reduction in kcat using cytidine as substrate. No change in secondary structure is evident in the circular dichroic spectrum. As measured by kcat/Km, Glu-91 thus appears to stabilize the transition state for cytidine deamination by an overall factor of 1.7 x 10(4), equivalent to -5.8 kcal/mol in free energy. To test the contribution of this interaction in the opposite sense, the 3'-OH group of the substrate was replaced by a hydrogen atom. Comparing 3'-deoxycytidine with cytidine, the native enzyme shows a 17-fold increase in Km and a 400-fold decrease in kcat, indicating that the 3'-hydroxyl group of cytidine stabilizes the transition state for deamination by an overall factor of 6.3 x 10(3), equivalent to -5.2 kcal/mol in free energy, as measured by kcat/Km. After one binding partner has been removed, however, the effect of removing the remaining partner is relatively slight. For the mutant enzyme E91A, removal of the 3'-hydroxyl group from substrate cytidine reduces kcat/Km by a factor of only 3. Complete removal of substituent ribose reduces the wild-type enzyme's kcat/Km by a factor of more than 10(8); thus, substituent ribose, although distant from the site of chemical transformation of the substrate, contributes at least 11 kcal to the free energy of stabilization of the transition state for cytidine deamination, matching the apparent contribution to transition state binding made by the 4-OH group of the pyrimidine ring, which is at the site of substrate transformation [Frick, L., Yang, C., Marquez, V. E., & Wolfenden, R. (1989) Biochemistry 28, 9423].