Application of microbial phytase and its influencing factors in vivo and in vitro
Five experiments were conducted in vivo to investigate the efficacy of phytase in improving the availability of phytate P as influenced by dietary Ca:total P (tP) ratios for pigs, broilers, and turkey poults. In pigs and poultry, microbial phytase was effective in improving performance, P and Ca digestibility, bone mineralization, and in decreasing fecal P excretion by enhancing hydrolysis of phytate P for young pigs, broilers and turkey poults fed a corn-soybean meal diet. Maximum responses were achieved at supplemental phytase levels of 750 to 1,050 units (U)/kg diet for young pigs and 600 to 900 U/kg diet for poultry. Based on nonlinear and linear response equations generated from the phytase and available P (aP) data in young pigs and non phytate P (nP) in broilers and turkeys, P-equivalency functions for phytase were developed. For pigs, the P-equivalency equation was Y = .2622(1 - .9706e⁻⋅⁰⁰¹⁸⁵ˣ); for broilers, the equation was Y = .2330(1 - .9818e⁻⋅⁰⁰⁰⁷⁴ˣ); and for turkey poults, the equation was Y = .1220(1 - 1.7721e⁻⋅⁰⁰⁵³ˣ). For these three equations, X = added phytase (U/kg diet) and Y = P-equivalency values (%). Based on these equations, 1 g of P as inorganic defluorinated phosphate could be replaced by 300 and 208 U of phytase/kg of diet for pigs fed diets containing .07 and .16% aP, by 937 U of phytase for broilers fed with .27% nP diet, or by 340 and 511 U of phytase/kg diet for turkey poults fed diets containing .27 and .36% nP, respectively. Phosphorus-equivalency values of phytase were also obtained by generating P-equivalency functions at each P level and each Ca:tP ratio. The phytase efficacy was influenced by dietary Ca:tP ratios, P, and vitamin D₃ levels. In pigs and poultry, a wide Ca:tP ratio decreased phytase efficacy because all measurements were decreased as the dietary Ca:tP ratio became wider. In young pigs, widening the ratios from 1.2 to 2.0:1 resulted in a decrease in phytase efficacy of 21.1 and 12.1% for .07 and .16% aP diets, respectively. In poultry, widening the ratio from 1.4 to 2.0 led to a decrease in phytase efficacy by 7.3% for broilers fed diets containing .27% nP, and by 6.3 and 4.2% for turkey poults fed diets containing .27 and .36% nP, respectively. A synergistic effect of vitamin D₃ addition and phytase supplementation was observed for broilers. Addition of vitamin D₃ indicated a potential for improving utilization of phytate P and Ca in the presence and absence of microbial phytase. Average daily gain, apparent P digestibility and bone ash content were the most sensitive measurements to assess microbial phytase efficacy for the replacement of inorganic P for pigs and poultry. These measurements were also sensitive for assessing the effects of varying Ca:tP ratios and levels of P. In summary, 1 g of P from defluorinated phosphate could be replaced by 250 to 400, 600 to 950 and 340 to 550 U of phytase/kg diet, respectively for young pigs, broilers and turkey poults when they were fed a com-soybean meal diet. Dietary Ca:tP ratio of 1.2:1 for young pigs and 1.1 to 1.4:1 for poultry resulted in maximum phytase efficacy. An in vitro study was performed for the evaluation of effects of cations on the characteristics of microbial phytase from A. niger. A discontinuous assay was applied to assay A. niger phytase. The enzyme was observed to have a high affinity for sodium phytate with a Km of 62 µM and a Vmax of 139 U of specific activity per mg of phytase protein. Malachite green was used as the color reagent in the discontinuous assay, which increased the sensitivity 50 fold over molybdovanadate as the color reagent. All cations tested in vitro (Mg²⁺, Mn²⁺, Ca²⁺, Cr³⁺, Fe³⁺, Cu²⁺ and Zn²⁺) inhibited phytase activity, and imposed a competitive or mixed inhibition; a binding of cations with phytate also was involved in the inhibition by decreasing the effective substrate concentration. The inhibition by Ca²⁺ and Mg²⁺ caused only a partial inhibition because the enzymatic reaction rate was never reduced to zero and replots of slopes for Ca²⁺ and Mg²⁺ were hyperbolic. Cations of Zn²⁺, Cu²⁺, Fe³⁺, Cr³⁺ and Mn²⁺ gave a pure inhibition. A decreasing order of the inhibitory effect from cations was observed on the phytase activity: Zn²⁺ > Cu²⁺ > Fe³⁺ > Cr³⁺ > Ca²⁺ > Mn²⁺ > Mg²⁺ based on the Ki value that increased from a low value for Zn²⁺ to a high value for Mg²⁺. In summary, cations possess a potential for decreasing A. niger phytase activity by a competitive or mixed-type inhibition system; binding of cations with the phytate substrate also inhibited the activity of A. niger phytase.