بهینه سازی شرایط کشت به منظور تولید آنزیم فیتاز توسط باکتری باسیلوس سوبتیلیس جدا شده از خاک
محورهای موضوعی : میکروب شناسی محیطی
مریم پرهام فر
1
(کارشناس ارشد، دانشگاه علوم پزشکی اراک، دانشکده پزشکی، گروه میکروب شناسی، مرکز تحقیقات پزشکی و مولکولی)
حمید ابطحی
2
(دانشیار، دانشگاه علوم پزشکی اراک، دانشکده پزشکی، گروه میکروب شناسی)
میلاد پرهام فر
3
(کارشناس ارشد، دانشگاه اسن-دویسبرگ، دانشکده شیمی)
کلید واژه: بهینه سازی, فیتات, فیتاز باکتریایی, باسیلوس سوبتیلس,
چکیده مقاله :
سابقه و هدف: فیتات منبع مهم فسفات در دانه های گیاهی می باشد. فیتات اثرات ضد تغذیه ای قوی در انسان و حیوانات (دام، طیور، ماهی ها) دارد. آنزیم فیتاز یک زیرگروه از فسفاتازها می باشد که هیدرولیز فیتات را کاتالیز می کند. فیتاز میکروبی، پتانسیل کاربرد بیوتکنولوژیکی در زمینه های مختلف مانند کشاورزی، تغذیه انسان و حیوانات دارد. این مطالعه با هدف بهینه سازی شرایط تولید فیتاز توسط باکتری باسیلوس سوبتیلیس جدا شده از خاک صورت گرفت.مواد و روش ها: در این مطالعه تجربی از خاک آلوده به فضولات دامداری در شهر اراک نمونه برداری صورت گرفت. نمونه ها در محیط اختصاصی PSM به مدت 48 ساعت در دمای 30 درجه سلیسیوس کشت داده شدند. غربالگری باکتری مولد فیتاز روی محیط PSM براساس وجود هاله شفاف انجام شد. بهترین گونه توسط آزمون بیوشیمیایی و مورفولوژیکی شناسایی گردید. میزان تولید آنزیم و فعالیت حل کنندگی فسفات در حضور گستره های pH مختلف و 4 نوع محیط کشت (PSM ,PVK NBRIP, NBRIY, دارای فیتات به عنوان تنها منبع فسفات) بررسی گردید.یافته ها: در این مطالعه حداکثر تولید آنزیم در باکتری های جدا شده در محیطPSM در 48-36 بعد از گرمخانه گذاری مشاهده شد. باکتری12S مولد فیتاز به عنوان باسیلوس سوبتیلیس شناسایی گردید. بر اساس نتایج، pH بهینه تولید فیتاز در محیطPSM معادل 7 بود. بررسی میزان تولید آنزیم در حضور محیط های مختلف نشان داد که محیط PVK واجد فیتات، مناسب ترین محیط می باشد.نتیجه گیری: باکتری باسیلوس سابتیلس جدا شده، فرصتی برای معرفی فیتاز جدید برای کاربرد در صنایع غذایی و محیطی فراهم می کند. همچنین از محیط PVK می توان به عنوان یک محیط موثر برای غربالگری باکتری مولد فیتاز و تولید آنزیم استفاده نمود.
Background & Objectives: Phytate is the primary storage form of phosphorus in plant seeds. It has an anti-nutritive effect in both human and animals. Phytase is a subgroup of phosphatases which catalyzes the hydrolysis of phytate. Microbial phytases have potential biotechnological application in various fields, such as agriculture, human and animal foods. This study was aimed to investigate the optimal conditions for the production of phytase by Bacillus subtilis isolated from the soil. Materials & Methods: Samples were collected from Arak area, where the soil was contaminated with animal faces. Samples were incubated in PSM medium at 30 ºC for 48 hours. The screening of phytase - producing bacteria on PSM media was performed based on the formation of the clear halo. The most suitable bacterial strain was identified according to its biochemical and morphological characteristics. The level of enzyme production and phosphate- solubilising activity of this strain was assessed in different pH range, and on various media types including PSM, PVK, NBRIP, as well as NBRIY. Results: In this study, maximum enzyme production by the bacterial isolates has been observed following 36-48h incubation in PSM medium. 12S phytase- producing bacterial strain was identified as Bacillus subtilis. Our results showed that the optimum pH for phytase production in PSM medium is pH of 7. Furthermore, investigating different media, phytate-containing PVK was recognised as the most appropriate medium for enzyme production. Conclusion: B. Subtilise isolate can provide an opportunity to introduce new phytase to food as well as environmental industries. Moreover, PVK can be used as an effective medium to produce phytase enzyme and to screen phytase-producing bacterial starins.
1. Brinch-Pedersen H, Dahl-Sǿrensen L, Holm PB. Engineering crop plants: getting a handle on phosphate. Plant Sci. 2002; 7(3): 118-120.
2. Reddy NR, Pierson MD, Sathe SK, Salunkhe DK. Phytates is cereals and legumes. CRC Press, Inc., Boca Raton, Fla. 1989; pp: 345-349.
3. Rousseau X, Létourneau-Montminy MP, Même N, Magnin M, Nys Y, Narcy A. Phosphorus utilization in finishing broiler chickens: effects of dietary calcium and microbial phytase. Poult Sci. 2012; 91: 2829-2837.
4. Sreedevi S, Reddy BN. Identification of phytase producing bacteria C43 isolated from cattle shed soil samples of Hydrabad, A.P. Helix. 2013; 1: 238-242.
5. Vohra A, Satyanarayana T. Phytases: microbial sources, production, purification and potential biotechnological applications. Crit Rev Biotech. 2003; 23(1): 29-60.
6. Sreedevi S, Reddy BN. Purification and characterization of phytase from Bacillus subtilis C43. Adv. Bio Tech. 2013; 12(8): 1-6.
7. Kies AK, De Jonge LH, Kemme PA, Jongbloed AW. Interaction between protein, phytate and microbial phytase. In vitro studies. J Agric Food Chem. 2006; 54: 1753-1758.
8. Hahn-Didde D, Purdum ES. The effects of an enzyme complex in moderate and low nutrient-dense diets with dried distillers grains with solubles in laying hens. J Appl Poult Res. 2014; 23(1): 23-33.
9. Singha B, Satyanarayanab T. Phytases from thermophilic molds: Their production, characteristics and multifarious applications. Process Biochem. 2011; 46(7): 1391-1398.
10. Sasirekha B, Bedashree T, Champa KL. Optimization and partial purification of extracellular phytase from Pseudomonas aeruginosa p6. Euro J Exp Bio. 2012; 2(1): 95-104.
11. Mittal A, Singh G, Goyal V, Yadav A, Aneja KR, Gautam SK, Aggarwal NK. Isolation and biochemical characterization of acido-thermophilic extracellular phytase producing bacterial strain for potential application in poultry feed. Jundishapur J Microbiol. 2011; 4(4): 273-282.
12. Meor Hussin AS, Farouk AE, Greiner R, Salleh HM, Ismail AF. Phytate-degrading enzyme production by bacteria isolated from Malaysian soil. World J Microbiol Biotechnol. 2007; 23: 1653-1660.
13. Fayazi Hosseini N, Fooladi J, Kiarostami K. Isolation of phytase-producing bacteria from rhizospher of Iranian wheat. Iran J Public Health. 2014; 43(2). [In Persian]
14. Ebrahimian M, Motamedi H, Shafie M. 2013. Isolation and characterization of phytase producing bacteria from environmental sources. MS.c. thesis. Faculty of Science, Shahid Chamran University of Ahvaz. [In Persian]
15. Hosseinkhani B, Emtiazi G, Nahvi I. Analysis of phytase producing bacteria (Pseudomonas sp.) from poultry faeces and optimization of this enzyme production. Afr J Biotechnol. 2009; 8(17): 4229-4232.
16. Parhamfar M, Badoei-Dalfard A, Khaleghi M, Hassanshahian M. Purification and characterization of an acidic, thermophilic phytase from a newly isolated Geobacillus stearothermophilus strain DM12. Progress Biological Sci. 2015; 5(1): 61-73.
17. Greiner R, Konietzny U, Jany, KLD. Purification and characterization of two phytases from Escherichia coli. Arch Biochem Biophys. 1993; 303: 107-113.
18. Aziz G, Nawaz M, Anjum AA, Yaqub T, Ahmed MD, Nazir J, Khan SU, Aziz K. Isolation and characterization of phytase producing bacterial isolates from soil. J Anim Plant Sci. 2015; 25(3): 771-776.
19. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. Bergey’s Manual of Determinative Bacteriology. 9th Ed. Williams & Wilkins, Baltimore, MS, USA, 1994.
20. Parhamfar M, Badoei-Dalfard A, Khaleghi M, Hassanshahian M. Isolation of phosphatase-producing phosphate solubilizing bacteria from Loriya hot spring: Investigation of phosphate solubilizing in the presence of different parameters. Biological J Microorganism. 2014; 3(9): 75-88. [In Persian]
21. Shekhar Nautiyal C. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett. 1999; 170(1): 265-270.
22. Sreeramulu G, Srinivasa DS, Nnd K, Joseph R. Lactobacillus amylovorus as a phytase producer in submerged culture. Lett Appl Micb. 1996; 23(6): 385-388.
23. Kumar D, Rajesh S, Balashanmugam P, Jeyanthi Rebecca L, Kalaichelvan PT. Screening, optimization and application of extracellular phytase from Bacillus megaterium isolated from poultry waste. J Modern Biotechnol. 2013; 2(2): 46-52.
24. Oh BC, Choi WC, Park S, Kim YO, Oh TK. Biochemical properies and substrate specificities of alkaline and histidine acid phytases. Appl Microbiol Biotechnol. 2004; 63: 362-372.
25. Kalsi HK, Singh R, Singh Dhaliwal H, Kumar V. Phytases from Enterobacter and Serratia species with desirable characteristics for food and feed applications. 3 Biotechnol. 2016; 6(1): 64.
26. Jareonkitmongkol S, Ohya M, Watanabe R, Takagi H, Nakamori S. Partial purification of phytase from a soil isolate bacterium, Klebsiella oxytoca M3. J Ferment Bioeng. 1997; 83(4): 393-394.
27. Kim HW, Kim YO, Lee JH, Kim KK, Kim YJ. Isolation and characterization of a phytase with improved properties from Citrobacter braakii. Biotechnol Lett. 2003; 25: 1231-1234.
_||_1. Brinch-Pedersen H, Dahl-Sǿrensen L, Holm PB. Engineering crop plants: getting a handle on phosphate. Plant Sci. 2002; 7(3): 118-120.
2. Reddy NR, Pierson MD, Sathe SK, Salunkhe DK. Phytates is cereals and legumes. CRC Press, Inc., Boca Raton, Fla. 1989; pp: 345-349.
3. Rousseau X, Létourneau-Montminy MP, Même N, Magnin M, Nys Y, Narcy A. Phosphorus utilization in finishing broiler chickens: effects of dietary calcium and microbial phytase. Poult Sci. 2012; 91: 2829-2837.
4. Sreedevi S, Reddy BN. Identification of phytase producing bacteria C43 isolated from cattle shed soil samples of Hydrabad, A.P. Helix. 2013; 1: 238-242.
5. Vohra A, Satyanarayana T. Phytases: microbial sources, production, purification and potential biotechnological applications. Crit Rev Biotech. 2003; 23(1): 29-60.
6. Sreedevi S, Reddy BN. Purification and characterization of phytase from Bacillus subtilis C43. Adv. Bio Tech. 2013; 12(8): 1-6.
7. Kies AK, De Jonge LH, Kemme PA, Jongbloed AW. Interaction between protein, phytate and microbial phytase. In vitro studies. J Agric Food Chem. 2006; 54: 1753-1758.
8. Hahn-Didde D, Purdum ES. The effects of an enzyme complex in moderate and low nutrient-dense diets with dried distillers grains with solubles in laying hens. J Appl Poult Res. 2014; 23(1): 23-33.
9. Singha B, Satyanarayanab T. Phytases from thermophilic molds: Their production, characteristics and multifarious applications. Process Biochem. 2011; 46(7): 1391-1398.
10. Sasirekha B, Bedashree T, Champa KL. Optimization and partial purification of extracellular phytase from Pseudomonas aeruginosa p6. Euro J Exp Bio. 2012; 2(1): 95-104.
11. Mittal A, Singh G, Goyal V, Yadav A, Aneja KR, Gautam SK, Aggarwal NK. Isolation and biochemical characterization of acido-thermophilic extracellular phytase producing bacterial strain for potential application in poultry feed. Jundishapur J Microbiol. 2011; 4(4): 273-282.
12. Meor Hussin AS, Farouk AE, Greiner R, Salleh HM, Ismail AF. Phytate-degrading enzyme production by bacteria isolated from Malaysian soil. World J Microbiol Biotechnol. 2007; 23: 1653-1660.
13. Fayazi Hosseini N, Fooladi J, Kiarostami K. Isolation of phytase-producing bacteria from rhizospher of Iranian wheat. Iran J Public Health. 2014; 43(2). [In Persian]
14. Ebrahimian M, Motamedi H, Shafie M. 2013. Isolation and characterization of phytase producing bacteria from environmental sources. MS.c. thesis. Faculty of Science, Shahid Chamran University of Ahvaz. [In Persian]
15. Hosseinkhani B, Emtiazi G, Nahvi I. Analysis of phytase producing bacteria (Pseudomonas sp.) from poultry faeces and optimization of this enzyme production. Afr J Biotechnol. 2009; 8(17): 4229-4232.
16. Parhamfar M, Badoei-Dalfard A, Khaleghi M, Hassanshahian M. Purification and characterization of an acidic, thermophilic phytase from a newly isolated Geobacillus stearothermophilus strain DM12. Progress Biological Sci. 2015; 5(1): 61-73.
17. Greiner R, Konietzny U, Jany, KLD. Purification and characterization of two phytases from Escherichia coli. Arch Biochem Biophys. 1993; 303: 107-113.
18. Aziz G, Nawaz M, Anjum AA, Yaqub T, Ahmed MD, Nazir J, Khan SU, Aziz K. Isolation and characterization of phytase producing bacterial isolates from soil. J Anim Plant Sci. 2015; 25(3): 771-776.
19. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. Bergey’s Manual of Determinative Bacteriology. 9th Ed. Williams & Wilkins, Baltimore, MS, USA, 1994.
20. Parhamfar M, Badoei-Dalfard A, Khaleghi M, Hassanshahian M. Isolation of phosphatase-producing phosphate solubilizing bacteria from Loriya hot spring: Investigation of phosphate solubilizing in the presence of different parameters. Biological J Microorganism. 2014; 3(9): 75-88. [In Persian]
21. Shekhar Nautiyal C. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett. 1999; 170(1): 265-270.
22. Sreeramulu G, Srinivasa DS, Nnd K, Joseph R. Lactobacillus amylovorus as a phytase producer in submerged culture. Lett Appl Micb. 1996; 23(6): 385-388.
23. Kumar D, Rajesh S, Balashanmugam P, Jeyanthi Rebecca L, Kalaichelvan PT. Screening, optimization and application of extracellular phytase from Bacillus megaterium isolated from poultry waste. J Modern Biotechnol. 2013; 2(2): 46-52.
24. Oh BC, Choi WC, Park S, Kim YO, Oh TK. Biochemical properies and substrate specificities of alkaline and histidine acid phytases. Appl Microbiol Biotechnol. 2004; 63: 362-372.
25. Kalsi HK, Singh R, Singh Dhaliwal H, Kumar V. Phytases from Enterobacter and Serratia species with desirable characteristics for food and feed applications. 3 Biotechnol. 2016; 6(1): 64.
26. Jareonkitmongkol S, Ohya M, Watanabe R, Takagi H, Nakamori S. Partial purification of phytase from a soil isolate bacterium, Klebsiella oxytoca M3. J Ferment Bioeng. 1997; 83(4): 393-394.
27. Kim HW, Kim YO, Lee JH, Kim KK, Kim YJ. Isolation and characterization of a phytase with improved properties from Citrobacter braakii. Biotechnol Lett. 2003; 25: 1231-1234.