Growth performance and amino acid digestibility responses of broiler chickens fed diets containing purified soybean trypsin inhibitor and supplemented with a monocomponent protease.

Trypsin inhibitors (TI) resident in soybeans affects protein utilization. While heat treatment influences residual TI, it simultaneously affects the structure and solubility of the soybean proteins and confounds any response to exogenous proteases. Using purified TI, the effect of exogenous protease to TI can be dissociated from changes in the soybean protein. Thus, the current study was designed to evaluate the growth performance and protein utilization responses of broiler chickens to purified TI and exogenous protease. Soybean meal (SBM) was preanalyzed for basal TI (2,996 TIU/g SBM), formulated into nutritionally adequate experimental diets to contain 1,033 TIU/g diet, and purified TI was added at 9,000 TIU/g diet. A total of 320 Cobb-500 broiler chicks were allocated to 4 diets, each with 8 replicate cages and 10 birds per replicate. The experimental diets were arranged as a 2 × 2 factorial with factors being dietary TI (1,033 or 10,033 TIU/g) and exogenous protease (0 or 15,000 PROT/kg). On day 7, 14, and 21 posthatching, protease supplementation improved the BW gain (P < 0.01) and gain to feed ratio (P < 0.05) of birds. On day 14 and 21 posthatching, the relative weight of pancreas increased (P < 0.05) with added TI but was reduced (P < 0.001) with protease supplementation. Apparent ileal digestibility of all amino acids, except methionine, decreased (P < 0.001) with added TI but increased (P < 0.05) with protease supplementation. Jejunal MUC-2 was downregulated (P < 0.01) and SCL7A-2 was upregulated (P < 0.05) by protease supplementation. Duodenal trypsin and chymotrypsin activities reduced (P < 0.05) with added TI but increased (P < 0.01) with protease supplementation. Exogenous protease produced longer villi (P < 0.05) and deeper crypts (P < 0.01) in the jejunal tissue. In conclusion, dietary addition of purified TI negatively affects nutrient utilization by broiler chickens. Furthermore, the study showed that the efficacy of the exogenous protease might be independent of dietary TI concentration.

Toward standardized amino acid matrices for exogenous phytase and protease in corn-soybean meal-based diets for broilers.

A total of 468 male Ross 308 broilers were used in a digestibility study to determine the additivity of apparent or standardized amino acid (AA) digestibility values for corn, soybean meal (SBM), or a mixture of corn and SBM that were supplemented, or not, with either phytase, protease, or a combination of phytase and protease. These treatments generated a total of 12 experimental diets that were arranged in a 3 × 4 design. A nitrogen-free diet was also fed to estimate endogenous AA loss. Apparent and standardized AA digestibility values were assessed on day 28 posthatch. The apparent digestibility of AA in the complete diet was higher (P < 0.05) than expected based on the digestibility of the corn and SBM individually. However, this overestimation was corrected by the adjustment to standardized values. Importantly, addition of protease or the combination of protease and phytase increased (P < 0.05) the digestibility of AA in corn and SBM. Furthermore, these effects were arithmetically coherent with respect to the measured effects of the enzymes in the mixture of corn and SBM, even improving the additivity of AA digestibility values when assessed on an apparent basis. This study demonstrates that the effect of exogenous protease and phytase on AA digestibility in complete diets is predictable based on measurements made in individual ingredients. In addition to improving digestibility values per se, exogenous protease and phytase may enhance precision in least cost formulation systems.

Contribution of individual broilers to variation in amino acid digestibility in soybean meal and the efficacy of an exogenous monocomponent protease.

A total of 72 male Ross 308 broilers were reared to day 34 on a standard wheat and soy-based diet and then offered one of the four semisynthetic experimental diets, comprising two different soybean meal sources either without or with exogenous protease (treatments therefore offered in a 2*2 factorial arrangement). Each experimental diet was fed to 18 individually housed birds from 34 to 37 D after which ileal digesta were collected and digestibility coefficients were calculated. The two soybean meal sources were found to be nutritionally divergent (P < 0.01), with one having the apparent ileal amino acid digestibility coefficient of 0.80 and the other 0.71. Exogenous protease increased (P < 0.01) apparent ileal amino acid digestibility coefficients from 0.74 to 0.77. There were no interactions between soybean meal origin and protease effect. On an individual bird level, there were substantial differences in the capacity to digest amino acids with the mean total amino acid digestibility coefficients from 0.54 to 0.80 for one of the soybean meal samples. Exogenous protease addition reduced the coefficient of variation for total amino acids from 11.4 to 9.1% in one soybean meal and from 7.7 to 6.3% in the other. It can be concluded that soybean meal digestibility varies and that some of this variance is associated with heterogeneity in the digestive capacity of broilers.

Additivity of apparent and standardized ileal amino acid digestibility of corn and soybean meal in broiler diets.

A total of 144 male Ross 308 broiler chickens were used in a digestibility bioassay to determine the additivity of apparent or standardized amino acid (AA) digestibility values for corn, soybean meal (SBM), or a mixture of corn and SBM. Following the receipt of a standard commercial starter diet from days 1 to 21, broilers were divided into 4 treatments (6 cages per treatment; 6 birds per cage) and received semi-purified diets based on corn, SBM, or a mixture of corn and SBM or a nitrogen-free diet (for the estimation of basal endogenous AA losses). Apparent and standardized ileal AA digestibility values were determined on day 28 and the measured values for the mixture of corn and SBM were statistically compared to calculated values based on the digestibility of the individual raw materials and their concentration in the mixed diet. The use of apparent ileal AA digestibility values for single ingredients resulted in an underestimation (P < 0.05) of the digestibility of the mixed feed for nitrogen, Lys, Arg, Thr, Asp, and Gly. Correction of apparent digestibility values to standardized values entirely corrected the underestimation in the mixed feed and resulted in a predictable linearity from single ingredients to the mixed diet. It can be concluded that standardized ileal AA digestibility values are more additive than apparent values and should be used, where possible, to enhance the accuracy of feed formulation and reduce both the diet cost and the environmental impact of nitrogenous pollution.

Disruption of an ionic network leads to accelerated thermal denaturation of D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima.

The role of an ionic network of four charged amino acid side-chains in the thermostability of the enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima (TmGAPDH) has been assessed by site-directed mutagenesis, replacing the central residue of the ionic network, arginine 20, by either alanine (R20A) or asparagine (R20N). The purified mutant enzymes display no differences to the wild-type enzyme regarding spectroscopic properties and enzymatic activity. However, denaturation kinetics reveal that the resistance towards thermal denaturation is strongly diminished in the mutant enzymes. This is reflected by a decrease in free energy of activation for thermal unfolding of about 4 kJ/mol at 100 degrees C and a shift of temperature of half denaturation after one hour incubation from 96 to 89 degrees C for both mutant enzymes. Due to a large decrease in activation enthalpy, the effects of the mutations are temperature dependent and become even more significant at the physiological temperature of Thermotoga maritima (approximately 80 degrees C). The importance of the arginine 20 side-chain for kinetic thermal stability is plausible in the light of its key role in the ionic network and the strategic positioning of this ionic network in the context of the overall protein structure.

Effect of preformed correct tertiary interactions on rapid two-state tendamistat folding: evidence for hairpins as initiation sites for beta-sheet formation.

The role of preformed correct side chain interactions, such as disulfide bonds, on protein folding kinetics is still not well understood. We investigated the effect of disulfide bond replacements on folding and stability of the small beta-sheet protein tendamistat. Tendamistat folds very fast (tau = 10 ms at pH 7 in water) and without detectable intermediates, which facilitates molecular interpretation of the kinetic data. Tendamistat contains two disulfide bonds, one between cysteines 11 and 27, which connects the ends of a beta-hairpin, and a second one between cysteines 45 and 73, which brings together the two outer strands of a three-stranded beta-sheet. Two single-disulfide variants of the protein were prepared by site-directed mutagenesis (tendamistat C11A/C27S and tendamistat C45A/C73A), and the effects on stability and on folding were monitored. Replacement of either disulfide bond leads to a large decrease in protein stability (DeltaDeltaG0 = 6.0 kcal/mol for the C11A/C27S variant and 5.1 kcal/mol for the C45A/C73A variant). This effect is caused both by entropic stabilization of the unfolded state and by enthalpic destabilization of the native structure. Kinetic experiments show that the main effect of fixed side chain contacts is on the unfolding rate. For both single-disulfide variants, unfolding is strongly accelerated (4250 times in the C11A/C27S variant and 250 times in the C45A/C73A variant) whereas the refolding rate constants are only slightly decreased. The activation parameters show that the observed small effect on the refolding reaction in the C11A/C27S variant is a consequence of large and compensating changes in the entropy and enthalpy of activation. Structural interpretation of the kinetic data suggests that formation of the beta-hairpin stabilized by the C11-C27 disulfide bond forms in the rate-limiting step of the refolding process. The interactions between the outer strands of the beta-sheet connected by the C45-C73 disulfide bond, in contrast, are made late in refolding. These results support the idea that beta-hairpins are initiation sites for beta-sheet formation and that additional strands are added late in the folding process.

Nonprolyl cis peptide bonds in unfolded proteins cause complex folding kinetics.

Folding of tendamistat, an inhibitor of alpha-amylase, is a fast two-state process accompanied by two minor slow reactions, which were assigned to prolyl isomerization. In a proline-free variant, 5% of the molecules still fold slowly with a rate constant of 2.5 s(-1). This reaction is caused by a slow equilibrium between two populations of unfolded molecules. The time constant for this equilibration process, its sensitivity to LiCl and its temperature dependence identify it as a cis-trans isomerization of nonprolyl peptide bonds. Although nonprolyl peptide bonds have the cis conformation populating only approximately 0.15% in unfolded proteins, their large number generates a significant fraction of slow-folding molecules. This emphasizes that heterogeneous populations in an unfolded protein can induce complex folding kinetics on various time scales.

Denaturant-induced movement of the transition state of protein folding revealed by high-pressure stopped-flow measurements.

The small all-beta protein tendamistat folds and unfolds with two-state kinetics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure between 0.1 and 100 MPa (1 to 1, 000 bar). GdmCl-induced equilibrium unfolding transitions reveal that the volume of the native state is increased by 41.4 +/- 2.0 cm(3)/mol relative to the unfolded state. This value is virtually independent of denaturant concentration. The use of a high-pressure stopped-flow instrument enabled us to measure the activation volumes for the refolding (DeltaVo/f) and unfolding reaction (DeltaVo/u) over a broad range of GdmCl concentrations. The volume of the transition state is 60% native-like (DeltaVo/f) = 25.0 +/- 1.2 cm(3)/mol) in the absence of denaturant, indicating partial solvent accessibility of the core residues. The volume of the transition state increases linearly with denaturant concentration and exceeds the volume of the native state above 6 M GdmCl. This result argues for a largely desolvated transition state with packing deficiencies at high denaturant concentrations and shows that the structure of the transition state depends strongly on the experimental conditions.

Improving the catalytic activity of a thermophilic enzyme at low temperatures.

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants.