PHYTASE FROM BACILLUS SP. STRAIN LA12: ISOLATION, PURIFICATION AND CHARACTERIZATION

Phytate take effect as an anti-nutrient element in food and feed materials. Thus, phytase, by catalyzing phytate, hydrolyzing the phosphomonoester bonds and releasing the inorganic phosphorous, decrease the phytate and enhance their nutritional value. Therefore, in this study, Bacillus sp. strain LA12was isolated from natural origins and the phytase production activity was evaluated. The novel extracellular phytase was produced and precipitated by saturated ammonium sulfate. The ion-exchange chromatography on DEAE-sepharose and the size-exclusion chromatography on Sephadex G-100 were used to purify the enzyme. The results showed that the purification yield and concentration of final enzyme were 5.9% and 18.4%, respectively. Based on SDS-PAGE results the molecular weight of the phytase was determined about 73 kDa. Optimal activity of the enzyme was obtained at pH of 5.5 and 60 oC. Kinetic parameters Km and Vmax were 0.197 mM and 1.174 µmol/min, respectively. Mg2+, Co2+ and EDTA accelerated the effect on phytase activity; whilst adding other metal ions such as Ca2+, Zn2+ and Fe2+ in both concentrations could decrease its activity. Moreover, ‏ Mn2+ ion didn’t show indicative effect on its activity. The purified phytase exhibits good thermal stability after incubation at 50-70°C for 30 min, whereas the phytase activity drastically decreased up to 61% at 80°C. This study indicated that the purified phytase has the desired characteristics and can promisingly be used for hydrolyzing of phytate in food and feed.


INTRODUCTION
Phytase are group of enzymes able to hydrolyze phytate to other component such as inositol phosphate and inorganic phosphate. The food/feed from plant origin contain phosphorus at notable level in which the main part of phosphorus is in form of phytate as the salt form of Phytic acid. Phytic acid (Myo-inositol 1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate) is a predominant phosphorus storage in plants (>70-80% of the total phosphorus). The amount of phytic acid, based on total weight count, comprises 3% to 5% of cereal, oilseed and legumes (Reddy et al., 1982a). As a matter of fact the application of phytase is one of the proper resources to overcome the phytic acid in food/feed. Phytic acid is recognized as anti-nutrient factor for monogastric animals which able to chelate divalent and trivalent cations such as calcium, magnesium, iron, manganese, copper, molybdenum and zinc therefore it has negative effect on their absorption and digestion Satyanarayana, 2006, Vohra andSatyanarayana, 2003). Phytate may adversely affect mineral bioavailability (Luo et al., 2007) along with it is not digest by monogastric animals, including human, due to lack of phytase performance. Thus, notable amount of ingested phytate phosphorus in these animals is excreted and is led to the environmental problems, e.g., water pollution (Bohn et al., 2008. Also being of phytate in foods is one of an important index in mineral related nutritional deficiency in human. In particular for individual who has tendency to plant food or follow vegetarian diet. In this regard as key role of phytase in human nutrition it could not only reduce phytate content but also prevent the mineral deficiency (Reddy et al., 1982b). From the past decade, attempt has been focused on the phytase enzyme due to the increasing concerns to wards phosphorus pollution in environment and also nutritional benefits . Phytases have a great advantage in raising the nutritional value of phytate in foods/ feed products. The extraction of Phytases from microorganisms (bacteria, yeast, fungi) or from the plants origin have been evaluated in numerous studies (De Angelis et al., 2003, Greiner and Carlsson, 2006, Kaur et al., 2007. Based on literature review, many isolated microbial strains containing efficient phytases have been introduced for industrial applications. In this study, the enzyme of phytase was purified from Bacillus sp. strain LA12 and after characterization the optimum condition of activity regarding pH and temperature also thermo stability properties were identified.

Chemicals
The salt of Phytic acid was obtained from Sigma Chemical Co. (St. Louis, MO, USA). All chemical agent and used media were purchased from Merck (Darmstadt, Germany) also the all reagents were analytical grade.

Bacterial Sample Collection
There are various reports on phytase producer strains of bacteria in poultry farm area soil samples (Sreedevi et al., 2013;Vihnudas et al., 2012& Tungala et al., 2012. Therefore, in this research more than fifty soil samples from the surface layer of different chicken farm zones in Iran (Fars, 15-50 km north of Shiraz) were used for study.

Isolation of phytase hydrolyzing bacteria
After thorough dilution of 10 g of each soil sample in 90 ml citrate sodium solution (2% w/v) using stomacher (Seaward Medical stomacher, London, UK). The Phytase Screening Medium (PSM) containing calcium phytate was used to select the phytase-producing bacteria and the inoculated plates were incubated at 37 °C for 24-72 h. Each of isolates bearing clear zones on PSM were selected for next examination (Bala et al., 2014).

DNA isolation
One of the best phytase producer isolates was selected for molecular characterization and identification. A loop of mature colony was transfer to nutrient broth and incubated for 24 h at 37°C. After centrifugation of culture (4,000 g for 10 min) the pellet was harvested and utilized for DNA extraction by the DNA kit (Thermo Fisher Scientific, Germany) based on defined protocol.
Phytate take effect as an anti-nutrient element in food and feed materials. Thus, phytase, by catalyzing phytate, hydrolyzing the phosphomonoester bonds and releasing the inorganic phosphorous, decrease the phytate and enhance their nutritional value. Therefore, in this study, Bacillus sp. strain LA12was isolated from natural origins and the phytase production activity was evaluated. The novel extracellular phytase was produced and precipitated by saturated ammonium sulfate. The ion-exchange chromatography on DEAEsepharose and the size-exclusion chromatography on Sephadex G-100 were used to purify the enzyme. The results showed that the purification yield and concentration of final enzyme were 5.9% and 18.4%, respectively. Based on SDS-PAGE results the molecular weight of the phytase was determined about 73 kDa. Optimal activity of the enzyme was obtained at pH of 5.5 and 60 ºC. Kinetic parameters Km and Vmax were 0.197 mM and 1.174 µmol/min, respectively. Mg 2+ , Co 2+ and EDTA accelerated the effect on phytase activity; whilst adding other metal ions such as Ca 2+ , Zn 2+ and Fe 2+ in both concentrations could decrease its activity. Moreover, Mn 2+ ion didn't show indicative effect on its activity. The purified phytase exhibits good thermal stability after incubation at 50-70°C for 30 min, whereas the phytase activity drastically decreased up to 61% at 80°C. This study indicated that the purified phytase has the desired characteristics and can promisingly be used for hydrolyzing of phytate in food and feed.

16S rRNA sequencing
The extracted DNA was used as a template for Polymerase chain reactions (PCR) amplification of 16S rDNA using primer pair Bacillus Primer-VivantisF (5´-TCACCAAGGCRACGATGCG-3´) and Bacillus Primer-VivantisR (5´-CGTATTCACCGCGGCATG-3´). The PCR program was done using a thermocycler (Eppendorf, Hamburg, Germany) at 94°C for initial denaturation (3 min), 94°C for denaturation by 35 cycles of 30 sec, 53°C for annealing and 72°C for 2 min extension, also 72°C for final extension (10 min). Then the electrophoresis of PCR products were carry out using a agarose gel (1% w/v) in Tris Boric acid EDTA buffer (100 V for 45 min). The determination of the size of DNA fragments was done using a 100 bp DNA Ladder (Fermentase, Canada). The aplified PCR product was fractionated on agarose gel (0.7 %) . the obtained fragments on the gel were cut and purified by gel extraction kit (Axygen Biosciences, Union City, CA, US). Then the purified product was sequence using a sequencer. 16S rDNA sequence of this strain was submitted to a Basic Local Alignment Search Tool (BLAST) search of the NCBI (National Center for Biotechnology Information) GenBank database (www.blast.ddbj.nig.ac.jp/) to identify the organism.

Enzyme production
The phytase enzyme was produced according to Singh and Satyanarayana (2008) (Singh and Satyanarayana, 2008) study with some modification. Briefly, the isolate was cultivated in Luria broth (LB) media for 24 h at 37°C and used as the seed culture. Phytase production media (in a final volume of 1 L) contained: 10 g D-glucose, 4 g Naphytate, 2 g NH4NO3, 0.5 g KCl, 0.5 g MgSO4_7H2O, 0.01 g FeSO4_7H2O and 0.01 g MnSO4_H2O. The prepared liquid fermentation inoculated with 1% of seed culture and incubated in a Benmarin shaker (250 rpm) set at 37°C. After 72 h, the culture was centrifuged at 10,000 g for 20 min. The obtained supernatant was used for the further enzyme purification stages (Gulati et al., 2007b).

Enzyme purification
Step 1: At first, ammonium sulfate was added to collected supernatant. Then the sample was centrifuged at 10,000 g for 30 min at 4°C. The pellet was removed and re-suspended with phosphate buffer 0.1 M (pH 7.0). The obtained sample was desalted using a dialysis bag (diameter: 0.45 μm).
Step 2: Then, the dialyzed fraction was subject to DEAE-sepharose anionexchange column chromatography (pH 7.0). The fractions with phytase activity were collected and concentrated using PEG 20,000.
Step 3: In the last step of purification, the concentrated sample was loaded onto a Sephadex G-100 chromatography column (2.5 × 35 cm) which was equilibrated with a phosphate buffer (pH7.0). Furthermore, sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was accomplished for determining the protein purity (Laemmli, 1970).

Measurement of protein content
After each steps of enzyme purification the total protein concentration of samples were calculated according to the method, of Lowry et al. (1951), using bovine serum albumin and reported as milligrams of protein per mL of sample.

Measurement of phytase activity
Phytase activity of the samples at each step of the purification was analyzed according to the standard method described by Engelen and Van Der Heeft (1994) (Engelen et al., 1994). Briefly, 100 µl of enzyme samples were suspended in 900 µl of 0.1 M acetate buffer (pH 6) and 500 µl of 0.1 M sodium phytate solution which was prepared in acetate buffer. The samples were incubated for 30 min at 37ºC. After that, 500 µl of color reagent was added to the sample to stop the reaction. The absorbance was measured at 415 nm by a spectrophotometer (UNICO, UV-2100, China).The blank sample was the substrate inoculated with deionized water. The phytase activity was calculated using absorbance data. One phytase unit (U) is defined as the amount of enzyme per milliliter of culture medium, which releases1 µg of inorganic phosphorus per minute (Vohra and Satyanarayana, 2003).

Phytase molecular weight estimation
The molecular weight of the purified phytase was determined by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) method as described by Laemmli (1970) (Laemmli, 1970).

Characterize of the purified phytase.
The favor temperature for phytase activity was optimized after incubation of the enzyme at different temperatures (30, 37, 40, 50, 60, 70, 80 and 90°C) for 30 min. The percent of residual activity was measured and then the optimum temperature was reported. The optimum pH for the phytase activity was measured using different buffers with pH ranging from 2.0 to 9.0 at 37 °C. The used buffers in the study were a citrate buffer for pH 3.0-5.8, a Tris-maleate buffer for pH 6.2-7.4 and a Tris-HCl buffer for pH 7.6-9.0 (Bogar et al., 2003).

Kinetic properties of phytase
The kinetic parameters of purified phytase were evaluated by measuring the rate of sodium phytate hydrolysis at the optimum pH and temperature conditions based on method of Lineweaver-Burk plot (Lineweaver and Burk 1934). Sodium phytate was used in concentration ranged from 0.2 to 10 mM. Michaelis-Menten constant (Km) and maximum reactionrate (Vmax) werecalculated from the slope and intercept of the regression line of Line weaver-Burk plots using Microsoft Excel 2010.

Effect of metal ions and EDTA on phytase activity
Effect of various cations (Ca 2+, Zn 2+, Mn 2+ and Fe 2+ ) and EDTA on phytase activity was evaluated by the method described by Escobin-Mopera (2012) (Escobin-Mopera et al., 2012). For this experiment, a 100 µl of the phytase solution was mixed with 100 µl of metal ions and EDTA solutions which was prepared in 0.05 M sodium acetate buffer with concentrations of 1 mM and 5 mM. After incubation of the solutions at 30ºC, the activity of the phytase was assayed.

Statistical analyses
Experiments were done in triplicates based on two different occasions (n=6) so the mean value was calculated. All obtained data was analyzed using One-way Analysis of Variance (ANOVA) and PostHoc Multiple comparison test (LSD) using SPSS version 19.0 (USA).

Microbiological analysis
Thirty one phytase producing bacteria were isolated from the soils samples according the formed clear zones on PSM medium. After further screening on liquid medium, a gram positive bacteria was choose as the best phytase producer strain. The result of nucleotide sequence of 16S rRNA revealed species of isolate as Bacillus. The accession number for the sequence was MG322293. Some bacteria belonging to both Gram-positive and Gram-negative group which isolated from soil e.g. Klebsiella sp. (Sajidan et al., 2004) Bacillus laevolacticus (Gulati et al., 2007a), Staphylococcus lentus (Hussin et al., 2007), Acromobacter sp. , Tetrathiobacter sp. and Bacillus sp. (Kumar et al., 2013) have been reported able to produce phytase.

Phytase purification
As presented in Table 1, the crude enzyme had the highest total protein and phytase activity and lowest specific phytase activity. After successive (NH4)2SO4 precipitation, the phytase was purified 1.4 fold andyield of 57.7%. The purification degree of phytase by ammonium sulfate precipitation in Bacillus sp. strain LA12was 100-fold less compared to Amycolatopsis vancoresmycina (Bandyopadhyay et al., 2017), but it was more than Bacillus megaterium EBD 9-1 strain (Demirkan et al., 2017). The enzyme purification followed by DEAEsepharose and Sephadex G-100 and the purification increased to 9 and 18.4 fold, respectively. The results showed that the specific activity of the enzyme increased after each purification step, while the yield of enzyme purification significantly decreased. DEAE cellulosecolumn was used for phytase purification from Amycolatopsis vancoresmycina with a 3.53 fold purification (Bandyopadhyay et al., 2017). Figure 1 shows the SDS-PAGE analysis of the resulted proteins from purification steps. The protein band with determined molecular weight of 73 kDa was observed in the last step of protein purification (G-100 size-exclusion chromatography). The obtained molecular weight in our study was higher than Amycolatopsis vancoresmycina S-12, Klebsiella pneumonia 9-3B, Bacillus megaterium and Enterobacter sp. ACSS which achieved in Bandyopadhyay et al.

Effect of pH and temperature on the phytase activity
Generally, the phytase derived from different sources has a wide range of features, while the industrial applications of enzymes strongly affected by its properties (Yao et al., 2012). Since the thermo stability of enzymes has a major role in their application, the thermal dependence of phytase activity has shown in Figure 2. Our results indicated that the activity of phytase was increased by increasing the temperature from 30 ºC to 60ºC, however more increase in thermal treatment resulted in slow decrease in phytase activity. This may be due to heat denaturation of enzyme with increasing temperature. Phytase displayed the highest activity at 60°C, so the optimum temperature of the enzyme activity was at 60°C. In the present study, the optimum temperature was closely similar to those observed for Enterobacter and Serratia species (Kalsi et al., 2016), Bacillus megaterium (Demirkan et al., 2017),Bacillus subtilis B.S.46 (Rocky -Salimi et al., 2016), Geobacillus (Jorquera et al., 2017), Penicillium simplicissimum (Boyce and Walsh, 2007)and Aspergillus niger (Vats and Banerjee, 2005),while it lower than the values reported for Rhizomucor pusilllus (Chadha et al., 2004) and Thermomyces lanuginosus (Gulati et al., 2007b). Our results indicated that a negligible change is observed in the phytase activity by incubation of the phytase solution in the temperature range of 50-70°C for 30 min. Furthermore, the activity after incubation of phytase solution at 80°C for 10, 20 and 30 min were 68, 51 and 39%, respectively. Therefore, the thermal stability was higher than the enzyme obtained from those Bacillus species that reported by (Jorquera et al., 2017). The purified phytase activity was significantly (p< 0.05) affected by the pH value of the medium (Fig.3).The phytase activity has increased by increasing the pH values from 2 to 5.5. By increasing the pH above 5.5, the phytase activity suddenly decreased. The enzyme activity was completely lost at pH values of 7.5 and higher. This may be related to denaturation of the enzyme in the alkaline condition. Phytase showed optimum activity at pH 5.5. Under high alkaline and acidic conditions, the phytase lost its activity which it may be due to structural change in the phytase proteins. Overall, the enzyme showed good stability at board pH spectra, since its activity preserved in board pH spectra. The optimum pH of phytase activity was comparable to other species presented in Table 3. Generally, the pH of the different part of digestive tract including salivary glands, stomach and small intestine are 5, 2-4 and 4-6, respectively (Simon and Igbasan, 2002). Therefore, obtained results demonstrated that the enzyme has a good activity in the digestive tract.

Kinetic properties of phytase
After purification of enzyme, the Kinetic parameters of phytase based on sodium phytate as substrate were estimated by Lineweaver-Burk plot. The Michaelis-Menten constant (Km) was found to be 0.197 mM and the Lineweaver-Burk plot demonstrated the maximum velocity (Vmax) was found 1.174 µmol/min. A lower Km indicates higher affinity of enzyme towards the substrate and Vmax is dependent on the affinity of the enzyme for the substrate and catalytic power. According to these results, it can be concluded that Bacillus sp. strain LA12 phytase had high affinity with low Km and Vmax towards sodium phytate. Furthermore, the evaluation of the kinetic constants indicated that the efficiacy of enzyme could be proper for industrial applications.

Effect of metal ions on phytase activity
Different metal ions and modulator such as EDTA had significant effects on purified phytase activity ( Table 2).The different phytase activities were reported after incubating metal ions and EDTA. Comparing to the control sample, EDTA is led to stimulate the phytase activity at 5 mM. The results showed that Ca 2+, Zn 2+, Mn 2+ and Fe 2+ at both concentrations decreased the phytase activity. The variation of phytase activity in presence of Ca 2+ was comparable to phytases of other fungi and yeast species (Table 3). Zn 2+ had the highest inhibition effect on the phytase activity (reduced phytase activity up to 63% at the concentration of 5 mM). It may be related to -SH groups in the active site of the phytase. Mg 2+ and Co 2+ slightly increased the phytase activity at the concentration of 1mM, while at concentration of 5 mM, these metal ions significantly inhibit the activity. These results were in agreement with those achieved by (Wang et al., 2004)that Co 2+ increased the phytase activity of K. pneumoniae subsp. pneumoniae XY-5. They concluded that the decrease in phytase activity could be due to the changing the enzyme conformation. Their finding was inconsistent with that observed in Bacillusin which the phytase activity was stimulated bycalcium ions (Greiner and Carlsson, 2006). Also, EDTA even lightly promoted the phytase activity at the concentration of 5mM. Kerovuo et al. (2000) (Kerovuo et al., 2000) concluded that the EDTA stimulation of phytase activity may be related to metal ions chelation in which form the accessibility of substrate be grater for enzyme. This means that chelating agent, e.g., EDTA can be used as an additive to enhance the efficacy of phytase, The screening of the best bacteria species which has high phytase production efficacy from soil was the major limitations of present study.