55 Phytase

Onno Misset
DSM Patents & Trademarks, Delft, The Netherlands

I.

INTRODUCTION

Phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) is the primary source of inositol and storage form of phosphorus in plant seeds. Approximately 75% of the total phosphorus in plants is in the form of phytic acid. Animal feed is primarily of vegetal origin (i.e., oilseed meals, cereal grains, and legumes) and will therefore contain a lot of phytate. In contrast to polygastric animals such as ruminants, monogastric animals like humans, pigs, and poultry have only a limited microbial population in the upper part of the digestive tract and are not able to use phytate as a phosphorus source. Consequently, to supply these animals with phosphate, inorganic phosphate has to be added separately to the feed. Phosphorus from phytate ends up in the manure, which gives rise to serious environmental pollution. Degradation of phytate by exogenous enzymes would therefore avoid or lower the necessity to add inorganic phosphate and reduce the amount of phosphorus from phytate in the manure. Phytate is also considered an antinutritional factor because of its strong chelating properties. The phosphate moieties of phytic acid are able to bind di- and trivalent metal ions such as calcium, magnesium, zinc, and iron. The chelating properties of inorganic phosphate are much less than those of phytate. Degradation of phytate in this case would considerably reduce the metal chelation properties of plant-derived animal feed. Phytases are enzymes that are capable of hydrolyzing the phosphate groups from phytate. Several com-

mercial phytase products have been developed and used as a feed additive since 1991. For reviews of phytate and phytases, the reader is referred to (1) and (2, 3), respectively. A. Chemical Reaction Catalyzed

Phytase catalyzes the hydrolysis of the 6-monophosphate esters in phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) via intermediary penta-, tetra-, tri-, di-, and monophosphates ultimately to inorganic phosphate and myo-inositol. B. Chemical Structure of Substrate

Phytic acid was described for the rst time in 1903, and its chemical structure (Fig. 1) was elucidated in 1914 (3). The chemical formula is C6 H18 O24 P6 and it has a molecular weight of 659.86 Da. Because phytate carries six phosphate groups, it can be regarded as a multisubstrate molecule. Upon successive hydrolysis, the resulting penta-, tetra-, tri-, di-, and monophosphate forms of myo-inositol are again substrates for phytase. C. Classication According to Enzyme Commission Nomenclature

Two different types of phytases are known and classied accordingly: EC 3.1.3.8 and EC 3.1.3.26. They belong to the class of hydrolases (group 3), acting on

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Table 1 Production of Intra- and Extracellular Phytases by Microorganisms Organism Fungi Agrocybe pediades Aspergillus spp. Aspergillus amstelodami Aspergillus awamori Aspergillus chevalieri Aspergillus candidus Aspergillus cuum Aspergillus fumigatis Aspergillus avus Aspergillus niger Aspergillus repens Aspergillus sydowi Aspergillus terreus Aspergillus vesicolor Aspergillus wentii Botrytis cinerea Emericella nidulans Geotrichum candidum Mucor species Mucor proformis Mucor racemosus Myceliophtora thermophila Neurospora crassa Paxillus involtus Penicillium species Penicillium caseicolum Peniophora lycii Rhizopus oryzae Rhizopus oligosporus Rhizopus stolonifer Talaromyces fumigatus Thermomyces lanugosis Trametes pubescens Ruminant microorganisms Selenomonas Prevotella Treponema Megasphaera Yeast Saccharomyces cerevisiae Schwanniomyces occidentalis Bacteria Aerobacter aerogenes Bacillus subtilis Bacillus natto N-77 Bacillus DS11 Escherichia coli Escherichia coli B Klebsiella aerogenes Pseudomona species Intra- or extracellular Reference Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra Extra 6 7 8 9 8 8 8 10, 11 7, 8 7, 8, 14 8 8 7, 12, 15 7, 8 8 8 13 8 7 8 8 10, 12 16 6 7 17 6, 18 8 8 8 10, 13 19, 20 6 21

Figure 1 Structure of phytic acid. Carbon 1 of the myoinositol ring has been indicated.

ester bonds (subgroup 1), in particular phosphomonoesters (subsubgroup 3). EC 3.1.3.8 is a 3-phytase (recommended name) and hydrolyzes rst the ester bond at the 3-position of the substrate. The systematic name is myo-inositol hexakisphosphate 3-phosphohydrolase. EC 3.1.3.26 is 6-phytase (recommended name) and hydrolyzes rst the ester bond at the 6-position of the substrate. The systematic name is myo-inositol hexakisphosphate 6phosphohydrolase. II. NATURAL OCCURRENCE OF PHYTASE

Phytases are found in both the plant and microbial kingdoms. While most plant phytases belong to the class of 6-phytases (EC 3.1.3.26), most microbial phytases are considered to be 3-phytases (EC 3.1.3.8). However, not all publications mention whether the phytase involved is a 3- or 6-phytase; the reason for this is that it is rather complicated to determine the nature of the myo-inositol pentaphosphate (1,2,4,5,6 or 1,2,3,4,5) generated by the enzymes. The occurrence of phytase in germinating plants has been reviewed recently (2, 5). The rst phytase preparations were of plant origin; Suzuki et al. in 1907 isolated them from rice and wheat bran (3). All present-day commercial phytase products are derived from lamentous fungi, in particular from Aspergillus spp. For this reason this chapter will further focus on the microbial, in particular fungal, phytases. Extensive screening programs have been carried out in the past and are still carried out today. These pro-

Extra Extra Intra Extra Extra Extra Intra Intra Intra Intra

8 2224 25 26, 27 28, 29 30, 31 32 33 34 35

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grams have resulted in a long list of bacteria, yeasts, and fungi that produce 3-phytase or 6-phytase either intra- or extracellular phytase (Table 1). Many fungal phytases were identied a few decades ago (7, 8), and resulted in most of the Aspergillus phytases known today. There is a tendency to screen more for phytases from thermophilic strains or from microorganisms that are present in the gastrointestinal tract of ruminants. As can be seen in Table 1, fungal phytases are more abundant than phytases derived from yeasts or bacteria. In addition to screening, protein engineering via site-directed mutagenesis of existing phytases has resulted in enzymes with increased specic activity (36) and thermostability (37).

In addition to its application as a feed additive, it has been suggested that phytase can also be used advantageously in the starch-processing industry during the liquefaction step of cornstarch (42). The function of phytase is mainly to degrade the phytate that has been shown to be an EIC (enzyme-inhibiting composition) inhibiting especially the alpha-amylase that is used in the liquefaction step.

IV. A.

PROPERTIES AS PROTEIN Molecular Weight

III.

INDUSTRIAL APPLICATION OF PHYTASE

The most important industrial application of phytase is in animal nutrition for reasons set out in Section I. When added to pig and poultry feed, the normal addition of inorganic phosphate can be lowered considerably, and the amount of undigested phytate in the manure of the animals is reduced substantially (30 60% depending on the type of diet composition and animal). These and other related subjects have been documented in great detail in the BASF Reference Manual entitled Phytase in Animal Nutrition and Waste Management (38) and will not be further dealt with here. At present only a limited number of phytase products are commercially available (Table 2). The rst phytase product, which entered the market in 1991, is manufactured by DSM (Gist-brocades) and sold by BASF under the trade name Natuphos. This product contains the phytase PhyA from Aspergillus niger (cuum) NRRL3135 and is available as powder, granulate, or liquid (e.g., see www.natuphos.com).

Most of the phytases from fungi and yeast are glycosylated. This means that their molecular weight is composed of the total of the amino acids of the (mature) protein and the molecular weight of the attached sugar chains. Furthermore, since most phytases are extracellular enzymes, their genes are encoding for a precursor protein that is slightly larger than the nal mature secreted enzyme, the difference being the signal peptide that is the responsible signal for the excretion process. Therefore, we can discriminate among the following molecular weights for phytases: 1. Precursor phytase, not glycosylatedcalculated from the amino acid sequence derived from the gene sequence. 2. Mature phytase, not glycosylatedcalculated from the amino acids (gene) sequence and the experimentally determined maturation site (e.g., via N-terminal amino acid sequencing of the puried enzyme). 3. Mature, glycosylated phytaseexperimentally determined by various methods (see below). Table 3 summarizes the availability of amino acid sequences derived from either the corresponding gene sequences or from Edman degradation. The fungal PhyA and PhyB genes encode precursor phytases with 439487 amino acids, corresponding to molecular weights ranging from 47 to 53 kDa (Table 4). In the

Table 2 Commercially Available Phytase Products Company DSM (Gist-brocades)/BASF Novozymes A/S Ro hm (formerly from Alko)
a

Trademark Natuphos Phytase Novo Finase P

Produced by GMOa Yes Yes Yes

Production organism Aspergillus niger

Phytase from

Reference 39 40 41

Aspergillus niger cuum NRRL 3135, van Tieghem Aspergillus oryzae Aspergillus niger Trichoderma Aspergillus awamori

Genetically modied organism.

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Table 3 Phytase Amino Acid Sequence Entries in the Protein Information Resource (PIR) and SWISS-PROT/TrEMBL Databases Available on the Web (http://www-nbrf.georgetown.edu/pirwww/pirhome.shtml and http://www.expacy.ch respectively) Gene Phy Phy PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA PhyA1 PhyB PhyB Organism Bacillus strains DS11 Bacillus subtilis Agrocybe pediades Aspergillus awamori Aspergillus cuum Aspergillus fumigatus Aspergillus nidulans Aspergillus niger (cuum) NRRL 3135 Aspergillusterreus 9A1 Aspergillus terreus CBS 116.46 Myceliophtora thermophila Peniophora lycii Talaromyces thermophilus Thermomyces lanuginosis Trametes pubescens Paxillus involtus Aspergillus awamori Aspergillus niger (cuum) NRRL 3135 Abbr. B.DS11 B.sub A.ped A.awa A.nig02 A.fum A.nid A.nig01 A.ter02 A.ter01 M.the P.lyc T.the T.lan T.pub A.awa A.nig PHYB_ASPAW PHYB_ASPNG SWISS PROT PHYT_BACSP PHYT_BACSU PHYA_ASPAW O00092 PHYB_EMENI PHYA_ASPNG O00085 O00100 O00107 O00096 JN0656 JN0482 JN0889 TrEMBL PIR Ref 30, 31 6 9 46 10, 11 14 43, 44 45 12 48 12 6, 18 13 19, 20 6 6 9 47

JN0890 JN0715

case of the PhyA phytase of Aspergillus cum (strain NRRL 3135), the molecular weight of the precursor phytase (51,086 daltons; Table 4) could be calculated from the gene-derived sequence (43, 44). In addition, the mature protein was also sequenced completely by Edman degradation (45). A comparison of the two sequences allowed identifying the position of the maturation site; it was found that the signal peptide consists of 23 amino acids, resulting in a mature protein of 444 amino acids with a calculated molecular weight of 48,846 daltons. The chemically and genederived sequences of the mature protein were found to be identical. In a recent study, Wyss et al. (49) determined the molecular weights of several fungal phytases by SDSPAGE, analytical ultracentrifugation, and gel ltration analysis (see Table 4 for a summary). The experimentally determined molecular weights are much larger than the values calculated on the basis of the amino acid sequences. The difference in molecular weight must be attributed to the glycosyl chains that are attached to the enzymes. The authors concluded that all phytases are monomers and that the contribution of the sugar chains to the molecular weight of the glycosylated mature enzyme could amount to 2065%.

B.

Primary, Secondary, Tertiary, and Quaternary Structures

The primary sequences of some 2025 phytases are known. Table 2 shows that many of these sequences are available at the Web from sequence databases such as SWISS-PROT, TrEMBL, and PIR. Yet, many (new) sequences are available from the patent literature only. Pairwise alignment of the amino acid sequences and calculation of the homology revealed that the phytase sequences can be derived into three groups: fungal phyA sequences, fungal phyB sequences, and others such as the sequences from Bacillus. The homology within a group can be as high as 4597% as is illustrated in Table 5 for the fungal PhyA phytases. Between the groups the homology is much lower; i.e., between phyA and phyB the homology is 2327% whereas no signicant homology exists between the Bacillus phytases and the fungal phyA and/or phyB phytases (not shown). Figure 2 shows a multiple alignment of fungal PhyA sequences. The N-terminal residues with a gray background have been shown or are suggested to belong to the signal peptide. Residues in white with a black background are conserved in at least ve sequences.

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Table 4 Molecular Weights of Phytases Precursor Calculated Calculated MW (Da); no MW (Da); no Abbr. #AA glycosylation #AA glycosylation A.awa 467 A.nig02 467 A.fum 465 A.nid 463 51,075 51,164 50,836 51,786 51,086 51,093 51,055 50,030 52,537 47,552 47,563 51,450 53,279 47,773 52,678 52,611 52,595 41,802 41,946 444 444 439 441 444 441 447 447 427 467 423 413 452 444 426 460 460 357 356 48,851 48,864 48,270 49,355 48,846 48,524 (45) 49,128 49,174 47,256 50,518 45,468 44,845 50,082 50,009 45,908 50,848 50,781 39,230 39,359 Mature Analytical ultracentrifuge References for #AA and calculated MW 9 46 10, 11 13 43 45 12

Gene PhyA PhyA PhyA PhyA PhyA

Organism Aspergillus awamori Aspergillus cuum Aspergillus fumigatus Aspergillus nidulans

SDS-PAGE

Gel ltration

72,360 520 (49) 60,770 2; 150 (49) 66,430 2; 300 (49) 66,360 2,440 (49) 85,000 (44)

70,740 (49) 67,400 (49) 64,890 (49)

90,500 1; 630 (49) 77,850 600 (49) 86,930 1,330 (49) 82,360 1,710 (49)

Aspergillus niger (cuum) NRRL 3135 PhyA Aspergillus terreus 9A1 PhyA Aspergillus terreus CBS116.46 PhyA Agrocybe pediades PhyA Myceliophtora thermophila PhyA1 Paxillus involtus PhyA Peniophora lycil PhyA Talaromyces thermophilus PhyA Thermomyces lanuginosis PhyA Trametes pubescens PhyB Aspergillus awamori PhyB Aspergillus niger (cuum) NRRL 3135 Phy Bacillus strain DS11 Phy Bacillus subtilis

A.nig01 467 A.ter02 A.ter01 A.ped M.the P.inv P.lyc T.the T.lan T.pub A.awa A.nig 446 466 454 487 442 439 466 475

62,890 1,210 (49)

66,150 (49)

73,800 200 (49)

6 12 6 6, 18 13 19, 20 6 9 45 30, 31

128,400 1,700 (49) 137,140 (49)

443 479 479 476 B.DS11 383 B.sub 382

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Table 5 Amino Acid Sequence Homology Between Various Fungal PhyA Phytases
A.nig01 A.nig02 A.awa A.fum A.nid A.ter01 A.ter02 T.the T.lan M.the P.lyc P.inv 1 P.inv 2 T.pub A.ped

A.nig01 A.nig02 A.awa A.fum A.nid A.ter01 A.ter02 T.the T.lan M.the P.lyc P.inv 1 P.inv 2 T.pub A.ped

96 97 66 62 62 60 61 54 46 40 41 38 41 38

95 65 62 62 60 61 53 46 41 41 39 39 38

66 62 63 61 61 54 46 41 40 38 41 38

67 60 59 60 52 49 40 40 39 40 37

59 57 59 54 48 40 40 39 39 42

87 59 49 45 43 41 39 39 38

87 48 47 43 41 39 39 38

59 45 43 37 36 39 37

46 38 40 39 40 37

43 41 40 41 39

55 55 51 51

85 62 58

63 58

61

The secondary and tertiary structures of phytase are known in great detail because the rst 3-D structure of phytase from A. niger (cuum) was recently determined by by x-ray crystallography at a resolution of 2.5 A researchers of HoffmanLa Roche (50). To be successful in crystallizing the phytase, the enzymes had to be deglycosylated completely. The structure has an =-domain similar to that of rat acid phosphatase and an -domain with a new fold (Fig. 3). The high homology that exists between fungal PhyA phytases suggests that all these phytases (Table 5 and Fig. 2) will have the same secondary structural elements and tertiary fold and topology. Not all the 444 amino acids could be detected in the electron density map: residues 16 and 249252 (numbering of the mature protein; add 23 to obtain the precursor protein numbering) were not visible, presumably because of too high a mobility. All 10 cysteine residues are involved in a disulde bridges: Cys817, Cys48391, Cys192442, Cys241259, and Cys413 421. The amino acid alignment in Figure 2 shows that, with the exception of the cysteines involved in bridge Cys817, the eight other cysteines and therefore the four disulde bridges are completely conserved in the fungal PhyA phytases. Amino acids involved in catalysis and substrate binding will be discussed later. C. Isoforms

3). The homology between these two types of phytases is relatively low, and the enzymological properties are very different (51). In relation to phytases, several publications also refer to acid phosphatases (EC 3.1.3.2). However, these enzymes show no amino acid sequence homology with phytases whatsoever, and also their substrate spectrum differs from phytase since they cannot use phytate as a substrate (51, 53). Therefore, fungal PhyA phytases can be regarded as very specialized acid phosphatases because of their unique property to efciently dephosphorylate phytic acid. 2. From Posttranslational Modication

1. From Different Genes In Aspergillus cuum and A. awamori, two phytase genes have been identied: PhyA and PhyB (Table

Extracellular phytases undergo two posttranslational modications: glycosylation and maturation. Glycosylation takes place in the Golgi apparatus of eukaryotic cells like fungi and yeast (and not in prokaryotic cells such as bacteria), and it involves only those proteins that are secreted by the cell. Glycosylation is known to generate a heterogeneous population of glycoproteins. Since the glycosyl chains contribute substantially to the molecular weight of phytase, the molecular weight will show a distribution around an average value instead of having one single value. Glycosylation of different phytases depends on the microbial expression system (49). In Aspergillus, glycosylation was found to be moderate, whereas it was excessive and highly variable in Humicola polymorpha

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Figure 2 Amino acid sequence alignment of fungal PhyA phytases. For abbreviations and references see Table 3. The sequences were aligned using the Multiple Alignment Tool from PIR at http://www.nbrf.georgetown.edu/pirwww/search/multaln.html which makes use of Clustal W. White text and a black background mark conserved amino acid residues in at least ve sequences. A gray background marks signal peptides. The -helices and -strands are denoted by  and , respectively.

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Figure 2

Continued

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and Saccharomyces cerevisae. On the basis of the consensus Asn-Xxx-Ser and Asn-Xxx-Thr sequences, potential N-linked glycosylation sites in A. niger phytase were identied at positions 27, 59, 105, 120, 207, 230, 339, 352, 376, and 388. However, the multiple alignment of the amino acid sequences shows that only the consensus sequences at two positions: Asn207 and Asn339 are conserved. Apparently, different phytases can become glycosylated at different positions. Although the effect of glycosylation on enzyme structure and function is always under debate, the dif-

ferent glycosylation patterns will certainly affect properties such as molecular weight, isoelectric point, solubility, and possibly also thermostability but much less likely the enzyme activity. Translocation of the protein over the cell membrane is mediated by the signal peptide. During this process, the signal peptide is cleaved off by signal peptidases. This process can also give rise to heterogeneity at the N-terminus of the mature protein (49). In Figure 2, a gray background marks the (putative) signal peptides of the various phytases.

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Figure 3 Schematic representation of the phytase structure. The cylinders represent the -helices and the arrows the strands. The left-hand side of the molecule is the =-domain and the right-hand side the -domain. (Picture courtesy of Dr. Jan-Metske van der Laan.)

3. Proteolysis When fungal phytases were expressed in an Aspergillus niger strain and stored as concentrated culture supernatants, Wyss et al. (49) found that the enzymes were susceptible to limited proteolytic cleavage. In some cases, this proteolysis was accompanied with activity loss (A. fumigatis) but not in others (E. nidulans). In the latter case, the fragments remained associated with each other as was shown by gel ltration experiments, and the complex was as active as the intact enzyme. Determination of the cleavage sites was done by Nterminal sequencing of the proteolytic fragments. Comparison with the 3-D structure of the A. niger enzyme showed that proteolysis occurred at exposed loops on the surface of the molecule. By site-directed mutagenesis of the cleavage site amino acid residues it was possible to reduce the proteolytic susceptibility without affecting the specic activity of the mutant enzyme (49). When expressed in Hansenula polymorpha, no proteolysis of the fungal phytases was observed.

to hydrolyze many other phosphate esters. Table 6 summarizes the kcat , KM , and kcat =KM values of the phytase from A. niger strain NRRL3135 for various phosphate ester substrates. Phytase has the highest afnity for phytate with six phosphate groups (IP6) as can be deduced from the low KM values compared with the values for the other substrates. The highest kcat values are found with the phytate substrates, in particular IP4 and IP3, for which kcat is even much higher than for the IP6 substrate. The highest specicity constants (kcat =KM ) are observed for the phytate substrates that are 10 to 1000-fold higher than for the other substrates. In conclusion, phytate is the best substrate for phytase. In a comparative study, Wyss et al. (53) measured the specic activities of nine fungal and bacterial phytases and acid phosphatases with 14 different substrates at a xed concentration of 5 mM and at pH 5.0 and 37 C (Fig. 4). The results clearly show that the phytases from A. niger, A. terreus, and E. coli have a narrow specicity compared to the other enzymes. Their highest specic activities are obtained with phytate while 30% or lower values of the phytate specic activity were found with the other 13 substrates. On the contrary, the phytases from A. fumigatis, E. nidulans, and M. thermophilus, and the acid phosphatases from A. niger (measured at pH 5.0 and pH 2.5) display higher activity toward a broader range of substrates. However, their activity toward phytate is much lower than the activity of A. niger, A. terreus, and E. coli phytase, or not even detectable (acid phosphatase).

B.

Effect of Environmental Factors

V. A.

PROPERTIES AS ENZYME Substrate Specicity

Phytases are known as acid phosphatases capable of cleaving phosphate monoester bonds. In addition to phytic acid, phytases have been reported to be able

Environmental factors such as pH and temperature exert their effect on two enzyme properties: activity and stability. Both properties are important since they determine the nal application properties of the enzyme. The pH dependence of the phytase activity has been studied extensively for various phytases from fungal and bacterial origin (Table 7). In general, the fungal phytases are active in the acid-neutral region, while the bacterial phytases behave the same (E. coli) or display their activity in the alkaline region (e.g., Bacillus). Various research groups found that the PhyA phytase from Aspergillus niger NRRL3135 has a broad pH optimum ranging from pH 2.5 to  6. The prole is remarkable in the sense that two optima are found one at pH 4 and another at pH 5.5.

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To be active at the pH of the digestive tract of animals (i.e., neutral in the chickens crop and acidic in the stomachs), a phytase is required to possess the pH activity prole as displayed by PhyA phytase from A. niger (cuum) NRRL3135. The activity of phytase increases with temperature until a maximum is observed. This maximum is usually caused by inactivation of the enzyme due to instability. Table 7 shows that the temperature optimum of the various phytases ranges from 45 C to 65 C. The stability of phytase, in particular its thermostability, is an important property in view of its application as a feed additive. Namely, during the feed pelleting step, steam is injected which results in an increase of the temperature up to 6595 C and sometimes even higher. Consequently, phytase, which has already been added to the feed at this stage, must be able to withstand these temperatures; otherwise, too much activity will be lost. This demand for thermostability explains the continuous screening efforts for more thermostable phytases and/or the protein engineering activities to improve the thermostability of existing phytases. Also, improved formulations of phytase comprising stabilizing additives are continuously developed in order to deliver stability to the enzyme during the feed pelleting step. Wyss et al. (55) compared the thermostability of A. niger phytase (the currently used commercial phytaseTable 2), the A. fumigatis phytase, and A. niger acid phosphatase. Both in solution studies and in feed pelleting trials, the A. fumigatis enzyme proved to be more thermostable than the A. niger enzyme. This better thermostability, however, is counteracted by a vefold lower specic activity of the A. fumigatis enzyme compared with the A. niger enzyme (Table 8). The same research group also used protein engineering to improve the thermostability of phytase (37). A so-called consensus phytase was designed that has an amino acid sequence consisting of the most conserved residues found at each position in the phytases from A. niger (two sequences), A. awamori, A. terreus (two sequences), A. fumigatis (ve sequences), A. awamori, A. terreus (two sequences), A. fumigatis (ve sequences), A. nidulans, T. thermophilus, and M. thermophila. This consensus phytase proved to have the highest thermostability among all the other phytases mentioned. The temperature optimum of the activity was  70 C (compare with Table 7) and also the melting temperature as measured with differential scanning calorimetry (78 C)

was higher than of the other phytases (up to 63:3 C for the A. niger phytase).

C.

Specic Mechanism of Action

Phytases hydrolyze phosphomonoester bonds in a variety of substrates and as such belong to the class of phosphatases. This class can be divided into three groups: alkaline, acid, and protein phosphatases (56). The acid phosphatases can be further subdivided into low- and high-molecular-weight ( 18 kDa and 5060 kDa, respectively). Examples of the last group are the extracellular fungal enzymes and the purple acid phosphatases with binuclear Fe-Fe and Fe-Zn centers in their active site. Phytases belong to the group of histidine highmolecular-weight acid phosphatases. They hydrolyze phytate and other phosphoesters in a two-step mechanism (Ping-Pong) involving a covalent phosphorylated histidine adduct enzyme intermediate (57, 58). In the rst partial reaction, the phosphorylated substrate molecule binds to the enzyme to give the noncovalent Michaelis complex which is then followed by transfer of the phosphate group to the active-site histidine. In the second partial reaction, a water molecule dephosphorylates the enzyme, resulting in free enzyme and inorganic phosphate. The consequence of this mechanism is that in case of chiral substrates, net retention of the conguration at the phosphorus atom will occur, consistent with two successive associative in-line phosphoryl transfers, each of which occurs with inversion of the conguration. Sequence alignment of phytases shows that there is a conserved R81 H82 G83 A84 R85 sequence (residues 81 85 in the A. niger precursor sequence numbering). On the basis of chemical modication studies of arginines and lysines, Ullah et al. suggested that this sequence contains an essential arginine and histidine residue (59, 60), which was conrmed by site-directed mutagenesis experiments in the E. coli acid phosphatase (57). The 3D structure of A. niger phytase indeed conrmed that this peptide was involved in substrate binding (50). Therefore, it is very likely that the histidine in the peptide mentioned is involved in the phosphorylation/dephosphorylation cycle. The conserved peptide, at least residues R81 H82 G83 , has been found to be conserved in other members of the group of acid phosphatases, some bisphosphatases, and some mutases which all make use of a phosphohistidine intermediate (59, 61).

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Table 6 Substrate Specicity of PhyA Phytase from Aspergillus niger (cuum) NRRL 3135 kcat (sec1 ) 216 348 171 138 222 477 1554 164 12 104 17 42 104 17 28 103 200 59 22 22 KM (mM) 0.040 0.027 0.250 < 0:005 0.025 0.070 0.16 1.00 0.20 4.50 0.27 1.66 0.29 0.35 0.50 8.30 0.53 0.72 1.60 0.39 0.53 kcat =KM (mM1 :sec1 ) 5400 12888 684 5520 3171 2980 1554 820 3 385 10 145 300 34 3 194 278 37 56 42 Spec. act. at Vmax (U/mg) 126 100 103 81 130

Substrate Phytate: Dodeca-sodium phytate (IP6) Dodeca-sodium phytate (IP6) Dodeca-sodium phytate (IP6) Dodeca-sodium phytate (IP6) Mono-potassium phytate (IP6) Dipotassium-tetramagnesium phytate (IP6) Myo-inositol-1,2,4,5,6pentakisphopate (IP5) Myo-inositol-1,2,5,6tetrakisphophate (IP4) Myo-inositol-triphophate (IP3) Myo-inositol 2-monophosphate (IP1) Para-nitrophenylphosphate Beta-glycerophosphate Na2 H2 pyrophosphate ATP Beta-NADP Glucose-6-phosphate Phenylphosphate 4-nitrophenylphosphate bis D-fructose-1,6-bisphosphate Adenosine 5 0 -diphosphate Beta-naphthylphosphate

Conditions 58 C, 58 C, 37 C, 37 C, 58 C, 58 C, pH 5:0 pH 5:0 pH 5:5 pH 5:0 pH 5:0 pH 5:0

Reference 52 51 44 53 52 52 51 51 51 52 52 52 52 52 52 52 52 52 52 52 52

58 C, pH 5:0 58 C, pH 5:0 58 C, pH 5:0 58 C, pH 5:0 58 C, 58 C, 58 C, 58 C, 58 C, 58 C, 58 C, 58 C, 58 C, 58 C, 58 C, pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0

7 60 10 24 60 10 2 6 117 34 13 13

The structure of phytase was compared with the structure of rat acid phosphatase (50). The substrate prole of phytase differs from the rat acid phosphatase in that phytase is able to hydrolyze the large phytate substrate compared to the smaller substrates of the rat enzyme. Inspection of the active site of the two enzymes reveals on one side of the substrate-binding pocket the following conserved residues (numbering of the A. niger precursor protein): Arg81 (Arg11 in the rat acid phosphatase), His82 (His12), Gly83 (Gly13), Arg85 (Arg15), Pro87 (Pro17) of the RHGxRxP peptide as well as Arg165 (Arg79), His361 (His257), and Asp362 (Asp258) which all belong to the =-domain of the enzyme. On the other side of the substrate-binding pocket (the -domain) no similarities were found. Here, the active site of the phytase is much larger than that of the rat acid phosphatase, thus allowing the accommodation of the larger phytate substrate. A molecular modeling study of the phytasephytate complex showed that all conserved charged

active-site residues (Arg81, His82, Arg85, Arg165, His361, and Asp362) are involved in electrostatic interactions with the scissile 3-phosphate group of the substrate. Furthermore, His82 is in a favorable position to make the nucleophilic attack at the phosphorus and Asp362 is in a position to protonate the leaving alcohol [for a further discussion see (50)].

VI.

PHYTASE ASSAYS

The (specic) activity of phytase is usually determined with its natural substrate phytate or the chromogenic substitute para-nitrophenylphosphate. The rst assay involves the detection of the liberated inorganic phosphate while the second one detects the yellow paranitrophenol spectrophotometrically. Many conditions have been reported in the literature for the phytase assay using phytate as a sub-

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Figure 4 Substrate specicities of wild-type phytases and A. niger pH 2.5 acid phosphatase. All substrates were used at a concentration of 5 mM. 1, Phytic acid; 2, para-nitrophenyl phosphate; 3, phenyl phosphate; 4, fructose 1,6-bisphosphate; 5, fructose 6-phosphate; 6, glucose 6-phosphate; 7, ribose 5-phosphate; 8, -glycerophosphate; 9, -glycerophosphate; 10, 3-phosphoglycerate; 11, phosphoenolpyruvate; 12, AMP; 13, ADP; 14, ATP. (A) A. fumigatis phytase; (B) A. nidulans phytase; (C) M. thermophila phytase; (D) A. niger pH 2.5 acid phosphatase; (E) A. niger pH 2.5 acid phosphatase (measurements obtained at pH 2.5 instead of pH 5.0); (F) A. niger phytase; (G) A. terreus CBS phytase; (H) A. terreus 9A1 phytase; (I) E. coli phytase. (Courtesy of the American Society for Microbiology.)

Table 7 pH Proles of Phytasesa Phytase from A. niger (cuum) NRRL 3135 pH optimum at 37 C 2.56 2.56 2.56 26 38 37.5 47 47 5.57 3.56.5 38 36 36 69 2.56 Ref. 54 44 53 19 53 11, 36 53 53 53 53 19 6 18 27 53 Temperature optimum (pH) 58 50 55 55 55 (5.0) (5.5) (5.0) (5.5) (5.0) Ref. 54 44 37 19 37 37 37 37 19 6 18

A. fumigatis A. terreus 9A1 A. terreus CBS E. nidulans M. thermophila T. lanuginosis A. pediades P. lycii B. subtilis E. coli
a

50 (5.0) 45 (5.0) 45 (5.0) 65 (5.5) 50 (5.5) 50 (5.5)

In all cases, phytate was used as substrate.

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Table 8 Specic Activities of Various Phytases with Phytate as Substrate Phytase from Aspergillus niger (cuum) PhyA PhyA PhyA PhyA Aspergillus fumigatis Aspergillus nidulans Aspergillus terreus 9A1 Aspergillus terreus CBS Myceliophtora thermophila Thermomyces lanuginosis Peniophora lycii Paxillus involtus PhyA1 Paxillus involtus PhyA2 Trametes pubescens Escherichia coli Bacillus subtilis B13 Spec. act. (U/mg) 103 20 180 100 126 27 5 29 4 142 6 196 18 42 4 987 810 1370 1450 811 216 88 Conditions 37 C, 37 C, 37 C, 58 C, 37 C, 37 C, 37 C, 37 C, 37 C, 37 C, 37 C, 37 C, 37 C, 37 C, 37 C, pH 5:0 pH 5:5 pH 5:5 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 5:0 pH 6:0 pH 5:5 pH 5:5 pH 5:5 pH 5:5 pH 5:0 pH 7:5 Ref. 53 19 44 62 36, 53 53 53 53 53 19 6, 18 6 6 6 53 27

strate. The basic principle, however, is the same. Variations exist with respect to the incubation temperature, pH, and choice of buffer, substrate concentration, and the like. A suitable assay is carried out as follows (44): 100 L enzyme solution or distilled water as a blank is added to 900 L of a solution composed of 0.25 M sodium phosphate buffer, pH 5.5, and 1 mM phytic acid (sodium salt). The resulting mixture is incubated for 30 min at 37 C. The reaction is stopped by the addition of 1 mL of 10% trichloroacetic acid. After the reaction has terminated, 2 mL of a reagent composed of 3.66 g of FeSO4 7 H2 O in 50 mL of ammonium molybdate solution (2.5 g (NH4 )6 Mo7 O24 4 H2O and 8 mL H2 SO4 , diluted to 250 mL with demineralized water), is added. The intensity of the blue color is measured spectrophotometrically at 750 nm. The measurements are indicative of the quantity of phosphate released in relation to a calibration curve of inorganic phosphate in the range of 01 mM. One unit of phytase is dened as the amount of enzyme that is capable of forming 1 mole of phosphate per minute. Specic activities of various phytases are summarized in Table 8. The activity of phytase can also be determined conveniently by using para-nitrophenylphosphate (PNPP) as a substrate. The conditions for this determination are as follows: 10 L of an enzyme solution is added to a solution containing 890 L of 0.25 M sodium phos-

phate, pH 4.0, and 100 L 10 mM PNPP. The absorbance of the liberated para-nitrophenol is measured spectrophotometrically at 30 nm as a function of time. The absorbance increase (A/min) can be converted to a rate expressed in M/min using an extinction coefcient of 3690 M1 cm1 . One unit of phytase is dened as the formation of 1 mole of para-nitrophenol per minute.

VII. PURIFICATION Numerous purication methods for the various phytases discussed here have been published in the literature; for a summary see Table 9. In general, the complexity of the method and number of steps involved depend on the expression level of the enzyme. This means that for the purication of phytase from nonrecombinant strains, more steps are required to obtain a homogeneous phytase preparation than pure phytase from overexpressing strains.

ACKNOWLEDGMENTS I would like to acknowledge my colleagues Dr. Rob Beudeker and Dr. Jan-Metske van der Laan for helpful discussions and critically reading the manuscript.

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

Table 9 Overview of Phytase Purication Methodsa Phytase from A. fumigatis ATCC13073 A. niger NRRL 3135 Original strain or overexpressed in A. niger NW205 Original strain Method 1. 2. 1. 2. 3. 4. 1. 2. 3. 4. 1. Remarks Reference 11 54

A. niger CB

S. cerevisaea

1. 2. 1. 2. 1. 2. 3. 4. 1. 2. 1. 2. 1. 2. 1. 2. 1. 2.

A. pediades

A. niger NW205 or H polymorpha S. cerevisiae W3124

A. terreus

S. cerevisaea

E. nidulans

A. niger NW205 or H. polymorpha S. cerevisaea

E. coli

A. niger NW205 or H. polymorpha E. coli BL21

Desalting Fast-Desaltin HR 10/10 Cation-ion exchange chromatography Poros HS/M Methanol treatment of the culture ltrate Mixture of phytase and Cation-ion exchange chromatography over SP-Trisacryl M acid phosphatase that Anion exchange chromatography over DEAE-Trisacryl M copuried along with Chromatofocusing the phytase (42) Filtration of the culture broth Cation-ion exchange chromatography S-Sepharose Fast-Flow Anion exchange chromatography Q-Sepharose Fast-Flow Isoelectric focusing (42) Simple one-step Immunoafnity column chromatography using monoclonal procedure antibodies against phytase that were covalently attached to to CNBr-activated Sepharose 4B. Elution of active phytase with a pH 2.5 buffer. Hydrophobic interaction chromatography Butyl Sepharose Fast Flow Gel permeation chromatography Sephacryl S-300 Desalting East-Desaltin HR 10/10 or Sephadex G-25 superne Anion exchange chromatography Poros HQ/M Anion exchange chromatography Q-Sepharose Fast ow Hydrophobic interaction chromatography Phenyl Toyopearl 650s Anion exchange chromatography Source 30Q Anion exchange chromatography Hightrap Q Hydrophobic interaction chromatography Butyl Sepharose Fast Flow Gel permeation chromatography Sephacryl S-300 Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 superne Anion-ion exchange chromatography Poros HQ/M Hydrophobic interaction chromatography Butyl Sepharose Fast Flow Gel permeation chromatography Sephacryl S-300 Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 superne Anion-ion exchange chromatography Poros HQ/M Sonication of cell suspension + centrifugation Enzyme expressed Afnity chromatography Ni2 chromatography intracellulary

44

44

49

49 6

49

49 49

49 49

continued

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Table 9 Continued Original strain or overexpressed in S. cerevisaea

Phytase from M. thermophila

Method 1. Hydrophobic interaction chromatography Butyl Sepharose Fast Flow 2. Gel permeation chromatography Sephacryl S-300 1. Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 superne 2. Anion exchange chromatography Poros HQ/M 1. Anion exchange chromatography Q-Sepharose Fast Flow 2. Hydrophobic interaction chromatography Butyl Toyopearl 650S 3. Desalting Sephadex G25 4. Anion exchange chromatography Q- Sepharose Fast Flow 5. Desalting Sephadex G25 6. Anion exchange chromatography Source 30Q 7. Hydrophobic interaction chromatography Phenyl Toyopearl 650S 1. Hydrophobic interaction chromatography Phenyl Sepharose Fast Flow 2. Hydrophobic interaction chromatography Butyl Toyopearl 650S 3. Desalting Sephadex G25 4. Anion exchange chromatography Q-Sepharose Fast Flow 5. Anion exchange chromatography Source 30Q

Remarks

Reference 49

A. niger NW205 or H. polymorpha P. involtus Phy 1 A. cryzae IFO4177

49

P. involtus Phy 2

A. oryzae IFO4177

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P. lycii

A. oryzae IFO4177

T. pubescens

A. oryzae IFO4177

T. lanuginosis

Fusarium venenatum

T. thermophilus

S. cerevisaea

A. niger NW205 or H. polymorpha

1. Anion exchange chromatography Q-Sepharose Fast Flow 2. Hydrophobic interaction chromatography Phenyl Toyopearl 650S 3. Desalting Sephadex G25 4. Anion exchange chromatography Q-Sepharose Fast Flow 5. Anion exchange chromatography Source 30Q 1. Anion exchange chromatography Q-Sepharose Fast Flow 2. Hydrophobic interaction chromatography Butyl Toyopearl 650S 3. Desalting Sephadex G25 4. Anion exchange chromatography Q-Sepharose Fast Flow 5. Desalting Sephadex G25 6. Anion exchange chromatography Source 30Q 1. Anion exchange chromatography Q-Sepharose Big Beads 2. Ultraltration 3. Anion exchange chromatography MonoQ HR10/16 column 4. Ultraltration 5. Cation exchange chromatography MonoS HR 5/5 column 1. Hydrophobic interaction chromatography Butyl Sepharose Fast Flow 2. Gel permeation chromatography Sephacryl S-300 1. Desalting Fast-Desaltin HR 10/10 or Sephadex G-25 superne 2. Anion exchange chromatography Poros HQ/M

18

19

49

49

a Most methods comprise as a rst step the removal of the cells by centrifugation or ltration. The clear, occasionally ultraltrated, culture supernatant is then subjected to the further stages as indicated.

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