The induction and characterization of phytase and beyond Bing-Lan Liu,* Amjad Rafiq, † Yew-Min Tzeng,* and Abdul Rob † *Institute of Biotechnology, National Dong Hwa University, Shoufeng, Taiwan and † Department of Biological and Chemical Science, University of Essex, Colchester, United Kingdom The hydrolysis of phytic acid, the principal storage form of phosphorus in seeds and pollen, to myo-inositol and phosphoric acid is a very important metabolic process in many biological systems. This dephosphorylation of free or bound inositol phosphate is believed to be mainly affected by phytase. Generally, phytase behaves like a monomeric protein of a molecular mass of approximately 40 –200 kDa and shows a broad substrate specificity with optimal degradation of phytate occurring around pH 4.5– 6.0 and a temperature at 45– 60°C. Furthermore, it is found that the Aspergillus ficuum phytase consists of 594 amino acid residues and the secondary structure contains 17.3% -helixes, 29% -sheet, 32.6% turns, and 24.7% coils. The N-linked mannose and galactose of intact phytase from A. ficuum account for 27.3% of the molecular weight. This implies that the enzyme is a glycoprotein. Recently, the crystal structure of this phytase has been determined at 2.5 Å resolution. In this review, the properties of various phytases are summarized and the digestion of phytate by phytase and its products are also discussed. © 1998 Elsevier Science Inc. Keywords: Phytic acid; phytase; myo-inositol; dephosphorylation Introduction Most cereals and legumes are rich in protein and fat but they have antinutritional factors which discourage their use in food. One such effect is phytic acid (myo-inositol- hexakisphosphate) in these produces. The phytic acid acts as an antinutrient due to its chelation of various metals and binding of protein, therefore diminishing the bioavailability of proteins and nutritionally important minerals. Phytase, or myo-inositol-hexakisphosphate phosphohy- drolase (EC 3.1.3.8), was first discovered by Suzuki et al. in the course of rice bran hydrolyzing studies. They found an enzyme present in the rice bran which catalyzed the hydro- lysis of phytic acid to inositol and orthophosphoric acid. 57 The principal end products of phytase action are phosphoric acid and myo-inositol, but the phosphatidylinositols repre- senting various degrees of dephosphorylation from inositol hexakisphosphate to inositol are generated as intermediates, or in some cases, as end products. Phytate, an inhibitor of iron absorption, can be degraded by phytase. The effects of two kinds of dietary phytase, cereal phytase and microbial phytase from Aspergillus niger on iron absorption have been investigated in some details. The results suggested that effective and complete degradation of phytate occur in the broiler’s gut when A. niger phytase was given in the feed. 67 In practice, phytase from A. niger increased the availability of phosphorus from feed for monogastric animals by releas- ing phosphate from its substrate, phytic acid. A phytase cDNA from A. niger has been expressed in transgenic tobacco plants. 97 Secretion of the protein to the extracellular fluid has been established by use of a signal sequence from the tobacco pathogen-related protein S. The specific phytase activity in isolated extracellular fluid was found to be 90-fold higher than in whole leaf extract, suggest- ing that the enzyme was secreted. This has been further confirmed by use of immunolocalization. Although there are differences in glycosylation, specific activities of tobacco and A. niger phytase are identical. This recombinant phytase was found to be biologically active and to accumulate in leaves up to 14.4% of total soluble protein during plant maturation. 80 Chemical modification study has indicated that the active Address reprint requests to Dr. Y.-M. Tzeng, National Dong Hwa Univer- sity, Institute of Biotechnology, Shoufeng, Hualien, Taiwan Received 15 May 1997; revised 11 September 1997; accepted 23 Septem- ber 1997 Enzyme and Microbial Technology 22:415– 424, 1998 © 1998 Elsevier Science Inc. All rights reserved. 0141-0229/98/$19.00 655 Avenue of the Americas, New York, NY 10010 PII S0141-0229(98)00210-X