Introduction

Phytate, the primary storage form of phosphate and inositol in many plants, serves as a strong chelator by binding positively charged proteins, amino acids, and minerals in insoluble complexes in the digestive tract [1]. The stepwise hydrolysis of phytate is catalyzed by phytase with minerals and orthophosphate being released and a series of lower isomers of myo-inositol phosphates and eventually myo-inositol being formed [2]. During the last few decades, phytases have been studied intensively because of the great interest in its application as feed additive [3, 4], processing of human food [5, 6], and synthesis of lower inositol phosphates [7, 8].

Phytases are widely distributed in nature and could be derived from a number of sources including plants, animals, and microorganisms. Based on their structural differences and various catalytic properties, phytases were categorized into three major classes including members of histidine acid phosphatases (HAPs), beta-propeller phytases (BPPs), and purple acid phosphatases (PAPs) [9]. Among these, BPPs are mainly isolated from Bacillus with higher thermal stability and optimum pH under neutral and exhibit unique Ca2+-dependent catalytic property and highly strict substrate specificity for the calcium–phytate complex. This review focuses attention on the present finding of Bacillus phytases and the future prospects.

Bacillus Species with Phytase Activities

Production of Bacillus Phytases

Bacillus species producing thermally stable phytase was mainly isolated from soil [10, 11] and cooked rice [12]. Submerged fermentation by B. amyloliquefaciens DS11 in the medium consisting of wheat bran and casein hydrolysate produced thermostable, extracellular phytase at 37 °C [10]. The maximal phytase activity produced by B. laevolacticus was achieved under shake flask culture and optimized conditions (2.957 U/ml) [11]. Bacillus sp. KHU-10 produced the highest level of 0.2 U/ml phytase after 4 days of fermentation under the optimized conditions [12].

Characterization of Bacillus phytases

Purification and Properties

There have been reports of partially purified Bacillus phytase preparations. Power and Jagannathan [13] reported the isolation of a phytate preparation from Bacillus subtilis with a molecular weight of 36.5 kDa. This enzyme preparation was shown to have a pH optimum between 7.0 and 7.5. This phytase exhibited a maximum activity at a temperature of 60 °C and was stable up to a temperature of 70 °C. The specific activity of the purified enzyme was reported as 8.5 to 9.0 U/mg protein.

B. subtilis (natto) phytase described by Shimizu [14] was purified to homogeneity and shown to have a molecular weight of between 36 and 38 kDa. This enzyme had a pH optimum between pH 6.0 and 6.5 and an optimum temperature of 60 °C. The specific activity of this purified phytase was reported as 8.7 U/mg protein. Kerovuo et al. [15] isolated, purified, and characterized phytase from B. subtilis VTT E-68013 showing optimum temperature and pH at 55 °C and 7.0, respectively.

Choi et al. [16] reported that an extracellular phytase from Bacillus sp. KHU-10 was purified and estimated to be 44 kDa. Its optimum pH and temperature were pH 6.5–8.5 and 40 °C without 10 mM CaCl2 and pH 6.0–9.5 and 60 °C with 10 mM CaCl2. About 50% of its original activity remained after incubation at 80 °C for 10 min in the presence of 10 mM CaCl2. The enzyme activity was fairly stable from pH 6.5 to 10.0.

Gulati et al. [11] described the partially purified phytase from B. laevolacticus was optimally active at 70 °C and pH 7.0–8.0. This enzyme exhibited thermal stability with 80% activity at 70 °C and pH 8.0 for up to 3 h in the presence or absence of 5 mM CaCl2.

Thermal Stability

The thermal stability of phytase is considered to be an important and useful criterion for application as an animal additive because of the process with high temperature and steam during pelleting [17, 18]. Bacillus phytases are quite stable at the high temperature range of 80–95 °C [10, 16, 19, 20]. Elkhalil et al. [21] revealed that Bacillus phytase is more resistant to heat treatments than E. coli and Klebsiella phytases. Considering this property, Bacillus phytases will no doubt be better candidates for feed supplements.

Effect of Calcium Ions

Bacillus phytases were found to be metal ion-dependent as they required calcium for activity and stability [10, 13, 14, 22]. Ca2+ binds two oxyanions from the phosphate groups of phytate to form an ideal positively charged distribution in the calcium–phytate complex. Because of the existence of three Ca2+-binding sites at the active site cleft, Ca2+ serves as an essential activator to reduce the negative charge around the active site cleft, whereas excess amounts of Ca2+ acts as a competitive inhibitor [22]. In addition, Ca2+ has an important effect on the stability against temperature and pH [16].

Kerovuo et al. [23] studied the metal ion requirement of B. subtilis phytase and reported that the removal of metal ions from the enzyme by EDTA resulted in complete inactivation. The loss of enzymatic activity was most likely because of a conformational change, as the circular dichroism spectra of holoenzyme and metal-depleted enzyme were different. Metal-depleted enzyme was partially able to restore the active conformation when incubated in the presence of calcium. Only minor reactivation was detected with other divalent metal ions and their combinations.

Substrate Specificity

Bacillus phytases exhibit highly strict substrate specificity for calcium–phytate complex and have no enzymatic activity on other phosphate esters [10, 13–16, 24]. Oh et al. [22] reported that no detectable phytate hydrolysis was observed when the phytase was added to phytate in the Ca2+-free environment at several different phytate concentrations. At fixed phytate concentration, increasing the concentration of Ca2+ enhanced the initial velocities of phytase in a saturating manner. This activation is mediated by a cooperative interaction of Ca2+ to the substrate and the Ca2+-dependent increase of reaction rate followed a sigmoidal curve. Other divalent cations such as Co2+, Cs2+, Cu2+, Mg2+, Mn2+, and Ni2+ have no effect on enzymatic activation. According to these results, the calcium–phytate complex is regarded as the true substrate for the BPPs, and Ca2+ plays an important role as a substrate activator in the formation of the true substrate.

The crystal structure of the phytase from B. amyloliquefaciens DS11 demonstrated that a negatively charged active site provides a favorable electrostatic environment for the positively charged calcium–phytate complex [20, 25].

Molecular Characteristics

Genes encoding Bacillus phytases from B. amyloliquefaciens [24, 26], B. licheniformis [19], and B. subtilis [15, 19] have been cloned and sequenced. They are composed of 383 amino acids and encoded an extracellular monomeric protein. The molecular mass is approximately 42 kDa; and SDS-PAGE gives an apparent molecular mass of approximately 38–44 kDa. The amino acid sequences of phytases from Bacillus are highly homologous to each other with 90–98% sequence identity. However, these amino acid sequences do not align with any other previously well-characterized HAPs nor with PAPs. Most significantly, the active site heptapeptide motif RHGXRXP and the catalytically active dipeptide HD, which are highly conserved sequence among HAPs [27], are absent. Besides, Bacillus phytases do not carry disulfide bonds, which are necessary for the conformational stability and catalytic activity in several fungal phytases [28–30]. There are 11, 11, and 3 residues that are involved in the phosphate binding sites and high- and low-affinity calcium-binding sites, respectively.

Crystal Structure

Ha et al. [31] carried out preliminary x-ray crystallographic analysis of a novel phytase from a B.amyloliquefaciens strain using the hanging-drop vapor-diffusion method. The enzyme consists of five 4-stranded and one 5-stranded antiparallel β sheets aligned around a pseudo sixfold symmetry axis lying on the shaft of the propeller, which is a distinct central channel filled with many ordered water molecules.

Ha et al. [20] further determined the crystal structure of B. amyloliquefacience phytase at 2.1 Å resolution in partially and fully Ca2+-loaded states. Two calcium ions (Ca1 and Ca2) form a biocalcium center where the Asp308 carboxylate serves as a bridging arm and tightens the “double clasp,” stabilizing the circular arrangement of the propeller structure. The binding of Ca1 and Ca2 to high-affinity calcium-binding sites results in a dramatic increase in thermal stability by tightening of several peptide segments at the strategic location remote in the amino acid sequence. Three calcium ions (Ca4, Ca5, and Ca6) at the active site form a triadic calcium center and neutralize an otherwise negatively charged calcium cage surrounded by a total of six aspartate and glutamate residues. The top of the molecular structure forms a shallow cleft that is lined predominantly with negatively charged side chains. The calcium-occupied cleft turns into a favorable electrostatic environment for the binding of phytate together with nearby Lys76, Lys77, Arg122, and Lys179. In addition, Ca4, Ca5, and Ca6 participate in catalysis directly by binding the phosphate group(s) of the substrate and stabilizing the pentavalent transition-state intermediate. Ca2+ reduces the negative charge around the active site cleft such that phytate neutralized by Ca2+ can easily fit to the active site.

Furthermore, the enzyme has two low phosphate binding sites, the “cleavage site,” which is responsible for the hydrolysis of a substrate, and the “affinity site,” which increases the binding affinity for the atom of the substrates containing adjacent phosphate groups [25].

Pathways of Phytate Dephosphorylation

A bidentate (P3-Ca2+-P4) of Ca2+-InsP6 initially binds to two phosphate-binding sites in the active site of Bacillus phytase, which preferentially hydrolyzes the phosphate group at the d-3 position of Ca2+-InsP6 to release Ins(1,2,4,5,6)P5 as an initial product [24]. After the hydrolysis of the first phosphate group, the enzyme binds another bidentate (P1-Ca2+-P2) of Ins(1,2,4,5,6)P5, followed by the hydrolysis of the phosphate group at the d-1 position to release Ins(2,4,5,6)-P4, as identified by 2-D NMR analysis of reaction intermediates. Finally, the enzyme binds a bidentate (P5-Ca2+-P4) of Ins(2,4,5,6)P4 and eventually hydrolyzes the phosphate group at the d-5 position to yield myo-Ins(2,4,6)P3 as a final product [32].

Greiner et al. [33] established the pathway of dephosphorylation of phytate by B. subtils 168, B. amyloliquefaciens ATCC15841, and B. amyloliquefaciens 45 using a combination of HPIC analysis and kinetic studies. The result demonstrated that these enzymes dephosphorylate phytate by sequential removal of phosphate groups via two independent routes: the routes via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 and finally, to D-Ins(2,4,6)P3. These enzymes prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does support previous phytate degradation models proposed by Kerovuo et al. [34] and Greiner et al. [35], but seems to fit with the structural model given by Shin et al. [25].

Expression of Bacillus Phytase

Expression of Bacillus Phytases in Microbial Hosts

Tye et al. [19] overexpressed the phytase gene (phyL) from B. licheniformis and phytase gene (168phyA) identified from B. subtilis strain 168 in B. subtilis using a ϕ105MU331 prophage vector system. Up to 35 U/ml were secreted into the culture media. Both phytases exhibited broad temperature and pH optima and showed high thermal stability. The phytase encoded by phyL exhibited higher thermal stability, even at a lower calcium concentration, as it was able to recover 80% of its original activity after denaturation at 95 °C for 10 min.

The gene-encoding phytase from Bacillus sp. DS11 was cloned in E. coli BL21 using the pET22b(+) vector with the inducible T7 promoter and produced up to 20% content of total soluble proteins [26]. The phytase gene with a native promoter derived from B. amyloliquefaciens was expressed in B. subtilis, which resulted in 100 times higher phytase yield in modified Luria broth medium [36].

Expression of Bacillus Phytases in Plants

Plants could be used as phytase producers and carriers for animal feeding. Bacillus phytases would be good candidates for producing transgenic plants because of their optimum pH under 7.0. The expression of a Bacillus phytase in transgenic plants may create a new biochemical pathway that mobilizes inorganic phosphate from phytate, so that more phosphorus is available for plant growth or physiology [19]. Engineering crop plants would produce heterologous phytase to reduce phosphate load on agricultural ecosystems with improving phosphate bioavailability [37].

Yip et al. [38] introduced a characterized phytase from B. subtilis into the cytoplasm of tobacco cells that resulted in equilibrium shift of inositol biosynthesis pathway, thereby making more phosphate available for primary metabolism. The transgenic line exhibited phenotypic changes like increased flowering, lower seed IP6/IP5 ratio, and enhanced growth under phosphate starvation conditions compared to wild type.

Lung et al. [39] reported that the phytase from B. subtilis (168phyA) was constitutively expressed in tobacco and Arabidopsis to generate transgenic plants capable of utilizing exogenous phytate. In tobacco, phytase activities in transgenic leaf and root extracts were seven to eight times higher than those in wild-type extracts; whereas, the extracellular phytase activities of transgenic plants were enhanced by four to six times. Similar results were observed from the transgenic Arabidopsis. These results may offer a new perspective on mobilizing soil phytate into inorganic phosphate for plant uptake.

Chan et al. [40] studied the biochemical properties and kinetic parameters of the recombinant phytase (t168phyA) from transgenic tobacco. The t168phyA was glycosylated with a 4-kDa increase in molecular size. Its temperature optimum shifted from 60 to 45–50 °C and its pH optimum shifted from pH 5.5 to 6.0; whereas, the thermal stability remained unchanged. Kinetic data showed that the t168phyA had a lower K cat, but a higher K m than the native enzyme. Despite these changes, t168phyA remained catalytically active and has a specific activity of 2.3 U/mg protein. These results verify the activity of recombinant Bacillus phytase that is expressed in plants.

Commercial Prospects

Applications in Animal Nutrition

Because of the absence or insufficient amount of phytase in monogastric animal gastrointestinal tract, phytases are frequently added to their feed to facilitate optimal growth and decrease the supplementation of inorganic phosphate besides consequently reducing the phosphorus pollution in the areas of intensive livestock units. The Bacillus phytase is very suitable to be used in animal feed particularly in common carp feed because of its optimum pH with excellent thermal stability. Zeng et al. [41] reported that Bacillus phytase supplementation of 300 U/kg could gain the same result as that of 1,000 U/kg supplementation of acidic phytase and neutral phytase supplementation of 1,000 U/kg could replace the inorganic phosphorus supplement.

Moreover, a combination of Bacillus phytases and other acidic phytases might induce a more effective hydrolysis of phytate in both the stomach and small intestine of animals in terms of the pH of the animal gastrointestinal tract [42].

Applications in Human Health

In addition to its major application in animal nutrition, phytase has also been found increasingly interesting for use in processing and manufacturing of food for human consumption. Supplemental phytases could play an important role in human nutrition, both for phytate degradation during food processing and in the gastrointestinal tracts [43]. Furthermore, phytase may find application in the production of functional foods or food supplements with health benefits [5, 44]. The effectiveness and limitations of food supplementation with phytases may also depend on their sensitivity to protease digestion [45]. The enzymes from Bacillus exhibit sensitivity to pancreatin, but a much higher susceptibility to pepsin [46]. Until now, Bacillus phytases are, however, not available for human consumption. With good thermal stability and the triphosphate as final product, future investigations into the application of Bacillus phytase in food are merited.

Application in Synthesis of Lower Inositol Phosphates

Lower inositol phosphates play crucial roles in energy metabolism, metabolic regulation, and signal transduction pathways in biological system [47]. The use of phytase has been shown to be very effective in producing different inositol phosphate species. Among the advantages of enzymatic hydrolysis over chemical synthesis are stereospecificity and mild reaction conditions [8]. The myo-inositol trisphosphate isomer is the end product of phytate degradation by Bacillus phytases [25, 32, 33]. Therefore, these enzymes can be used for the preparation of myo-inositol triphosphates.

Conclusions and Future Perspectives

Along with the increasing concerns in relation to phosphorus pollution in the areas of intensive livestock, phytases have considerable potential in commercial and environmental applications. Potential applications of phytases in improving human health and in synthesis of lower inositol phosphates have increasingly gained attention. Under these conditions, significant progress has been made in phytase research during the last few decades. Bacillus phytases, which exhibit desirable activity profile under neutral pH, excellent thermal stability, and strict substrate specificity, are more promising for commercial exploitations. In addition, engineered transgenic plants harboring the phytase gene from Bacillus could also be used to improve soil fertilization and nutrient uptake by plants.

With the collaborative efforts of phytase scientists from different fields, effective solutions to the biotechnological development of Bacillus phytases for animal nutrition, human health, and environmental protection will be available in the near future.