Purification and characterization of a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsoniiZJ0702
© Yu and Chen; licensee BioMed Central Ltd. 2013
Received: 25 February 2013
Accepted: 25 September 2013
Published: 28 September 2013
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.
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.
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.
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 [3–6]. 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 [7–9]. 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 Ca2+, Mg2+, Fe2+ and Zn2+, 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, 10–15]. Thus, it is necessary to remove phytic acid and phytates in food and feed processing to avoid the above-mentioned problems.
Phytase (myoinositol hexakisphosphate phosphohydrolases EC22.214.171.124) 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 . In addition, phytase has important applications in ameliorating human nutrition [17–19], as well as in some other areas, including aquaculture . 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 [21–31]. However, the focus has been on fungus-derived phytases that are active at low pH values and show low thermal stability [31–34]. 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 . 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.
Purification of the phytase from B. nealsoniiZJ0702
Purification results of the phytase from a newly isolated strain B. nealsonii ZJ0702
DEAE-sepharose Fast Flow
Homology analysis of the phytase
Enzymatic properties of the purified phytase
Effect of metal ions on the activity of the purified phytase
Relative activity (%)b
Substrate specificity of the purified phytase
Relative activity (%)
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), Aspergillus oryzae (60°C), Bacillus subtilis (55–60°C), Escherichia coli (55–60°C), Penicillium simplicissimum (55°C), Aspergillus niger (55–58°C) and Klebsiella terrigena (58°C) [38–40]. Compared with acidic phytases from Saccharomyces cerevisiae (pH3.6), Cladosporium sp. FP-1 (pH4.0), Pichia anomala (pH4.0), Candida krusei (pH4.6), Lactobacillus sanfranciscensis (pH4.0), Pantoea agglomerans (pH4.5), Klebsiella terrigena (pH5.0) and Penicillium simplicissimum (pH4.0) [40–42], the phytase from B. 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. The strong inhibition of the activity of the phytase by Ba2+, Cu2+ and Co2+ 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 from B. nealsonii ZJ0702 is highly specific for inositol polyphosphate.
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 . Phytic acid, sodium phytase, ATP, glucose-1-phosphate, 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 NH4NO3, 2 g/l CaCl2, 0.5 g/l KCl, 0.5 g/l MgSO4 · 7H2O and 1 g/l KH2PO4, pH7.0. The other reagents used in the experiments were of analytical grade and used according to the specifications provided by the manufacturer.
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.
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 .
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.  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 NH4Mo7O24 · 4H2O:5N H2SO4:acetone (1:1:2) was added. The reaction was allowed to proceed for 30s. The reaction was halted by adding 0.1 ml of 1 M citric acid. The color of the reaction of the Pi (inorganic phosphate)-Mo complex was read at A380. A reference standard (KH2PO4,0.1-0.4 μM) was simultaneously assayed with the samples. A unit (U) of phytase activity was defined as the amount of enzyme required to release 1 nM of Pi per minute at 55°C. The protein concentration was measured according to the Bradford method using bovine albumin as the standard .
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, P1: 5′-ATGGGAGCGATCGATACATGTCCAAAC-3′ and P2: 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. , except that the detergent Triton X-100 was replaced by octyl-β-D- glucopyranoside (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: Ba2+, Ca2+, Cu2+, Co2+, Mg2+, Mn2+, Ni2+ and Zn2+. 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.
The authors thank the Zhejiang Province Science and Technology Committee (No. 2011C22074) for generous support of this study.
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