Purification and characterization of a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702

Background Phytic acid and phytates can interact with biomolecules, such as proteins and carbohydrates, and are anti-nutritional factors found in food and feed. Therefore, it is necessary to remove these compounds in food and feed processing. Phytase can hydrolyze phytic acid and phytates to release a series of lower phosphate esters of myoinositol and orthophosphate. Thus, the purification and characterization of novel phytases that can be used in food and feed processing is of particular interest to the food and feed industries. Results A novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702 was purified to homogeneity with a yield of 5.7% and a purification fold of 44. The molecular weight of the purified phytase obtained by SDS-PAGE was 43 kDa. The homology analysis based on N-terminal amino acid and DNA sequencing indicated that the purified phytase was different from other known phytases. The optimal thermal and pH activity of the phytase was observed at 55°C and 7.5, respectively. Seventy-three percent of the original activity of the phytase was maintained following incubation at 90°C for 10 min. The phytase was stable within a pH range of 6.0 − 8.0 and showed high substrate specificity for sodium phytate. Cu2+, Co2+, Zn2+, Mn2+, Ba2+ and Ni2+ ions were found to inhibit the activity of the phytase. Conclusions A novel phytase purified from B. nealsonii ZJ0702 was identified. The phytase was found to be thermally stable over a wide temperature range at neutral pH. These properties suggest that this phytase is a suitable alternative to fungal phytases for the hydrolysis of phytic acid and phytates in food and feed processing industries.


Background
Phytic acid and phytates exist widely in edible legumes, cereals, oil seeds, pollens and nuts, and account for about 1 − 5% of the dry weight of plant seeds [1,2]. The presence of phytic acid and phytates in plant food and feed has been well documented [3][4][5][6]. They are a primary source of inositol and an important storage form of phosphorus in plant seeds that are often used as animal feed ingredients [7][8][9]. Because of their ability to interact with biomolecules, such as proteins and carbohydrates, they can act as anti-nutritional factors in several ways: (i) chelating cations, such as Ca 2+ , Mg 2+ , Fe 2+ and Zn 2+ , to form insoluble metal phytate complexes under gastrointestinal pH conditions; (ii) reducing the digestibility of protein, starch and lipids; and (iii) inhibiting the activity of enzymes, including amylase, trypsin, acidic phosphatase and tyrosinase [2,[10][11][12][13][14][15]. Thus, it is necessary to remove phytic acid and phytates in food and feed processing to avoid the abovementioned problems.
Phytase (myoinositol hexakisphosphate phosphohydrolases EC3.1.3.8) cleaves phosphor-monoester bonds in phytic acid and phytates. This results in the sequential release of a series of lower phosphate esters of myoinositol and orthophosphate. Therefore, phytase has gained rapid acceptance as an animal feed or a food additive worldwide [16]. In addition, phytase has important applications in ameliorating human nutrition [17][18][19], as well as in some other areas, including aquaculture [20]. Thus, it is highly desirable to reduce the content of phytic acid and phytates in food and feed processing by the hydrolysis of phytase.
A wide variety of phytases have been isolated from different organisms [21][22][23][24][25][26][27][28][29][30][31]. However, the focus has been on fungus-derived phytases that are active at low pH values and show low thermal stability [31][32][33][34]. Moreover, although the addition of phytase is widely used to improve the release of plant phosphorus in poultry and swine, the use of phytase in feed for aquatic species has not been developed [35]. Some aquaculture species are agastric and their digestive system pH is neutral. Thus, fungus-derived acidic phytases are of limited use to these species because these phytases show low activity at neutral pH. Bacterial phytases are an important alternative to fungal ones because of their high thermal stability, phytate substrate specificity, wide pH profile and proteolysis resistance [33,36,37]. Therefore, the purification and characterization of novel phytases with high thermal stability and activity at neutral pH from bacteria is of particular interest for future industrial applications in food and feed processing.
In the present study, a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702 from the soil was sequentially purified to homogeneity by ammonium sulfate precipitation, DEAE-sepharose Fast Flow column chromatography and Sephadex G-100 size-exclusion chromatography. The enzymatic properties of the purified phytase were investigated in detail.

Results
Purification of the phytase from B. nealsonii ZJ0702 The supernatant obtained by centrifugation of the culture broth at 12,000rpm for 20 min was used as the enzyme source. The purification of the phytase was sequentially performed by (NH 4 ) 2 SO 4 precipitation, DEAE-sepharose anion-exchange column chromatography and Sephadex G-100 size-exclusion column chromatography. The purification results are presented in Figure 1 and Table 1. After (NH 4 ) 2 SO 4 precipitation, 76% of the total protein was removed. The residual protein was subject to DEAE-sepharose anion-exchange column chromatography (Figure 1a), and the protein containing the activity of phytase was collected and pooled, giving 6% of the total protein. The protein was subject to further purification by Sephadex G-100 size-exclusion chromatography. Here, fractions 2-8 showed the highest phytase activity (Figure 1b). These fractions were collected and pooled, giving 1% of the total protein. The purification folds of phytase from the above three purification steps were 2, 10 and 44, respectively. Corresponding recovery rates of the total activity of phytase were 49, 6 and 5.7%, respectively ( Table 1). The SDS-PAGE analysis of the protein samples from the above three purification steps is shown in Figure 1c. Only a single protein band with an estimated molecular weight of 43 kDa was present following the Sephadex G-100 size-exclusion chromatography step. This result indicates that the obtained phytase is electrophoretically pure and can be used for the analysis of enzymatic properties.

Homology analysis of the phytase
The homology tree of phytase based on the DNA sequence is presented in Figure 2. A very low sequence homology was found between the DNA sequence of the phytase from B. nealsonii ZJ0702 and phytases from selected microorganisms. The determined N-terminal amino acid sequence of the purified phytase from B. nealsonii ZJ0702 is: MGAIDTCPNKYSTIRRVLIMN KKTQMIHGGH. A similarity comparison was also carried out between this protein sequence and the corresponding regions of other known phytases; however, no similarity was found. These results strongly suggest that the phytase from B. nealsonii ZJ0702 is different from other known phytases ( Figure 2).

Enzymatic properties of the purified phytase
Enzymatic properties of the purified phytase are shown in Figure 3. The activity of the phytase increased when the temperature was increased from 20 to 50°C, and reached a maximal value at 55°C. Thereafter, it decreased rapidly as the temperature increased beyond 55°C. This shows that the optimal temperature of the purified phytase is 55°C (Figure 3a). The activity of the phytase varied as a function of the pH. The highest activity of the phytase was observed at pH7.5 ( Figure 3b). For the phytase from B. nealsonii ZJ0702, strong thermal stability was observed at 37 and 55°C. The activity of the phytase showed negligible change when incubated at either of these temperatures for 30 min. The residual activities of the phytase were 75, 62 and 41% when the protein was incubated at 80°C for 10, 20 and 30 min. The residual activities of the phytase at 90°C were 73, 51 and 21% when the protein was incubated for 10, 20 and 30 min, respectively (Figure 3c). A high pH stability of the phytase was observed when it was incubated at pH7.0 and 8.0. At pH4.0, the activity of the phytase decreased dramatically as the incubation time increased, and only 1% of the original activity of the phytase remained after incubation at pH4.0 for 30 min (Figure 3d).
The effect of different metal ions on the activity of the purified phytase (Table 2) indicated that Ca 2+ at 1 mM and 5 mM did not alter its activity. In addition, the activity of the purified phytase was not altered when 1 mMMg 2+ was added. However, the activity decreased by 38% when the concentration of Mg 2+ increased to 5 mM. Cu 2+ , Co 2+ , Zn 2+ , Ba 2+ , Mn 2+ and Ni 2+ at 1 mM and 5 mM were found to inhibit the activity of phytase significantly. The relative hydrolytic rate of different phosphorylated compounds showed that the purified phytase exhibited very narrow substrate specificity ( Table 3). The phytase showed high activity only towards the substrate sodium phytate. No phytase activity was detected when other phosphorylated substrates were used.

Discussion
In this study, a neutral and heat-tolerate phytase from B. nealsonii ZJ0702 was purified to homogeneity, and the homology analysis based on N-terminal amino acid sequencing and DNA sequencing revealed that the phytase is novel. This enzyme display the optimal activity at 55°C, which is similar to that of other known phytases from particular microorganisms, such as Mucor hiemalis (55°C),   nealsonii ZJ0702 showed the optimal activity at pH7.5, indicating that it is neutral and should be suitable for the application in some aquaculture species whose digestive system pH is neutral. The thermal stability of the phytase suggests that it has stronger thermal stability than the phytase from Yersinia kristensenii [21]. The strong inhibition of the activity of the phytase by Ba 2+ , Cu 2+ and Co 2+ at 5 mM shows that the active site of phytase may include -SH moieties. The high substrate specificity of the phytase for sodium phytate indicates that the phytase

Conclusions
The results from this study demonstrate that the phytase from B. nealsonii ZJ0702 shows optimal activity at neutral pH, strong thermal stability and high substrate specificity for sodium phytate. These unique properties make this phytase an attractive alternative to replace fungus-derived phytases for the hydrolysis of phytic acid and phytates. Extensive studies on the application of the purified phytase in food and feed processing are ongoing.

Strain and reagents
The strain B. nealsonii ZJ0702 was isolated from the soil at the Xihu district of Zhejiang Province, China, and was kept in our laboratory. This strain is deposited in the China General Microbiological Culture Collection (CGMCC, No.5396), and produces the extracellular phytase [43]. Phytic acid, sodium phytase, ATP, glucose-1phosphate, fructose-1, 6-diphosphate, β-glycerophosphate and p-nitrophenylphosphate (pNPP), DEAE-sepharose and Sephadex G-100 were purchased from the Sigma Co., Ltd., LA, USA. PCR reagents and the PCR product purification kit were purchased from TaKaRa Biotech Co., Ltd., Japan. The UNIQ-10 DNA extraction kit was purchased from Sangon Co., Ltd., Shanghai, China. The strain culture medium contained 35 g/l wheat bran, 20 g/l tryptone, 5 g/l NH 4 NO 3 , 2 g/l CaCl 2 , 0.5 g/l KCl, 0.5 g/l MgSO 4 · 7H 2 O and 1 g/l KH 2 PO 4 , pH7.0. The other reagents used in the experiments were of analytical grade and used according to the specifications provided by the manufacturer.

Strain culture
A single colony of the strain B. nealsonii ZJ0702 was transferred from a slant culture to an Erlenmeyer flask (1000 ml) containing 500 ml of the culture medium, followed by incubation at 34°C with vigorous agitation in a shaking incubator at 165 rpm for 72 h for producing the extracellular phytase.

Enzyme purification
All purification steps were carried out at 4°C unless otherwise stated. The 72 h culture broth was centrifuged at 12,000 rpm for 20 min to remove the cells. The supernatant was collected and ammonium sulfate was first added until 30% saturation. The resultant precipitation was removed by centrifugation at 10,000 rpm for 30 min at 4°C. Proteins were then fractioned from the supernatant by adding ammonium sulfate until different saturation levels were reached (40, 50, 60, 70 and 80%). The obtained precipitates were pelleted by centrifugation at 10,000 rpm for 30 min at 4°C, combined and resuspended in a 0.1 M phosphate buffer (pH7.0). The combined precipitate was desalted using a dialysis bag (diameter: 0.45 μm) and then loaded onto a DEAE-sepharose Fast Flow ion-exchange column. Proteins were eluted at 0.8 ml/min using a phosphate buffer (pH7.0) containing a linear NaCl-gradient with the concentration ranging between 0 and 2 M. The fractions with phytase activity were combined and concentrated by PEG 20,000. The concentrate was loaded onto a Sephadex G-100 chromatography column (2.5 × 35 cm) pre-equilibrated with a phosphate buffer (pH7.0). Proteins were eluted at 0.5 ml/min using the same buffer. The fractions with phytase activity were collected and combined. The protein purity was determined by SDS-PAGE analysis. SDS-PAGE was carried out as described by Laemmli [44].
Determination of the activity of phytase and the concentration of the total protein The phytase activity of samples collected from each purification step was analyzed. The phytase activity was determined as described by Engelen et al. [45] with minor modifications. Samples were diluted accordingly before the analysis. 0.5 ml diluted samples and 25 mM sodium phytate in 0.2 M phosphate buffer (pH7.0) were incubated separately at 55°C for 10 min. Then, 0.5 ml of the substrate was added to the sample and the mixture was incubated for another 10 min. Thereafter, 2 ml of 10 mM NH 4

PCR amplification of the phytase gene and determination of the N-terminal amino acid sequence
The genomic DNA from B. nealsonii ZJ0702 was extracted using the UNIQ-10 DNA extraction kit according to the specifications provided by the manufacturer. The primer set, P 1 : 5′-ATGGGAGCGATCGATACATGTCCAAAC-3′ and P 2 : 5′-TTAGATCGACCCCTGTATGACCACT-3′, was designed for the amplification of the phytase gene by PCR with the genomic DNA as the template. PCR conditions consisted of an initial denaturation at 95°C for 5 min, 35 cycles of the amplification consisted of denaturation at 95°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min. Then a further extension at 72°C was performed for 10 min. PCR products were purified by the PCR purification kit and sequenced by Sangon Co., Ltd., Shanghai, China. The homology analysis of phytases based on the DNA sequences was carried out with the DNAMAN 7.0 software. For the analysis of the N-terminal amino acid sequence of the purified phytase, the proteins on the SDS-PAGE gel were transferred to a PVDF membrane at 200V for 1 h. After the proteins were stained, the membrane corresponding to the protein band of the purified phytase was cut out and digested with sequencing grade trypsin, as described by Fernandez et al. [47], except that the detergent Triton X-100 was replaced by octyl-β-Dglucopyranoside (Boehringer Mannheim). Phytase was analyzed with a HP G1005A protein automated sequencing system (Hewlett-Packard Co., Ltd.).

Enzymatic properties of the purified phytase
The temperature stability of the phytase was determined by subjecting aliquots of phytase solutions to different temperatures for 30 min. Temperatures used were 37, 55, 80 and 90°C. The residual activity of the phytase was detected once every 5 min. The effect of temperature on the activity of the phytase was determined at different temperatures ranging from 20 to 80°C. Similarly, the optimal pH for the activity of the phytase was determined by mixing equal volumes of buffers at different pH values ranging from 3.0 to 11.0 at 55°C, while the pH stability was examined by subjecting aliquots of phytase solutions to different pH values for 30 min. The residual activity of the phytase was determined once for 5 min. Buffers used were: 0.1 M glycine-HCl buffer (pH3.0); 0.1 M acetic acid buffer (pH4.0 − 5.0); 0.1 M Tris-HCl buffer (pH6.0 − 9.0); and 0.1 M glycine-NaOH buffer (pH10.0 − 11.0). The phytase sample at 4°C and pH7.0 was used as the control and its activity was defined as 100%. The effect of metal ions on the activity of the phytase was studied by incubating metal ions with a purified enzyme solution (0.5 ml) for 10 min at 55°C. The following metal ions at 1 and 5 mM were used: Ba 2+ , Ca 2+ , Cu 2+ , Co 2+ , Mg 2+ , Mn 2+ , Ni 2+ and Zn 2+ . The substrate specificity of the purified enzyme was evaluated by following the standard assay procedure, except that the substrate was replaced with different phosphorylated compounds: pNPP, glucose-1-phosphate, ATP, fructose-1,6-diphosphate and β-glycerophosphate.

Nucleotide sequence accession number
The DNA sequence of the phytase from B. nealsonii ZJ0702 is in the GenBank database under accession number HQ843995.