Phytase or myo-inositol-hexakis phosphate phosphor hydrolase (EC 18.104.22.168) was first discovered by Suzuki et al. (1907). They found an enzyme in the rice bran which catalyzed the hydrolysis of phytic acid to inositol and orthophosphoric acid (Nagai and Funahashi, 1962). It is an important reaction for energy metabolism, metabolic regulation and signal transduction pathways (Vats and Banerjee, 2004). Phytases are subgroup of phosphatases. The complete hydrolysis of phytate results in the formation of one molecule of inositol and six molecules of inorganic phosphate (Shamsuddin, 2002). So from last two decades, phytase enzyme has attracted scientists and entrepreneurs in the areas of nutrition, environmental protection and biotechnologists.
The International Union of Biochemists categorizes the phytase enzyme into two classes namely 3-phytase and 6-phytase based on where dephosphorylation initiated on phytate molecule. The 3-phytases begins by removing the phosphate in the carbon 3rd position and yields 1,2,4,5,6 pentakisphosphate, while the 6-phytases in the carbon 6th position yields 1,2,3,4,5 pentakisphosphate as the first product along with Pi (inorganic phosphorus). The 3-phytases are produced from microorganisms and the 6-phytases present in plants (Reddy et al., 1982 ; Nayini and Markakis, 1986). Based on the structure and catalytic mechanism, phytases are classified into three classes. The three classes include histidine acid phosphatases (HAPs), ?-propeller phytases (?PP) and purple acid phosphatases (PAP) (Mullaney and Ullah, 2003).
i. Histidine acid phosphatase (HAP)
The HAP group is studied broadly because it has a high specific activity for phytic acid. This group found in both prokaryotes (app A phytase from E.coli) and in eukaryotes (phyA and phyB from Aspergillus species, from plant and yeasts). All HAP shares a conserved active site hepta-peptide motif RHGXRXP and catalytically active dipeptide HD (Van Etten et al., 1991). This group enzyme catalyzes the phytic acid in two step mechanism: a nucleophilic attack on the phosphorus atom by the histidine in the active site and followed by hydrolysis of the resulting phospho-histidine intermediate (Vincent et al., 1992). They do not need any co-factor for optimal activity (Konietzny and Greiner, 2004)
ii. ?-propeller phytases (?PP)
The ?PP groups were identified in wide group of micro-organisms. The three dimensional structure of ?PP is made of six blades. ?PP are the major phytate degrading enzymes in water and soil and play a major role in phytate- phosphorus cycle. It has two phosphate binding sites (Shin et al., 2001).
iii. Purple Acid Phosphatases(PAP)
The PAPs catalyze the hydrolysis of a wide range of phosphomonoester and amide substrates. These enzymes identified and characterized from numerous plant and animal sources.
This class of metalloenzyme has been widely studied. PAPs are active in the pH range between 3.0 and 8.0 and have been purified and characterized from a number of mammals and plants.
2.3.1. Sources of phytases
Phytases are produced to a greater extent by microorganisms and plants and to a lesser extent by animals (Pandey et al., 2001). The major phytase producers are fungi, bacteria and yeast (Pandey et al., 2001). Most of them in this group are 3-phytases (Maenz et al.,, 2001) and very few are 6-phytases (Greiner and Egli, 2003). Over 2000 microorganism screened for phytase production from soil source (Kerovou and Tynkkyen, 2000).
i. Plant phytases
Most plant phytases belong to either an acidic group with a pH optimum around 5.5 or to an alkaline group with a pH optimum around 8.0. Plant phytases are present in almost all germinating seeds. The levels of phytase in plants have shown to increase by several orders of magnitude during germination. Courtois and Perez (1948) examined the seeds from a number of different species of plants and found more or less phytase activity in Phoenix dactylifera (date), hard wheat, soft wheat, oats, barley, Bromuspratensis, Dactylisglomerata, Ricinuscommunis, radish whitemustard, Citrus nobilis, C. vulgaris, C. aurantium, Pistaciaatlantica, Fabavulgaris, Phaseoluslunatis, Lens esculenta and Cicerarietinum. Wheat and barley were highest in activity, with hard wheat more active than soft wheat. Dates had least activity and the activity in all seeds increased on germination. Phytase has also been detected in spinach leaf, tobacco leaf (Shaw, 1966), orange juice and in the roots of higher plants (Saxena, 1964; Szember, 1960; Wild and Oke, 1966).
The phytate P%, total phytate P and phytase activity (U/kg) of cereals and seeds in Table 1.
Table 1: Phytate P%, total phytate P and phytase activity (U/kg) of cereals and seeds
ii. Microbial Phytase
Phytase activity has been detected in all microbes including bacteria, fungi and yeast. Microbial sources are more promising for the production of phytases on a commercial scale (Reddy et al., 1982; Pandey et al., 2001).
Fungal sources are more promising for the production of phytases on a commercial scale (Pandey et al., 2001; Vohra and Satyanarayana, 2003; Singh et al.,2011). Fungal phytase are generally extracellular in nature and produced in large amounts (Vats and Banerjee, 2004; Singh et al., 2011). Many fungal phytase work are reported on Aspergillus species like A. ficuum, A. niger, A. fumigatus, A. oryzae (Sheih and Ware, 1968; Shimizu, 1993). The production of phytase from fungus has been achieved using different cultivation methods like solid-state which are very cost effective using various agro-industrial residues as substrates or submerged fermentation (Papagianni et al., 1999; Singh et al., 2011). These fungus secretes many other enzymes (amylase, cellulose, xylanase, etc) which improves the nutritional qualities and digestibility of the animal feed (Bogar et al., 2003a, b; Singh and Satyanarayana, 2006).Shieh and Ware (1968) identified A.niger NRRL 3135 as the most potent phytase producer in corn starch media and Howson and Davis (1983) in semisynthetic media. From 68 soil samples, Sheih and Ware (1968) screened about 2000 cultures for phytase production. Ahmad et al. (2000) and Ebune et al. (1995) used maize starch-based and canola meal medium for the production of phytase in submerged fermentation (SmF) and solid state fermentation (SSF) using Aspergillus spps. The phytase had a higher optimum temperature for its activity than the commercial enzyme, Natuphos, from A.ficuum NRRL 3135 (Kim et al., 1999).Vats and Banerjee, 2002 isolated A. niger van Teigham from wood logs that produced 184 nkat/ml phytase activity at 30 °C and pH 6.5.
Thermophilic fungi, Thermomyces lanuginosus phytase exhibited optimum activity at 65 °C and at a pH of 6.0 (Berka et al., 1998), Sporotrichum thermophile produced phytase optimally at 45 °C and at pH 6.0 in submerged as well as solid-state fermentations (Singh and Satyanarayana, 2006, 2008). Thermoascus aurantiacusTUB F43 synthesized phytase in a medium containing glucose and starch as carbon sources and peptone as a nitrogen source at 45 °C, 150 rpm and pH 5.5 after 72 hour of fermentation (Nampoothiri et al., 2004). Aspergillus fumigatus secreted a heat-stable phytase in submerged fermentation (Pasamontes et al., 1997). Hassouni et al., (2006) studied phytase production by M. thermophila in SSF using sugarcane bagasse, and maximum phytase production was achieved at 45 °C and pH 6.0, after 36 h of incubation at a moisture level of 70%. Phytase production by the T. lanuginosus TL-7 was optimized using wheat bran as a substrate in SSF using a Box–Behenken factorial design of response surface methodology, which resulted in maximum phytase production (Gulati et al., 2007).
Bacteria secreted enzyme mostly are cell associated and the only bacteria that produces extracellular phytases are of the genera Bacillus (Choi et al., 2001; Kerovou et al., 1998; Kim et al., 1998). Bacterial phytases are detected in many species like Bacillus, Pseudomonas, E.coli, Klebsiella, Lactobacillus species (Quan et al., 2001; Pandey et al., 2001; Kim et al., 1999, 1998; Griener et al.,1993; Shimuzu, 1992; Yoon et al.,1996). Bacterial phytases are generally produced by submerged fermentation at pH 7.0. Some bacterial phytases, especially those of the genera Bacillus and Enterobacter, exhibit pH optima in the range from 6.0 to 8.0 (Shimizu, 1992). Therefore, they would be more beneficial as feed additives for poultry as their pH optimum is close to the physiological pH of the poultry crop. The phytases of E. coli have been reported to be periplasmic enzymes (Greiner et al., 1993) and phytase activity in Selenomonas ruminantium, Bacillus spp. and Mitsuokella multiacidus was found to be associated with the outer membrane (D,Silva et al., 2000). Phytases are also found in enteric bacteria such as Pseudomonas spp., Bacillus spp., Raoultella spp., E. coli, Citribacter braakii, Enterobacter; anaerobic rumen bacteria,Selenomonas ruminantium, Prevetella spp. and Megasphaer aelsdenii (Konietzny and Greiner, 2004).Sreeramulu et al., (1996) evaluated 19 strains of lactic acid-producing bacteriaof the genera Lactobacillus and Streptococcus for the production of extra-cellular phytases. A genetically modified B. subtilisKHU-10 also produced extracellular phytase which constituted over90 % of the total protein. The yield was 100-fold higher than the wild type B.amyloliquefaciensDS11 (Kim et al.,, 1999). Kim et al., (1998), Shimizu (1992) and Griener et al., (1993) studied bacterial strains, Bacillus spp and E.coli isolated from soil near the roots of leguminous plants. These bacteria produced high levels of extracellular phytase under optimized conditions in a maltose, peptone and beef extract medium. Gut microorganisms also produces little phytase activity, however these microbes do not secret the enzyme and the pH of the intestine is not favourable for this enzyme (Garrett et al., 2004). Some bacteria (wild or genetically modified) such as Lactobacillus amylovorus, E. coli, Bacillus subtilis, B. amyloliquefaciens, Klebsiella spp., etc., have been employed for phytase synthesis (Pandey et al., 2001).
Yeast has found to be important source of phytase (Vohra and Satyanarayana, 2001). Yeast phytase production was carried out in SmF systems such as Schwanniomyces castellii, S.occidentalis, Hansenula polymorph, Arxula adeninivorans, Rhodotorula gracilis (Pandey et al., 2001). In a continuous culture using a strain of S. castellii, phytase production increased with pH and dilution rate. It decreased when phytic acid or phosphate content increased (Pandey et al., 2001). Mayer et al. (1999) developed an efficient process for the low-cost production of phytases using Hansenula polymorpha. Glucose or glucose syrups were used as main carbon sources during fermentation. Compared with the process using glycerol, glucose led to a reduction of more than 80 % in the raw material costs. In addition, exceptionally high concentrations of active enzyme (up to 13.5 g.l-1) were obtained in the medium, with phytase representing over 97 % of the total accumulated protein (Pandey et al., 2001).