Biotechnology Letters 25: 1787–1794, 2003.
© 2003 Kluwer Academic Publishers. Printed in the Netherlands.
1787
Review
Phytase enzymology, applications, and biotechnology
Xin Gen Lei1,∗ & Jesús M. Porres2
1 Department of Animal Science, Cornell University, Ithaca, NY 14853, USA
2 Departamento de Fisiologı́a, Instituto de Nutrición, Universidad de Granada, Campus Universitario de Cartuja
s/n, Granada 18071, Spain
∗ Author for correspondence (Fax: 607-255-9829; E-mail: XL20@cornell.edu)
Received 2 July 2003; Revisions requested 11 July 2003; Revisions received 1 September 2003; Accepted 2 September 2003
Key words: biotechnology, environmental pollution, mineral nutrition, phytase, phytic acid
Abstract
Phytases are phosphohydrolases that initiate the step-wise removal of phosphate from phytate. These enzymes
have been widely used in animal feeding to improve phosphorus nutrition and to reduce phosphorus pollution of
animal waste. The potential of phytases in improving human nutrition of essential trace minerals in plant-derived
foods is being explored. This review covers the basic biochemistry and application of phytases, and emphasizes
the emerging biotechnology used for developing new effective phytases with improved properties.
Introduction a poor absorption of the bound metals in small in-
testine (Cheryan 1980). This is partially attributed to
During ripening, cereal and legume seeds accumulate the wide-spreading human nutritional deficiencies of
a substantial amount of phytic acid (myo-inositol- calcium, iron, and zinc in developing countries where
1,2,3,4,5,6-hexakis dihydrogen phosphate) (Honke the staple foods are plant origin (Ferguson et al. 1989,
et al. 1998). As a result, most of these seeds and their Manary et al. 2002). As a whole, challenges in the
co-products contain 1–2% phytic acid that represent above three areas of animal nutrition, environmental
> 60% of their total phosphorus (Reddy et al. 1982). protection, and human health have prompted the fast
Presumably, a large portion, if not all, of phytic acid emerging of phytase science and biotechnology.
in seeds is in the form of salts called phytate. Al-
though phytate serves as the major source of energy
and phosphorus for seed germination, the bound phos- Nomenclature of phytase
phorus is poorly available to simple-stomached anim-
als. Thus, inorganic phosphorus, a non-renewable and Phytases are meso-inositol hexaphosphate phospho-
expensive mineral, is supplemented in diets for swine, hydrolases that catalyze the stepwise phosphate split-
poultry, and fish to meet their nutrient requirement ting of phytic acid (IP6 ) or phytate to lower inositol
for phosphorus. Meanwhile, the unutilized phytate phosphate esters (IP5 -IP1 ) and inorganic phosphate
phosphorus from plant feeds is excreted, becoming (Figure 1). A number of phytase genes and proteins
an environmental pollutant in areas of intensive an- have been identified from plants and microbes includ-
imal agriculture. Excessive phosphorus in soil runs ing bacteria, yeast, and fungi. The first and probably
off to lakes and the sea, causing eutrophication and the best characterized phytase is Aspergillus niger
stimulating growth of aquatic organisms that may pro- PhyA that is encoded by a 1.4 kb DNA fragment
duce neurotoxins injurious to humans. Furthermore, and has a molecular mass of 80 kDa, with 10 N-
the negatively charged phytic acid chelates with pos- glycosylation sites (Han & Lei 1999, Van Hartingveldt
itively charged divalent cations (Figure 1), rendering et al. 1993). Average molecular masses of bacterial
1788
Fig. 1. Phytate hydrolysis by phytase into inositol, phosphate, and other divalent elements. Phytate is myo-inositol-1,2,3,4,5,6-hexakis dihydro-
gen phosphate that contains approx. 14 to 28% phosphorus and 12–20% calcium. Phytate also chelates trace elements of iron and zinc (1 to
2%) between phosphate groups within a single phytate molecule or between two phytate molecules. Phytase is the only known enzyme that
can initiate the phosphate hydrolysis at carbon 1, 3 or 6 in the inositol ring of phytate. The removal of phosphate group by phytase results in
releasing of calcium, iron, zinc, and other metals.
phytases are smaller than those of fungal phytases (Ostanin & Van Etten 1993). However, exceptions
(40–55 vs. 80–120 kDa), mainly due to glycosylation are the phytases isolated from different Bacillus sp.
differences (Choi et al. 2001, Golovan et al. 2000, whose gene sequences are not homologous to any
Han & Lei 1999, Kerovuo et al. 1998, Rodriguez of the histidine acid phosphatases in the data bank
et al. 2000b, Ullah et al. 2000, Van Hartingveldt or do not display the active site hepta-peptide mo-
et al. 1993). The molecular masses of plant phytases tif RHGXRXP or the catalytically active dipeptide
isolated from corn, wheat, lupine, oat, and barley HD. Those enzymes have a six-bladed folding scaf-
range from 47 to 76 kDa (Greiner 2002, Greiner fold for phytase activity and two distinct features:
et al. 2000a,b, Greiner & Larsson Alminger 1999, metal-assisted increase in thermostability and metal-
Maugenest et al. 1997). mediated activation of activity (Ha et al. 2000, Ker-
Phytases can be divided into two groups based on ovuo et al. 1998, 2000, Kim et al. 1998). Meanwhile,
the initiation site of phosphate hydrolysis in the car- a soybean phytase is a purple acid phosphatase with
bon ring of inositol. Microbial phytases, especially a dinuclear Fe-Fe or Fe-Zn center in the active site
those of fungal origin (E.C. 3.1.3.8), often split the (Hegeman & Grabau 2001). All phytases have pro-
phosphate group at the C1 or C3 (carbon) of the in- nounced stereospecificity and a strong preference for
ositol ring (D-and L-configuration), and are called equatorial phosphate groups, while they are virtually
3-phytases. Plant phytases (E.C. 3.1.3.26) act prefer- unable to cleave axial phosphate groups.
entially at the C6 carbon, and are called 6-phytase.
However, phytases from Escherichia coli (Greiner
et al. 1993) or Peniophora lycii and basidiomycete Characteristics of phytase
fungi (Lassen et al. 2001) are exceptions. Catalytic-
ally, most of phytases belong to the family of histidine Phytase activity is usually measured by the amount of
acid phosphatases that is characterized by a conserved inorganic phosphate released per min from a selected
active site hepta-peptide motif RHGXRXP and the substrate under certain pH and temperature. Just like
catalytically active dipeptide HD. This group of en- other enzymes, phytase activity or function is affected
zymes catalyze the phytic acid hydrolysis in two steps: by the inherent properties of the enzyme and the action
a nucleophilic attack from the histidine in the active conditions. The following properties of phytase are of
site of the enzyme to the scissile phosphoester bond practical significance:
of phytic acid (Ostanin et al. 1992, Van Etten 1982,
Vincent et al. 1992) and protonation of the leaving
group by the aspartic acid residue of the HD motif
