Phytase Stability Research Articles

Brewer's yeast efficiently degrades phytate phosphorus in a corn‐soybean meal diet during soaking treatment

Microbes such as yeast and Aspergillus are known to produce phytase, and Aspergillus phytase has been used as a feed additive for improving phytate-phosphorus bioavailability in monogastric animals. We measured phytase activity in some by-products from fermented food and beverage productions by yeast and Aspergillus. The phytase activity was as high as 3577 and 2225 PU/kg DM in raw and dried brewer's yeasts, respectively. On the other hand, the phytase activity was approximately 400 PU/kg DM in white-wine yeast and red-wine yeast. The phytase activity was further low in natto (fermented soybean) residue, soy sauce cake, rice brewer's grain and the activity was not detected in dried corn-barley distiller's grain with soluble and sweet-potato distiller's residue. The stability of phytase against pepsin was much lower in the brewer's yeast than in an Aspergillus phytase preparation. On the other hand, the addition of raw brewer's yeast effectively degraded phytate phosphorus in a corn-soybean meal diet during soaking. These results suggest that phytase in the examined by-products is not suitable for the phytase source of conventional diets, but that the soaking treatment with a raw brewer's yeast is an alternative method for improving phytate-phosphorus bioavailability in corn-soybean meal diets for pigs.

Stabilization of Beta-Propeller Phytase by Introducing Xaa→Pro and Gly→Ala Substitutions at Consensus Positions

Phytases play important roles in agricultural and feed industries. In this study, the stability of a beta-propeller phytase, PhyL, from Bacillus licheniformis was successfully improved by introducing Xaa-->Pro and Gly-->Ala substitutions at consensus positions. Our results suggest that Gly-->Ala substitution is a more promising strategy to improve protein stability.

Heat stability of endogenous and microbial phytase during feed pelleting

Heat stability of commercial preparations of phytase has been of concern for quite some time, and efforts have been made to develop new preparations of this enzyme. A study was conducted to determine the stability of commercially available phytase product (granulate; Phytase A) and the new experimental product (powder; Phytase B). In the in vitro study, incubation of 100 mg of each of Phytase A and B with 200 μl of buffer for 2 min (30 s at desired temperature) resulted in 27.6 and 10.4% loss of activity at 60 °C and 80.6 and 53.9% at 70 °C, respectively. Both enzyme products were further subjected to steam pelleting in feed mills located in Manitoba and British Columbia. In the Manitoba study, the temperature of pellets as recorded at the discharge averaged 67 °C and was similar to that determined in British Columbia (70 °C). Under such temperatures, which may have been lower than the actual temperatures within the pellet mill, the loss of endogenous phytase activity averaged 58.5% (from 451 to 187 U kg− 1) and 42.5% (from 287 to 165 U kg− 1) in the two mills, respectively. Following correction for endogenous phytase activity, Phytase A and B recovery averaged 36.4 and 49.9%, respectively, at the Manitoba site and 44.1 and 49.4% at the British Columbia site. It appears evident from this study that the heat stability of enzyme (protein) per se rather than the granulation technology is a primary factor determining the stability of microbial phytase during steam pelleting.

Differential interaction of Aspergillus niger and Peniophora lycii phytases with soil particles affects the hydrolysis of inositol phosphates

The effect of the soil environment on the mobility, stability and catalytic activity of phytase from two sources was compared, as these factors have important implications for the efficacy of enzyme function in soil. Phytase from an ascomycete fungus ( Aspergillus niger) and a basidiomycete fungus ( Peniophora lycii) was added to soil suspensions from three contrasting soils and activities in the solution and solid phase were monitored. The two enzymes were compared because the P. lycii phytase was known to have greater specific activity and a more acidic isoelectric point (pI) than A. niger and therefore predicted to have different adsorption characteristics. When added to soil suspensions buffered at pH 7.5, both phytases remained in solution in all of the soils. In contrast at near natural soil pH (pH 5.5), only the P. lycii phytase remained in solution, while the A. niger phytase was rapidly adsorbed to the soil solid phase. The extent of this adsorption was reduced, however, in a soil-dependent manner by prior addition of bovine serum albumin (BSA) to the soil suspensions. At the natural pH of the soil, the stability of the P. lycii phytase in soil solution was improved under sterile conditions, whereas degradation of the A. niger phytase was unaffected. Subsequently, P. lycii phytase was shown to be more effective at hydrolysing myo-inositol hexakisphosphate added to the soil. Moreover, the P. lycii phytase also hydrolysed more organic phosphate that was endogenous to a range of soils. This research indicates that the physicochemical properties of fungal phytases affect their mobility and temporal stability and their capacity to hydrolyse inositol phosphates in soil environments.

Culture Conditions and Characterizations of a New Phytase-Producing Fungal Isolate, Aspergillus sp. L117

A novel fungal strain Aspergillus sp. L117 that produced acid-stable and thermostable phytase was isolated on basis of the clearing zone on PSM plate and the ability of Na-phytate hydrolysis. The phytase of isolate showed a 3-fold higher activity than that of A. ficuun NRRL3135. The Aspergillus sp. L117 produced maximal level of phytase at initial pH of 5.0 and 30°C. The optimal pH and temperature for phytase activity were 5.5 and 50°C, respectively. The phytase showed totally stable activity after 20 min of exposure between 30 and 90°C, and even at 100°C. The highest level of residual phytase activity was obtained at pH 5.5, and still retained the stability at the broadest pH ranges (2.0 to 7.0) of all the aforementioned phytases. Storage stability of phytase was preserved over 96% of initial activities for 60 days at 4, -20, and -70°C and to retain even 70% of the initial activity at room temperature.

Study on stability of fungal phytase as poultry feed additive

Study on stability of fungal phytase as poultry feed additive

The role of disulfide bonds in the conformational stability and catalytic activity of phytase.

Previous studies have predicted five disulfide bonds in Aspergillus niger phytase (phy A). To investigate the role of disulfide bonds, intrinsic fluorescence spectra, far-ultraviolet circular dichroism (CD) spectra, and an enzyme activity assay were used to compare the differences of catalytic activity and conformational stability of phytase during denaturation in urea in the presence and absence of dithiothreitol (DTT). In the presence of 2 mM DTT, the inactivation and unfolding were greatly enhanced at the same concentration of denaturant. The fluorescence emission maximum red shift and decreases of ellipticity at 222 nm were in accord with the changes of catalytic activity. The kinetics of the unfolding courses were a biphasic process consisting of two first-order reactions in the absence of DTT and a monophasic process of a first-order reaction in the presence of DTT. The results suggested that the loss of enzymatic activity was most likely because of a conformational change, and that disulfide bonds played an important role in three-dimensional structure and catalytic activity.

Efficacy of yeast phytase in improving phosphorus bioavailability in a corn-soybean meal-based diet for growing pigs.

Crossbred barrows (n = 66; 6 wk old) were used in a 6-wk experiment to evaluate the efficacy of phytase from yeast or Aspergillus niger on performance, tibial characteristics, and serum inorganic P concentration. We also investigated the stability of these phytases in acidic solutions with pepsin, which simulated gastric conditions. Pigs were fed a P-adequate diet containing .34% nonphytate-P or a low-P diet containing .20% nonphytate-P. The low-P diet was supplemented with 0, 1,000, 2,000, or 4,000 phytase units (PU; the activity at optimal pH, i.e., pH 4.2 for yeast phytase and pH 5.5 for phytase from Aspergillus niger)/kg of yeast phytase, or 1,000 PU/kg phytase from Aspergillus niger. The graded level of yeast phytase linearly increased ADG (P = .047), tibial weight (P = .091), tibial density (P < .001), and P concentration in tibial cortex (P = .018). Aspergillus niger phytase also increased ADG (P = .022), serum inorganic P concentration (P < .001), tibial density (P = .007), and tibial P concentration (P = .025). The pigs given 1,000 PU/kg Aspergillus niger phytase showed greater ADG (P = .091), tibial density (P= .001), and tibial P concentration (P = .062) than those given 1,000 PU/kg yeast phytase. No measurements differed (P > .31) between the pigs given 1,000 PU/kg Aspergillus niger phytase and those given 4,000 PU/kg yeast phytase. These results suggested that yeast phytase improves bioavailability of P in the diet for growing pigs but the efficacy of yeast phytase is less than that of Aspergillus niger phytase. During incubation in acidic solutions with pepsin, yeast phytase (P < .001) lost more of its activity than Aspergillus niger phytase. This lesser stability of yeast phytase may be responsible for the poorer efficacy of yeast phytase than that of Aspergillus niger. In summary, supplementation of swine diets with yeast phytase is beneficial, but its efficacy is less than that of Aspergillus niger phytase.

Biophysical characterization of fungal phytases (myo-inositol hexakisphosphate phosphohydrolases): molecular size, glycosylation pattern, and engineering of proteolytic resistance.

Phytases (myo-inositol hexakisphosphate phosphohydrolases) are found naturally in plants and microorganisms, particularly fungi. Interest in these enzymes has been stimulated by the fact that phytase supplements increase the availability of phosphorus in pig and poultry feed and thereby reduce environmental pollution due to excess phosphate excretion in areas where there is intensive livestock production. The wild-type phytases from six different fungi, Aspergillus niger, Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, Myceliophthora thermophila, and Talaromyces thermophilus, were overexpressed in either filamentous fungi or yeasts and purified, and their biophysical properties were compared with those of a phytase from Escherichia coli. All of the phytases examined are monomeric proteins. While E. coli phytase is a nonglycosylated enzyme, the glycosylation patterns of the fungal phytases proved to be highly variable, differing for individual phytases, for a given phytase produced in different expression systems, and for individual batches of a given phytase produced in a particular expression system. Whereas the extents of glycosylation were moderate when the fungal phytases were expressed in filamentous fungi, they were excessive when the phytases were expressed in yeasts. However, the different extents of glycosylation had no effect on the specific activity, the thermostability, or the refolding properties of individual phytases. When expressed in A. niger, several fungal phytases were susceptible to limited proteolysis by proteases present in the culture supernatant. N-terminal sequencing of the fragments revealed that cleavage invariably occurred at exposed loops on the surface of the molecule. Site-directed mutagenesis of A. fumigatus and E. nidulans phytases at the cleavage sites yielded mutants that were considerably more resistant to proteolytic attack. Therefore, engineering of exposed surface loops may be a strategy for improving phytase stability during feed processing and in the digestive tract.

Enzymatic evaluation of Bacillus amyloliquefaciens phytase as a feed additive

Phytases from Bacillus amyloliquefaciens DS11 and Aspergillus ficuum for feed enzyme were compared on the basis of phosphate inhibition and thermal stabilities. The apparent half-life of the former enzyme at 80 °C was 42 min. The activity of B. amyloliquefaciens phytase was retained up to 5 mM phosphate while 3 mM phosphate inhibited 50% of the original activity in case of A. ficuum phytase. Addition of 5 mM CaCl2 significantly broadened the active pH range and also increased the pH stability of DS11 phytase.

Thermal stabilization ofAspergillus phytase by L-arginine

Phytase fromAspergillus species is a very heat unstable enzyme which inactivates to a great extent during the thermal processing of animal feed formulation. Various protein stabilization additives were tested to improve its heat stability. Among them, a basic amino acid, L-arginine remarkably increased the thermal stability of phytase in an aqueous solution state.