Characterization of Phytase of Beans (Phaseolus Vulgaris)

Abstract

Phytase from California Small White beans was extracted, concentrated and enriched by heat treatment and ammonium sulfate fractionation. Crude enzyme fractions tenaciously retained endogenous phytate which could be removed by autolysis at 45°C and p H 5.2. Characterization o f the phytase preparation showed: (1) increase in activity with increasing temperature from 37 to 60°C, ( 2 ) an activation energy of approximately 9,200 cal/mole f o r the hydrolysis of inositol hexaphosphate; ( 3 ) p H optimum o f 5.2; (4) a Km value of 2.22 X 1C4M;( 5 ) apparently partial inhibition at high substrate concentrations; ( 6 ) fully competitive inhibition by one o f the reaction products, inorganic phosphate, with Ki = 3.1 X 104M. These properties and kinetic constants are comparable to those reported f o r phytase preparations from similar sources and they adequately account for in situ autolytic loss of phytic acid from these beans.

Canola Phytase: Isolation and Characterization

“…In the case of phytase 2, however, no inhibition was evident in the presence of up to 3.0 mM substrate concentration. Complete or partial inhibition of phytase by substrate has been reported by other researchers (Chang and Schwimmer, 1977;Gibbins and Norris, 1963;Lolas and Markakis, 1977).…”

EFFECTS OF AUTOLYSIS ON THE NUTRITIONAL QUALITIES OF BEANS (Phaseolus vulgaris)

“…Incubation of the bean sample in water for 16-20 hr at 60°C followed by cooking in boiling water for 20 min (IBS) at 60°C increased the inorganic phosphorus concentration from 0.06 to 0.19% (Table l), amounting to 50% rather than the, 80% hydrolysis of bean phytate obtained in our previous studies (Chang et al, 1977). Owing to the limitation' of facilities, in the large scale preparation we reduced the quantity of'incubating medium from 10 ml to about 3 ml per gram of beans: Using proportionally less incubating medium may, have decreased the rate of inorganic phosphate diffusing out of'the, tissue, thus decreasing phytase activity' via substrate and product inhibition (Chang and Schwimmer, -1977).…”

“…As indicated in our previous study (Chang et al, 1977) about 70% of the phytate in California Small White beans is water-soluble. According to the study of Waldroup et al (1964), the solubility of phytate significantly affects the availability of its phosphorus.…”

“…Owing to the limitation' of facilities, in the large scale preparation we reduced the quantity of'incubating medium from 10 ml to about 3 ml per gram of beans: Using proportionally less incubating medium may, have decreased the rate of inorganic phosphate diffusing out of'the, tissue, thus decreasing phytase activity' via substrate and product inhibition (Chang and Schwimmer, -1977).…”

“…In our previous studies (Chang et al, 1977;Chang and Schwimmer, 1977), it was shown that phytate can be efficiently removed from dry beans by soaking in aqueous media at 60°C or by exposing them to water-saturated atmosphere at that temperature. These treatments initiate endogenous phytase action (Chang and Schwimmer, 1977;Lolas and Markakis, 1977) as evidenced by loss of bean phytate accompanied by a concurrent increase in inorganic phosphate. Some phytate was also lost by diffusion.…”