Comparison of phytase from genetically engineered Aspergillus and canola in weanling pig diets.
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Ninety-six crossbred pigs with an average weight of 9.0 kg were used in a 5-wk trial to compare the efficacy of genetically engineered Aspergillus ficuum phytase, expressed in Aspergillus niger (Natuphos) or in canola seed (Phytaseed), for enhancing the utilization of phytate P in corn-soybean meal-based diets fed to young pigs and to evaluate the safety of Phytaseed phytase. Three levels of the two sources of phytase (250, 500, or 2,500 U/kg of diet) were added to a corn-soybean meal basal diet containing .35% total P, .09% available P, and .50% Ca. There were six pens per treatment (one barrow and one gilt/pen), except that the diet without added phytase was fed to 12 pens of pigs. Pen feed consumption and BW were recorded weekly. During wk 5, pen fecal samples were collected for determination of apparent digestibilities of DM, Ca, and P. At the end of wk 5, all barrows were killed, and the 10th rib on both sides was removed for determination of shear force and energy. Thirty pigs (six from the diet without added phytase and the diets with 500 and 2,500 U/kg phytase from both sources) were randomly selected for gross necropsy and histologic evaluation of liver, kidney, and bone tissues. Both sources of phytase were equally effective in increasing (P < .05) daily gain, gain:feed, apparent digestibilities of DM, P, and Ca, and 10th rib measurements. Fecal P excretion was reduced with phytase addition. Feed intake was increased by phytase levels during wk 4 to 5. No significant abnormalities were seen in any of the 30 pigs necropsied. The fit of a nonlinear function revealed that most measurements were reaching a plateau at 2,500 U/kg phytase. In summary, based on performance, bone measurements, and digestibilities of P, Ca, and DM of young pigs, the efficiency of Phytaseed was similar to that of Natuphos for enhancing the utilization of phytate P in corn-soybean meal-based diets. General necropsy and histologic examination of tissues indicated no toxic effect of phytase. (+info)
Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism.
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Phytic acid (myo-inositol hexakisphosphate, InsP(6)) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P(3) and Ins(1,3,5)P(3). These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7, 147-153], and (2) computer-modelling analyses of enzyme-substrate complexes, a novel mode of phytic acid hydrolysis is proposed. (+info)
Exchanging the active site between phytases for altering the functional properties of the enzyme.
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By using a novel consensus approach, we have previously managed to generate a fully synthetic phytase, consensus phytase-1, that was 15-26 degrees C more thermostable than the parent fungal phytases used in its design (Lehmann et al., 2000). We now sought to use the backbone of consensus phytase-1 and to modify its catalytic properties. This was done by replacing a considerable part of the active site (i.e., all the divergent residues) with the corresponding residues of Aspergillus niger NRRL 3135 phytase, which displays pronounced differences in specific activity, substrate specificity, and pH-activity profile. For the new protein termed consensus phytase-7, a major - although not complete - shift in catalytic properties was observed, demonstrating that rational transfer of favorable catalytic properties from one phytase to another is possible by using this approach. Although the exchange of the active site was associated with a 7.6 degrees C decrease in unfolding temperature (Tm) as measured by differential scanning calorimetry, consensus phytase-7 still was >7 degrees C more thermostable than all wild-type ascomycete phytases known to date. Thus, combination of the consensus approach with the selection of a "preferred" active site allows the design of a thermostabilized variant of an enzyme family of interest that (most closely) matches the most favorable catalytic properties found among its family members. (+info)
The consensus concept for thermostability engineering of proteins.
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Previously, sequence comparisons between a mesophilic enzyme and a more thermostable homologue were shown to be a feasible approach to successfully predict thermostabilizing amino acid substitutions. The 'consensus approach' described in the present paper shows that even a set of amino acid sequences of homologous, mesophilic enzymes contains sufficient information to allow rapid design of a thermostabilized, fully functional variant of this family of enzymes. A sequence alignment of homologous fungal phytases was used to calculate a consensus phytase amino acid sequence. Upon construction of the synthetic gene, recombinant expression and purification, the first phytase obtained, termed consensus phytase-1, displayed an unfolding temperature (T(m)) of 78.0 degrees C which is 15-22 degrees C higher than the T(m) values of all parent phytases used in its design. Refinement of the approach, combined with site-directed mutagenesis experiments, yielded optimized consensus phytases with T(m) values of up to 90.4 degrees C. These increases in T(m) are due to the combination of multiple amino acid exchanges which are distributed over the entire sequence of the protein and mainly affect surface-exposed residues; each individual substitution has a rather small thermostabilizing effect only. Remarkably, in spite of the pronounced increase in thermostability, catalytic activity at 37 degrees C is not compromised. Thus, the design of consensus proteins is a potentially powerful and novel alternative to directed evolution and to a series of rational approaches for thermostability engineering of enzymes and other proteins. (+info)
Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR.
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Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P(6)] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P(5), Ins(1,2,3,4,6)P(5), D/L-(1,2,4,5,6)P(5), D/L-(1,2,3,4)P(4), D/L-Ins(1,2,5,6)P(4) and D/L-Ins(1,4,5,6)P(4) in PLP1B mutants as well as the parent variety. In grain extracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D/L-Ins(1,3,4,5)P(4) were observed whereas phytate and the above-mentioned inositol phosphates were present in relatively small amounts. Assignment of D/L-Ins(1,3,4,5)P(4) was corroborated by precise co-chromatography with a commercial Ins(1,3,4,5)P(4) standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and D/L-Ins(1,3,4,5)P(4), which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened further by phytate degradation assays showing that D/L-Ins(1,3,4,5)P(4) did not belong to the spectrum of degradation products generated by endogenous phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P(4) in barley low-phytate mutants are discussed. (+info)
Report of occupational asthma due to phytase and beta-glucanase.
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OBJECTIVES: Occupational asthma is the principal cause of respiratory disease in the workplace. The enzymes phytase and beta-glucanase are used in the agricultural industry to optimise the nutritional value of animal feeds. A relation between these enzymes and occupational asthma in a 43 year old man was suspected. METHODS: Inhalation challenge tests were performed with the enzymes phytase, beta-glucanase, and amylase. Skin prick tests were performed with the enzymes diluted to a concentration of 1 mg/ml and 5 mg/ml. Specific IgE to phytase and beta-glucanase were measured with a radioallergosorbent test. RESULTS: Baseline spirometry values were normal. A histamine challenge test showed bronchial hyperreactivity. Exposure to phytase and beta-glucanase led to significant reductions in forced vital capacity and forced expired volume in 1 second. No significant differences were noted after exposure to amylase. Skin tests showed a positive reaction to beta-glucanase (5 mm) at a concentration of 1 mg/ml and positive reactions to beta-glucanase (7 mm) and phytase (5 mm) at a concentration of 5 mg/ml. Similarly specific IgE was present against both phytase and beta-glucanase, at 2.5% and 9.3% binding respectively (2% binding is considered positive). CONCLUSIONS: This is the first description of occupational asthma due to the enzymes phytase and beta-glucanase. Their addition to the ever increasing list of substances associated with occupational asthma will have notable implications for those exposed to these enzymes. (+info)
Microbial phytase does not improve protein-amino acid utilization in soybean meal fed to young chickens.
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Three growth trials were conducted with young chicks to evaluate crude protein (CP) utilization in soybean meal (SBM) as affected by dietary addition of microbial phytase. In assay 1, chicks were fed two CP-deficient (50 or 150 g CP/kg) levels of dehulled SBM, and each SBM level was then supplemented with equimolar amounts of cystine or methionine (Met) or with 1200 U phytase/kg. At 50 g CP/kg, cystine or Met supplementation improved (P < 0.05) measures of growth performance, but when 150 g CP/kg from SBM was fed, only Met addition improved (P < 0.05) weight gain, food efficiency and protein efficiency ratio (PER). Thus, Cys was more limiting than Met in the diet that contained 50 g CP/kg, but Met was clearly first-limiting in the diet that contained 150 g CP/kg. Phytase supplementation did not improve (P > 0.10) chick performance at either level of CP. Chicks in assay 2 were fed 100 g CP/kg furnished by SBM, casein or corn gluten meal in the absence and presence of 1200 U phytase/kg. Weight gain, gain/food and PER values were greater (P < 0.05) in chicks fed SBM than in those fed casein, and greater (P < 0.05) in chicks fed casein than in those fed corn gluten meal. Phytase supplementation had no effect (P > 0.10) on any measure of chick performance, regardless of the protein source fed. In assay 3, three deficient levels of CP (50, 100 and 150 g/kg) from SBM were fed in the absence and presence of 1200 U dietary phytase/kg. Weight gain, food efficiency and protein accretion increased linearly (P < 0.05) as a function of protein intake, but phytase supplementation had no effect (P > 0.10) on slopes of the body weight and protein accretion curves. Likewise, phytase addition did not affect (P > 0.10) measures of protein utilization, i.e., weight gain/protein intake and protein gain/protein intake at any of the CP levels that were fed. Because sulfur amino acids are the growth-limiting factors when protein-deficient levels of SBM are fed to young chicks, we conclude that dietary addition of phytase does not improve sulfur amino acid utilization in SBM. (+info)
Novel method using phytase for separating soybean beta-conglycinin and glycinin.
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A novel method for separating soybean beta-conglycinin and glycinin from defatted soymilk by a phytase treatment was developed. Phytase was added to defatted soymilk (1000FYT/100 g of protein) at pH 6.0, and the mixture incubated for 1 h at 40 degrees C. This procedure separated beta-conglycinin and glycinin without needing a reducing agent or cooling into the soluble and insoluble fractions, respectively. Simultaneously, most of the phytate in both proteins was removed. (+info)