Expression, Gene Cloning, and Characterization of Five Novel Phytases from Four Basidiomycete Fungi: Peniophora lycii, Agrocybe pediades , a Ceriporia sp., and Trametes pubescens

Abstract

Phytases catalyze the hydrolysis of phosphomonoester bonds of phytate (myo-inositol hexakisphosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate. In this study, cDNA expression libraries were constructed from four basidiomycete fungi (Peniophora lycii, Agrocybe pediades, a Ceriporia sp., and Trametes pubescens) and screened for phytase activity in yeast. One full-length phytaseencoding cDNA was isolated from each library, except for the Ceriporia sp. library where two different phytase-encoding cDNAs were found. All five phytases were expressed in Aspergillus oryzae, purified, and characterized. The phytases revealed temperature optima between 40 and 60°C and pH optima at 5.0 to 6.0, except for the P. lycii phytase, which has a pH optimum at 4.0 to 5.0. They exhibited specific activities in the range of 400 to 1,200 U ⅐ mg, of protein ؊1 and were capable of hydrolyzing phytate down to myo-inositol monophosphate. Surprisingly, 1 H nuclear magnetic resonance analysis of the hydrolysis of phytate by all five basidiomycete phytases showed a preference for initial attack at the 6-phosphate group of phytic acid, a characteristic that was believed so far not to be seen with fungal phytases. Accordingly, the basidiomycete phytases described here should be grouped as 6-phytases (EC 3.1.3.26).Phytases (myo-inositol hexakisphosphate phosphohydrolases) belong to the family of histidine acid phosphatases sharing the sequence consensus pattern(27; http://www.expasy.ch /cgi-bin/get-prodoc-entry?PDOC00538). They are capable of catalyzing the hydrolysis of phosphomonoester bonds of phytate (salts of myo-inositol hexakisphosphate or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate), thereby creating lower forms of myo-inositol phosphates and inorganic phosphate (22,24). Phytases are grouped according to the specific position of the phosphate ester group on the phytate molecule at which hydrolysis is initiated, i.e., as 3-phytases (EC 3.1.3.8) or as 6-phytases (EC 3.1.3.26).Phytate is the primary source of inositol and the primary storage form of phosphate in plant seeds (23). Seeds, cereal grains, and legumes are important components of food and, in particular, of animal feed preparations. However, monogastric animals such as poultry and swine are incapable of utilizing the phosphorus bound in phytate due to low levels of phytase activity in the digestive tract. Furthermore, phytate acts as an antinutrient by chelating divalent cations and preventing the uptake of minerals, e.g., Zn (9). Thus, phytases are used as a cereal feed additive that enhances the phosphorus and mineral uptake in monogastric animals and reduces the level of phosphate output in their manure. Recently, there have been several reports on the cloning of fungal PhyA phytases from Aspergillus niger (16,20, 28), Aspergillus fumigatus (19), Aspergillus terreus, Myceliophthora thermophila (14), Emericella nidulans, Talaromyces thermophilus (18), and Thermomyces lanuginosus (2). Based on their characteristics and on their s...

Glycoprotein-surfactant interactions: A calorimetric and spectroscopic investigation of the phytase-SDS system

“…Peniophora lycii phytase was expressed in Aspergillus oryzae, purified according to previously published procedures [27], and stored at -25 °C in buffer solution (20 mM Tris-acetate, pH 6). The molecular mass of the glycosylated phytase (∼65 kDa) and the purity of this enzyme (> 95%) were determined by SDS-…”

“…This is a glycoprotein (N-glycosylated on ten glycosylation sites) consisting of a 47.5 kDa peptide and a total of ~18 kDa carbohydrate, which mainly consists of oligomeric mannose [25][26][27]. To address the effects of glycosylation on the SDS-induced destabilization, and to determine the relative affinity of the detergent for the peptide and glycan moieties, we compared data for Phy with results for an enzymatically deglycosylated form (dgPhy).…”

Disruption and Overexpression of Acid Phosphatase Gene (aphA) from a Miso Koji Mold, Aspergillus oryzae KBN630, and Characterization of the Gene Product

“…Analysis using the SignalP 3.0 program (ii) predicted that the first 19 N-terminal amino acids of AphA functioned as a signal sequence. Therefore, amino acids 20 to 27 of AphA were presumed to represent a propeptide similar to other fungal phytases (Wyss et al, 1999a, Lassen et al, 2001. The N-terminal amino acid sequence of AphA was quite different from those of two acid phosphatases and two phytases from A. oryzae RIB128.…”

Current and Potential Use of the Desert Fungus Podaxis pistillaris (L.) Fr. (Agaricaceae)

“…22,23 Three species of microscopic ascomycetes have been commercially used as the source of these enzyme products for ruminant diets: Aspergillus niger, A. oryzae and Trichoderma longibrachiatum. 24 However, basidiomycetes such as Peniophora lycii, Agrocybe pediades or Trametes pubescens 25 also have potential in this biotechnology area.…”