Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

Abstract

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.

Introduction

Phytate (myo-inositol hexakisphosphate) is the major phosphorus-containing compound in plants and is particularly abundant in legumes, cereals, and oil seed; it is formed during the maturation of seeds and cereal grains [1]. Phytate is considered to be an antinutritive compound for humans and animals because it may chelate nutritionally important cations such as Ca²⁺, Mg²⁺, Fe²⁺, and Zn²⁺, and basic amino groups in the proteins, thus decreasing the dietary bioavailability of these nutrients [2], [3]. Aside from these nutritional factors, phytate plays an important role in environmental pollution. Phytate is not metabolized by monogastric animals, since they have no or very low levels of phytate-hydrolyzing enzymes in their digestive tracts. Subsequently, in order to supply sufficient phosphate, it is necessary to supplement their feeds with inorganic phosphate, even though this method increases the phosphorus burden in manure, causing environmental problems by the eutrophication of surface water resources [4]. In order to increase the bioavailability of essential dietary minerals and decrease environmental pollution, the degradation of phytate in foods and feeds is of nutritional and environmental importance.

Phytase (myo-inositol hexakisphosphate phosphohydrolase, EC 3.1.3.8) catalyzes the hydrolysis of phytate to inorganic phosphate and less-phosphorylated myo-inositol derivatives [5]. Phytases are therefore considered to be enzymes of great value in enhancing the nutritional quality of phytate-rich foods and feeds [3].

Phytase is widely distributed in plants, certain animal tissues, and microorganisms; however, it has been most intensively studied in plant seeds [6], [7]. Phytase activity in microorganisms has been found most frequently in fungi [8], [9], bacteria [10], [11], [12] and yeast [13], [14], [15]. Many fungal and bacterial phytases have been purified, and their biochemical characteristics have been reported. Furthermore, a number of phytase genes have been cloned and sequenced [16], [17]. There have been few reports on the enzymatic properties of yeast phytases [13], [18], [19]. Among the bacterial phytases, the optimum pH for these extracellular and intracellular enzymes is 6.0–7.0 and 4.5–6.0, respectively. A phytase with a pH activity profile that is ideally suited for maximal activity in the digestive tract of monogastric animals is desirable for industrial applications. There is therefore an ongoing interest in isolating new and safe microbial strains that produce novel and efficient phytases due to their practical importance.

Recently, a yeast strain producing phytase, the Saccharomyces cerevisiae CY strain, was isolated from the mash of traditional Korean Yakju [20]. In this study we have purified and characterized the enzyme activity of an extracellular phytase that was produced by a S. cerevisiae CY strain, and found that it exhibited maximum activity in the acidic pH range.