Microbiology (1 997), 143, 245-252 Printed in Great Britain The phytase subfamily of histidine acid phosphatases: isolation of genes for two novel phytases from the fungi Aspergillus terreus and Myceliophthora thermophila David B. Mitchel1,t Kurt Vogel, Bernd J. Weimann, Luis Pasamontes and Adolphus P. G. M. van Loon Author for correspondence: Adolphus P. G. M. van Loon. Tel: +4161 688 7027. Fax: +41 61 688 1645. e-mail : Adolphus.van-Loon@Roche.com Biotechnology Section, Vitamins and Fine Division, F. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland Phytases catalyse the hydrolysis of phytate (myo-inositolhexakisphosphate) to myo-inositol and inorganicphosphate. In this study genes encoding novel phytases from two different filamentous fungi, Aspergillus terreus strain 9A-1 and Myceliophthora thermophila were isolated. The encoded PhyA phytase proteins show 60% (A. terreus) and 489'0 (M. thermophila) identity, respectively, to the PhyA of Aspergillusniger and have 21-29% identity compared to other histidine acid phosphatases. All three PhyA proteins, in contrast to the A. niger pH 25-optimum acid phosphatase, prefer phytic acid as substrate and show enzyme activity at a broad range of acidic pH values. Based on their enzyme characteristics and protein sequence homology, the phytases form a novel subclass of the histidine acid phosphatase family. Keywords : Aspergillus terreus, Myceliophthora thermophila, gene isolation, histidine acid phosphatase, phytase INTRODUCTION Phytases (myo-inositol-hexakisphosphate 3-phospho- hydrolase ; EC 3.1.3.8) are acid phosphatase enzymes which efficiently cleave phosphate moieties from phytate (myo-inositol hexakisphosphate) , thereby generating myo-inositol phosphates, myo-inositol and inorganic phosphate. Phytases belong to the family of histidine acid phosphatases, a subclass of phosphatases, all utilizing a phosphohistidine intermediate in their phosphoryl transfer reaction (van Etten, 1982). Phytate is a major phosphate storage form in plants. However, since phytate is not utilized by non-ruminants, these animals miss out on a major source of naturally occurring phosphorus. Inorganic phosphate has thus to be added to the feed to secure sufficient phosphate supply for the animal. Phytase was originally proposed as an animal feed additive to enhance the value of plant material in animal feed by liberating inorganic phos- phate (Shieh & Ware, 1968). As phytate can also act as t Present address: Life Systems Design, Kundmannweg ZA, CH- 4147 Aesch, Switzerland. The GenBank accession numbersfor the nucleotide sequences reported in this paper are U59805 (A. terreus phyA) and U59806 (M. thermophila phyA). an anti-nutrient factor in animal feed by chelating minerals (DeBoland et al., 1975; Reddy et al., 1982), addition of phytase would also increase the feed value by removing this anti-nutrient factor. More recently, phytase has been seen as a way to reduce the level of phosphorus pollution that results from the excretion of phytic acid and phosphate supplements : less inorganic phosphate has to be added to feed when additional phytase is present. A number of studies have already shown that the addition of phytase enhances phosphate utilization from phytic acid and drastically reduces inorganic phosphate excretion (Nelson et al., 1971 ; Nasi, 1990; Simons et al., 1990). The cloning and expression of the gene for phytase (phyA) from Aspergillus niger has been reported (Piddington et al., 1993 ; van Hartingsveldt et al., 1993). Ehrlich et al. (1993) also reported the cloning of the phyB gene from A. niger. However, since this protein shares over 99% amino acid sequence identity with the previously reported pH 2.5-optimum acid phosphatase encoded by the aph gene (Piddington et al., 1993), both may refer to the same acid phosphatase. We are interested in novel phytases for use in animal nutrition. We identified 27 strains of fungi expressing extracellular phytase and isolated the phytase genes 0002-1005 0 1997 SGM 245