Comparative Enzymatic Hydrolysis of Phytate in Various Animal Feedstuff with Two Different Phytases.

Abstract

ABSTRACT. Bacillus amyloliquefaciens DS11 phytase (DS11 phytase) and Aspergillus ficuum phytase (AF phytase) activities were investigated by measuring the release of phosphate from phytate in animal feedstuff such as wheat bran, corn meal, soybean meal and rice flour at pH 5 and 7. In all the tested feedstuff, the enzymatic activity of DS11 phytase was more active at pH 7, but that of AF phytase was more active at pH 5. From these results, the phytate in the gastrointestinal tract could be degraded in the small intestine or stomach by DS11 or AF phytase, respectively. In conclusion, the results presented in this paper indicated that different combination ratios of DS11 and AF phytase, depending on the kind of feedstuff, might effectively induce more enzymatic activity both in the stomach and small intestine in terms of the pH of the gastrointestinal tract.-KEY WORDS: fermentation, phytase, phytate.J. Vet. Med. Sci. 61(11): 1257-1259, 1999 MgSO 4 ·7H 2 O, 1% casein hydrolysate, 0.05% KCl, 0.001% FeSO 4 ·7H 2 O and 0.01% MnSO 4 ·4H 2 O. After fermentation, the culture supernatant was taken from the culture broth by centrifugation and assayed for phytase activity. Phytase activity was assayed by measuring the rate increase of inorganic orthophosphate (Pi), using the ascorbic acid method of Fiske and Subbarow [1]. A reaction mixture containing 100 µl DS11 or AF phytase preparation and 400 µl of 2 mM sodium phytate in 0.1 M Tris-HCl buffer (pH 7.0) containing 2 mM CaCl 2 or 0.1 M sodium acetate buffer (pH 5.0) was incubated at 37°C for 30 min. The reaction was stopped by adding 500 µl of 15% trichloroacetic acid. The released inorganic phosphate was measured at A 820 nm by incubating with 4 ml of reagent A (1:1:1:2 ratio of 6 N H 2 SO 4 /2.5% ammonium molybdic acid/10% ascorbic acid/ H 2 O) at 37°C for 30 min. One unit (U) of DS11 and AF phytase activity was defined as the amount of enzyme required to release one mole of phosphate per min under the assay condition.Ten grams each of finely ground grains of wheat bran, soybean meal, rice flour and corn meal were used as substrates. Each substrate was suspended in 100 ml of 0.1 M Tris-HCl (pH 7) and 0.1 M sodium acetate buffer (pH 5), and both phytases were added to the substrate at a dose rate of 50 U/ml. The reaction was carried out at the temperature of 37°C for 300 min in a waterbath. The phosphate and phytic acid levels were measured in each sample taken at 0,10, 20, 30, 60, 120, 180 and 300 min after the reaction had started. Phytic acid was extracted with 3% TCA at 30°C for 4 hr and assayed by the method of Wolfgang and Lantzsch [12], which is based on the decrease level of iron. By adding 25 mg of sodium phytate (Sigma) per 5 g of grains at the very beginning of the extraction procedure, the recovery rates of phytic acid from various grains were determined.B. amyloliquefaciens DS11 producing a thermostable DS11 phytase was isolated from the soil of a Korean cattle shed. An extracelluar DS11 phytase from the bacterium was purified and biochemically characterized...

Gene Cloning and Characterization of a Thermostable Phytase from Bacillus subtilis US417 and Assessment of its Potential as a Feed Additive in Comparison with a Commercial Enzyme

“…It can be used as feed additive, alone or in combination with phytase predominantly acting in the gizzard-like Phyzyme, for further improving phytic-P utilization both in the gizzard and small intestine as recommended by Elkhalil et al [15] and Park et al [39]. Fig.…”

Phytase production by Candida melibiosica 2491 alkalophylic strain

“…As an enzyme active at acidic pH, the phytase is suitable for supplementing monogastric animals' dietaries. Acidic phytases might induce a more effective hydrolysis of phytate in both the stomach and small intestine of animals in terms of the pH of the animal gastrointestinal tract (Park et al, 1999). They show broad substrate specificity and hydrolyze metal-free phytate at the acidic pH range, producing myo-inositol monophosphate as the final product.…”

Characterization of a thermostable alkaline phytase from Bacillus licheniformis ZJ-6 in Pichia pastoris

“…Besides, a combination of BPPs and HAPs might induce a more effective hydrolysis of phytate in both the stomach and small intestine of animals in terms of the pH of animal gastrointestinal tract (Park et al 1999). The data were from rePhyCm6 culture supernatant at the end of 96-h induction.…”