Purification and Characterization of an Extracellular Phosphatase Enzyme From Bacillus spp.

Journal of Cell and Molecular Research

مقاله 7، دوره 8، شماره 2 - شماره پیاپی 16، اسفند 2016، صفحه 90-97   

نوع مقاله: Research Articles

شناسه دیجیتال (DOI): 10.22067/jcmr.v8i2.58676

نویسندگان

Maryam Parhamfar^{* 1}؛ Arastoo Badoei-Dalfard¹؛ Milad Parhamfar²؛ Shohreh Fahimi Rad³

¹Shahid Bahonar University of Kerman

²Duissburg-Essen University

³Campus of Agriculture and Natural Resources, University of Tehran

چکیده

Phosphorus is one of the most important nutrients for plant growth and development. Chemical Pi fertilizer is used to provide the phosphorus for the plants, but it is mostly fixed in the soil into insoluble form and become unavailable to the plants. Phosphate-solubilizing bacteria have lots of application in agriculture as biological fertilizer. Consumption of biofertilizers instead of chemical fertilizers can lead to environmental pollution reduction and crop production enhancement using sustainable farming. In this study, a phosphatase-producing bacterium was isolated from agricultural soil in Kerman. Screening of phosphate solubilizing bacteria was performed on the PVK medium, based on clear area diameter. The best bacterium (AG41) was identified based on 16s rDNA gene. The optimum condition for production of phosphatase was also determined and it was purified and characterized. Sequence alignment and phylogenetic tree results show that AG41 is closely related to Bacillus subtilis, with 98% homology. Phosphatase activity was determined by end point method. The best carbon, nitrogen and phosphate sources for enzyme production were 1.0% glucose, 0.5% ammonium sulfate and (0.25%) sodium phytate +(0.25%) tricalcium phosphate, respectively. Bacterial phosphatase was partially purified using ammonium sulfate fractionation followed by dialysis. Results showed that the optimum temperature for the purified enzyme activity was 40oC and it was stable at temperatures below 60°C. This enzyme was stable between pH 3.0-7.0, and the optimal pH activity was found to 5.0. These results indicated that this strain can be a notable candidate for using as biofertilizers.

کلیدواژه‌ها

Screening؛ Biofertilizer؛ Phosphate-solubilizing bacteria؛ Phosphatase

مراجع

1. Boyce A. and Walsh G. (2007) Purification and characterization of an acid phosphatase with phytase activity from Mucor hiemalis Wehmer. Journal of Biotechnology 132(1): 82-7.

2. Chang C. H. and Yang S. S. (2009) Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation. Bioresource Technology 100: 1648–1658.

3. Chen K. S., Lin Y. S. and Yang S. S. (2007) Application of thermotolerant microorganisms for biofertilizer preparation. Journal of Microbiology, Immunology and Infection 40(6): 462-73.

4. Ferreira-Nozawa M. S., Nozawa S. R., Martinez-Rossi N. M. and Rossi A. (2003) The dermatophyte Trichophyton rubrum secretes an EDTA-sensitive alkaline phosphatase on high-phosphate medium. Brazilian Journal of Microbiology 34(2): 161-164.

5. Fitriatin B. N., Arief D. H., Simarmata T., Santosa D. A. and Joy B. (2011) Phosphatase-producing bacteria isolated from Sanggabuana forest and their capability to hydrolyze organic phosphate. Journal of Soil Science and Environmental Management 2(10): 299-303.

6. Gawas-Sakhalkar P., Singh S. M., Naik S. and Ravindra R. (2012) High-temperature optima phosphatases from the cold-tolerant Arctic fungus Penicillium citrinum. Polar Research.

7. Hu X. J., Li Z. J., Cao Y. C., Zhang J., Gong Y. X. and Yang Y. F. (2010) Isolation and identification of a phosphate-solubilizing bacterium Pantoea stewartii subsp. stewartii g6, and effects of temperature, salinity, and pH on its growth under indoor culture conditions. Aquaculture International 18(6): 1079–1091.

8. Jatoth K., Shantipriya A., Mangilal T. and Junapudi S. (2015) Optimization for the production of extracellular alkaline phosphatase from Bacillus subtilis. International Journal of Current Microbiology and Applied Sciences 4(10): 829-838.

9. Kerovuo J. and Tynkkynen S. (2000) Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755. Letters in Applied Microbiology 30: 325-329.

10. Mahesh M., Neha G., Rajesh T. S., Somashekhar R. and Puttaiah E. T. (2010) Isolation and characterization of extracellular thermostable alkaline phosphatase enzyme from Bacillus spp. International Journal of Applied Biology and Pharmaceutical Technology 1(1): 21-33.

11. Mclachlan K. D. (1980) Acid phosphatase activity of intact roots and phosphorus nutrition in plants. I. Assay conditions and phosphatase activity. Australian Journal of Agricultural Research 31: 429-440.

12. Nautiyal C. S., Bhadauria S., Kumar P., Lal H., Mondal R. and Verma D. (2000) Stress induced phosphate solubilization in bacteria isolated from alkaline soils. FEMS Microbiology Letters 182(2): 291-6.

13. Nenwani V., Doshi P., Saha T. and Rajkumar S. (2010) Isolation and characterization of a fungal isolate for phosphate solubilization and plant growth promoting activity. Journal of Yeast and Fungal Research 1: 9-14.

14. Nopparat C., Jatupornpipat M. and Rittiboon A. (2007) Isolation of phosphate solubilizing fungi in soil from Kanchanaburi, Thailand. KMITL Science and Technology Journal 7: 137-146.

15. Parhamfar M., Badoei-Dalfard A., Khaleghi M. and Hassanshahian M. (2014) Isolation of phosphatase-producing phosphate solubilizing bacteria from Loriya hot spring: Investigation of phosphate solubilizing in the presence of different parameters. Biological Journal of Microorganism 3(9): 75-88.

16. Pradhan N. and Sukla L. B. (2005) Solubilization of inorganic phosphates by fungi isolated from agriculture soil. African Journal of Biotechnology 5(10): 850-854.

17. Rodriguez H. and Fraga R. (1999) Phosphate solubilizing bacteria and their role in plant growth promotion. Biotechnology Advances 17: 319–339.

18. Rombola T. H., Pedrinho E. A., de Macedo Lemos E. G., Gonçalves A. M., dos Santos L. F. and Pizauro J. M. (2014) Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli. BMC Research Notes 7: 221.

19. Sambrook J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York.

20. Sharma S. B., Sayyed R. Z., Trivedi M. H. and Gobi T. A. (2013) Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springer Plus 2:587.

21. Shekhar Nautiyal C. (1999) An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiology Letters 170(1): 265-270.

22. Shen J., Yuan L., Zhang J., Li H., Bai Z., Chen X., Zhang W. and Zhang F. (2011) Phosphorus dynamics: From soil to plant. Plant Physiology 156: 997–1005.

23. Sievers F., Wilm A., Dineen D. G., Gibson T. J., Karplus K., Lopez W., Li R., McWilliam H., Remmert M., Söding J., Thompson J. D. and Higgins D. G. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7: 1-9.

24. Tamura K., Dudley J., Nei M. and Kumar S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596–1599.

25. Vassilev N. and Vassileva M. (2003) Biotechnological solubilization of rock phosphate on media containing agro-industrial wastes. Applied Microbiology and Biotechnology 61: 435–440