سیتروباکتر فارمری phas 32، جدایه‌ای از خاک مزرعه کشت لوبیا با توانایی بسیار در تولید آنزیم فیتاز

Introduction

Phosphorus is one of the important necessary elements in cell metabolism and growth but insoluble phosphate resources can’t be utilized by humans, animals and plants (1). Phytic acid (myo-inositol hexa kis phosphate) is one of these sources that act as a storage source of phosphate, myoinositol and variety of mineral cations for seedling growth and seed germination in plants. Phytate represents approximately 75-80% of total phosphorus of plant seeds (2). Through binding and form ing insoluble complexes with important metals such as iron, zinc, magnesium and calcium as well as proteins and vitamins, phytate reduces their absorption, digestion and biological activities (3, 4).

 Phytase i.e. myoinositol hexa kisphosphate phosphohydrolase (EC 3.1.3.8), is an important food additive for enhancing the bioavailability of phosphorus and other nutritive minerals in monogastric animals such as pigs, poultries and fish that hydrolyzes phytic acid to phosphoric acid and myoinositol (3). Plants, animals and microorganisms are sources of phytases while microbial resources such as bacteria, fungi and yeasts are the most important resources for phytase production (5). In order to food process and enhance minerals availability, human's and animal's diets are mostly enriched with microbial phytase (6). Phytases are also interested due to their function in remedy of iron deficiency in humans caused by phytate. The efficiency of myoinositol phosphates in prevention or treatment of cardio-vesicular disorders and malignancies has been investigated, too (7).

With regard to the importance and applications of phytase for humans and animals and advantages of bacterial phytases (6), screening of different habitats in order to find new isolates with higher production yield and unique characteristics is of great importance. Some of these studies are: Selvamohan et al. that have isolated Pseudomonas sp. as phytase producing bacterium from poultry feces (8) and Roy et al. isolated Shigella sp. CD2 from wheat rhizosphere (9). Yoon et al. isolated Enterobacter sp.4 as a phytase producing bacterium from soil near the roots of leguminous plants (10). Mopera et al, have introduced Klebsiella pneumoniae 9-3B with the ability of phytase production (11). Esakkiraj et al (5), reported that Pseudomonas AP-MSU 2 strain isolated from Etroplus suretensis can produce phytase enzyme (5). In the research of Singh et al Sporotrichum thermophile was reported with the ability to produce phytase following growth on sesame cake (12). Phytase production using agricultural byproducts was also reported in the study of Roopesh et al as Mucor racemosus identified with high levels of phytase production during growth on wheat bran and oilseed cakes (13). Shamna et al, have reported Bacillus subtilis BPTK4 with highest phytase production upon wheat bran (14).

Shushtar region in Khouzestan, Southwest of Iran, has numerous agricultural farms with long history of crop production that makes it as a good candidate to find phytase producing bacteria. Hot and humid climate of this region can harbor strains which can tolerate harsh environments and also produce more stable enzymes. The aim of this study was isolation of phytase producing bacteria from Shushtar's bean farm and optimization of growth and enzyme production conditions by the best isolate.

Materials and Methods

Screening of phytase producing bacteria: Soil samples were collected from three parts of plowed bean farm located in Shushtar (32° 2' 37" N / 48° 51' 24" E), Khouzestan, Iran. Samples were immediately transferred to lab and refrigerated till experiments. One gram of soil sample was transferred to 25 ml of 2% wheat bran medium and incubated (30˚C, 150 rpm) for 3 days. Then 100 μl of supernatant was cultured on phytase screening medium (PSM) containing (^g/_l) glucose (15), sodium phytate (1.5), NH₄NO₃ (2), KCl (0.5), MgSO₄.7H₂O (0.5), MnSO₄ (0.3), FeSO₄.7H₂O (0.3) and agar (2) (pH 7.5) at 30˚C for 24 h. A subculture was subsequently prepared on PSM medium from those colonies with halo zone to gain pure culture. Pure colonies were stored on nutrient agar at 4˚C (15).

Enzyme production assay: In order to confirm phytase production, the isolates with clear zone on PSM agar were cultivated in 25 ml of PSM broth and incubated (30˚C, 150 rpm, 72 h). Following a centrifugation (6000 ×g, 15 min), qualitative enzyme assay was performed on the supernatant and the best isolate was selected for growth and enzyme production optimization (15). In qualitative enzyme assay, the reaction mixture contained 2 ml of supernatant and 4 ml of substrate solution (0.84% solution of sodium phytate in buffer solution, pH 5.5). The buffer solution was prepared through dissolving 0.18 g acetic acid, 3 g sodium acetate.3H₂O and 0.15 g CaCl₂.2H₂O in 100 ml distilled water, pH = 5.5. The reaction was done at 37˚C for 65 min and then stopped by 4 ml freshly prepared color reagent. The color reagent was prepared by mixing 25 ml ammonium molybdate solution (10 g ammonium molybdate.4H₂O and 1 ml NH₃ (25%) in 100 ml distilled water) with 25 ml ammonium vanadate solution (0.235 g ammonium vanadate, 40 ml distilled water at 60˚C, then slowly added 2 ml nitric acid before dilution to 100 ml) and stirring while slowly adding 16.5 ml nitric acid (65%). Then the mixture cooled to room temperature and adjusted to 100 ml. The color changed following phytase activity was measured at 415 nm. In blank reaction, the color reagent was added prior to the enzyme sample. One unit of enzyme was defined as the amount of enzyme that can release 1 μmol inorganic phosphate in 1 min (16). Based on the quantitative assay, the best isolate was selected for further analysis.

Optimization of growth condition in PSM broth medium:To select the suitable temperature and pH for growth of selected bacterium, the growth curve of bacterium was drawn at 30˚C, 37˚C and 45˚C in PSM medium and following determination of optimum temperature, the optimum pH was obtained from pH 5.5, 7 and 8.5. Growth curve was gained by inoculating 1% inoculum equal to 0.5 McFarland turbidity to PSM, incubation while agitating at 150 rpm and reading the turbidity of culture medium at 580 nm (Bio-Rad, USA) every 2 h for 60 h. This process was done in triplicates and the absorption was read twice, i.e. 6 times of reading in each absorption determination.

Effects of different parameters on phytase production: In order to find the effects of culture conditions on phytase production a series of experiments was designed as follow. All of them were performed as triplicates and changing one factor in each experiment was applied. The tested parameters were temperature (30, 37 and 45˚C, (21)), pH (5.5, 7 and 8.5, (8)), phytic acid concentration (0.15, 0.25 and 0.35%, (17)) and different agricultural wastes (wheat bran, rice bran and powdered rice straw at 1% (^w/_v) while phytic acid and FeSO₄.7H₂O was deleted, (8, 18)). The enzyme activity was assessed at 12, 24, 48, 72 and 96 h of incubation.

Determining optimum conditions for phytase activity: The effect of temperature, pH and protease on enzyme activity was determined in triplicate experiments. To determine the optimum temperature of phytase, enzyme assay was performed by treating the enzyme at 30˚C, 37˚C, 45˚C, 55˚C, 65˚C and 75˚C. Similarly, phytase activity was assessed in pH 5.5, 7 and 8.5. The stability of phytase against proteases including pepsin and trypsin were also investigated. The supernatant of phytase enzyme was incubated with 0.1 ^mg/_ml of pepsin (Merck, Germany) or trypsin (Sigma, USA) at 37˚C for 60 min. Then, enzyme assay was performed and compared with the enzyme activity obtained from untreated supernatant (9).

Investigation of nitrogen fixation: Nitrogen fixation test was done according to Dobereiner et al. (19).

Partial purification of phytase: The supernatant obtained from cultivation of the isolate in PSM broth at optimum condition was centrifuged at 14000 ×g for 30 min. The proteins were precipitated through adding ammonium sulfate up to 85% saturation and incubation at 4˚C for 4 h by agitation. The pellet was then harvested by centrifugation at 14000 ×g for 30 min. The obtained pellet was resuspended in 20 mM Tris-HCl buffer (pH 8) and was dialyzed for one day in same buffer (20).

Protein concentration and molecular weight of enzyme: To determine the protein concentration in the supernatant obtained from optimum enzyme production conditions, the Bradford method was used. In this method, standard protein solution (Bovine Serum Albumin) 1 ^mg/_ml was used (21). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Sigma- Aldrich, SDS (L3771), acrylamide/bisacrylamide (a2917), temed (t9281), APS (A3678)) was performed in order to determine the molecular weight of produced phytase, as described by Laemmli using 10% acrylamide gel. Proteins were stained with Coomassie brilliant blue R-250 (22).

Identification of phytase producing isolate: The selected isolate was identified based on molecular and phenotypic methods. In molecular identification, DNA was extracted by DNA extraction kit (Cinnagen, Iran) and the 16S rRNA gene was amplified by FD1 (5-CCGAATTCGTCGACAACAGAGTTTGATCCTGGCTCAG-3) and RP1 (5-CCCGGGATCCAAGCTTACGGTTACCTTGTTACGACTT-3) primers (23). PCR reaction mixture was composed of 1X buffer concentration, 0.7 ^ρmol/_μl primers, 0.2 mM dNTPs, 3 mM MgCl₂, 1.5 u Taq DNA polymerase and 2 μl of template DNA in 25μl final volume. PCR reaction was performed in thermal cycler (Bio-Rad, USA), based on the following thermal cycling program:  denaturation (94˚C, 5 min), 30 cycles composed of denaturation (94˚C, 60s), annealing (58.1˚C, 40s) and extension (72˚C, 150s) and a final extension at 72˚C for 20 min.  The PCR product was electrophoresed in 1% agarose containing DNA safe stain and 1kb DNA ladder (Sinaclon (Cat. No. PR901645) and then sequenced (Macrogen, Korea) and edited by Bioedit software version 7.2.2. Then, its similarity with existing data in gene bank was searched by BLAST algorithm of NCBI.  Phenotypical identification was done based on Bergeyˊs manual of systematic bacteriology (24).

Results

As a result of primary screening, 6 phytase producing bacteria were isolated from plowed bean farm in Shushtar, but following their cultivation in PSM broth and phosphate content measurement, only four isolates had the ability of releasing phosphate. In enzyme assay analysis on 3 days culture of bacteria (Table 1), it was determined that phas32 isolate has the best potential of enzyme production, so it was selected for further analysis.

Table 1- Enzyme assay of 4 phytase producing isolates.

Figure 1 represents the growth curve of phas32 strain at 30˚C, 37˚C and 45˚C. This isolate could not grow at 45˚C and its optimum temperature was 37˚C. In such a manner that maximum growth was achieved after 22 h of incubation.

In the experiment of culturing at different pH (Figure 2), it was concluded that phas32 could grow in acidic, alkaline and neutral pH, but alkaline pH was more favorable for bacterial growth than others.

Enzyme production by phas32 isolate was investigated at 30˚C, 37˚C and 45˚C during 96 h. As represented in Figure 3, this isolate has the most enzyme production at 30˚C after 24 h and 72 h with 31 ^U/_ml yield of enzyme at these conditions. Furthermore, phas32 had the most enzyme production at this temperature and pH 7 after 24 h (Figure 4).

Fig. 1-Growth curve of phas32 isolate at different temperatures

 Fig. 2-Growth curve of phas32 isolate at different pH

 Fig. 3- Enzyme production by phas32 isolate at different temperatures

 Fig. 4- Phytase production by phas32 isolate at different pH