تولید و تعیین خصوصیت بیوشیمیایی فیتاز گرمادوست جدا شده از باکتری باسیلوس آمیلولیکوفاسینس LOR10

نویسندگان

گروه زیست شناسی، دانشکده علوم، دانشگاه شهید باهنر کرمان، کرمان، ایران

چکیده
فیتاز ارزش تغذیه­ای غذاهای گیاهی را با افزایش هضم پذیری پروتئین و در دسترس سازی مواد معدنی از طریق هیدرولیز فیتات در طی هضم در معده یا در طی پردازش غذا بهبود می­بخشد. منابع میکروبی برای تولید تجاری فیتاز پتانسیل قابل­توجهی دارند. بنابراین، هدف این تحقیق غربالگری و جداسازی باکتری­های مولد فیتاز از چشمه آب گرم با پتانسیل قابل توجه می­باشد. شناسایی بهترین سویه با روش مولکولی 16S rDNA انجام شد. بهینه سازی تولید آنزیم در حضور منابع مختلف کربن، نیتروژن و فسفات انجام گردید. فعالیت و پایداری آنزیم در pH ها، دماها و یون­های مختلف انجام شد. مقایسه توالی ژن 16S rDNA سویه LOR10 با سایر سویه­ها در بانک ژنی با استفاده از Clustal omega میزان 98 درصد همولوژی با باسیلوس آمیلولیکوفاسینس را نشان داد. نتایج بهینه­سازی محیط نشان داد که گالاکتوز، عصاره مخمر و تری­کلسیم فسفات بعنوان بهترین منبع کربن، نیتروژن و فسفات برای تولید فیتاز می­باشند. دمای بهینه فعالیت در دمای 70 درجه سانتی­گراد به دست آمد. بهترین پایداری فیتاز نیز در pH حدود 5 تا 8 به دست آمد. فعالیت فیتازی در حضور کلرید­کلسیم، کلرید­روی و سولفات­منیزیم حدود 4/1، 3/2 و 6/1 برابر به ترتیب افزایش یافت. خاطر نشان می­شود که فعالیت فیتازی در حضور EDTA و SDS حدود 30 درصد کاهش یافت. این نتایج خاطر نشان می­سازد که فیتاز LOR10 پتانسیل قابل­توجهی برای استفاده­های تجاری از جمله مکمل غذایی را داراست.



کلیدواژه‌ها


عنوان مقاله English

Production and biochemical characterization of a thermostable phytase from Bacillus amyloliquefaciens LOR10

نویسندگان English

Arastoo Badoei-dalfard
Maryam Parhamfar
Department of Biology, Faculty of Sciences, Shahid Bahonar University of Kerman, Kerman, Iran
چکیده English

Phytase can improve the nutritional value of plant-based foods by enhancing protein digestibility and mineral availability through phytate digestion in the stomach and the food processing industry. Microbial sources are more promising for the production of phytases on a commercial scale. The objectives of this exploration were to screening and isolation of phytase-producing bacteria from hot spring with commercial interest. Molecular identification of the best isolate was achieved by the 16S rDNA gene. Optimization of phytase production was prepared in the presence of different phosphate, nitrogen, and carbon sources. Enzyme activity and stability were also explored in the presence of different pHs, temperatures, and ion compounds. Comparing the 16S rDNA gene sequence of the isolate LOR10 with those in GenBank using Clustal omega shows 98% sequence homology with Bacillus amyloliquefaciens. Medium optimization studies showed that galactose, yeast extract, and tricalcium phosphate were the best sources of carbon, nitrogen, and phosphate for phytase production, respectively. The optimum temperature activity was also observed to be 70 oC. Phytase stability was at its optimum in a pH range of 5.0–8.0. Phytase activity increased in the presence of CaCl2, ZnCl2, and MnSO4 about 1.4, 2.3 and 1.6 folds, respectively. It could be mentioned that phytase activity decreased by about 30 % in the presence of EDTA and SDS. On the basis of the results, it could be concluded that LOR10 phytase has a great potential for commercial interest as an additive to animal plant-based foods.



کلیدواژه‌ها English

bacterial identification
hot spring
isolation
phytate-degrading bacteria
Afinah, S., Yazid, A.M., Anis Shobirin, M.H. & Shuhaimi, M. 2010. Phytase: application in food industry. Food Research International Journal 17: 13-21.
Azadian, F., Badoei-dalfard, A., Namaki-Shoushtari, A. & Hassanshahian, M. 2016. Purification and biochemical properties of a thermostable, haloalkaline cellulase from Bacillus licheniformis AMF-07 and its application for hydrolysis of different cellulosic substrates to bioethanol production. Molecular Biology Research Communications 5: 143-155.
Badoei-Dalfard, A. & Karami, Z. 2013. Screening and isolation of an organic solvent tolerant-protease from Bacillus sp. JER02: activity optimization by response surface methodology. Journal of Molecular Catalysis B: Enzymatic 89: 15-23.
Badoei‑Dalfard, A., Parhamfar, M. & Karami, Z. 2019. Characterization of a thermostable, acidic‑phytase from Bacillus tequilensis Dm018; medium optimization by response surface methodology. Catalysis Letters 149: 2961-2972.
Baruah, A., Sahu, N.P., Pal, A.K., Debnath, D., Yengkokpam, S. & Mukherjee, S.C. 2007. Interactions of dietary microbial phytase, citric acid and crude protein level on mineral utilization by Rohu, Labeo rohita (Hamilton), Juveniles. Journal of the World Aquaculture Society 38: 238-249.
Cao, L., Wang, W., Yang, C., Yang, Y., Diana, J. & Yakupitiyage, A. 2007. Application of microbial phytase in fish feed. Enzyme and Microbial Technology 40: 497-507.
Casey, A. & Walsh, G. 2004. Identification and characterization of a phytase of potential commercial interest. Journal of Biotechnology 110: 313-322.
Choi, Y.M., Suh, H.J. & Kim, J.M. 2001. Purification and properties of extracellular phytase from Bacillus sp. KHU-10. Journal of Protein Chemistry 20: 287-292.
Chunshan, Q., Linghua, Z., Yunji, W. & Yoshiyuki, O. 2001. Production of phytase in slow phosphate medium by a novel yeast candida krusei. Journal of Bioscience and Bioengineering 92: 154-160.
Demir, Y., Dikba, N. & Beydemir, Ş. 2018. Purification and biochemical characterization of phytase enzyme from Lactobacillus coryniformis (MH121153). Molecular Biotechnology 60: 783-790.
Dokuzparmak, E., Sirin, Y., Cakmak, U. & Saglam-Ertunga, N. 2017. Purification and characterization of a novel thermostable phytase from the thermophilic Geobacillus sp. TF16. International Journal of Food Properties 20: 1104-1116.
Farahmand, S., Fatemi, F. & Hajihosseini, R. 2019. Sequencing of the rus gene before and after the mutation with DES in the bacterial Acidithiobacillus sp. FJ2. Nova Biologica Reperta 6: 50-60. (In Persian).
Farhat, A., Chouayekh, H., Farhat, M.B., Bouchaala, K. & Bejar, S. 2008. Gene cloning and characterization of a thermostable phytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme. Molecular Biotechnology 40: 127-135.
Fugthong, A., Boonyapakron, K., Sornlek, W., Tanapongpipat, S., Eurwilaichitr, L. & Pootanakit, K. 2010. Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia Pastoris. Protein Expression and Purification 70: 60-67.
Gessler, N.N., Serdyuk, E.G., Isakovaa, E.P. & Deryabinaa, Y.I. 2018. Phytases and the prospects for their application. Applied Biochemistry and Biotechnology 54: 352-360.
Greiner, R., Haller, E., Konietzny, U. & Jany, K.D. 1997. Purification and characterization of a phytase from Klebsiella terrigena. Archives of Biochemistry and Biophysics 341: 201-206.
Greiner, R., Konietzny, U. & Jany, K.D. 1993. Purification and characterization of two phytases from Escherichia coli. Archives of Biochemistry and Biophysics 303: 107-113.
Hong, S.W., Chu, I.H. & Chung, K.S. 2011.Purification and biochemical characterization of thermostable phytase from newly isolated Bacillus Subtilis CF92. Journal of the Korean Society for Applied Biological Chemistry 54: 89-94.
In, M., Jang, J.E.S., Kim, Y.J. & Oh, N.S. 2004. Purification and properties of an extracellular acid phytase from Pseudomonas fragi Y9451. Journal of Microbiology and Biotechnology 14: 1004-1008.
Kerovuo, J., Lappalainen, I. & Reinikainen, T. 2000. The metal dependence of Bacillus subtilis phytase. Biochemical and Biophysical Research Communications 268: 365-369.
Kim Y.O., Kim, H.K., Bae, K.S., Yu, J.H. & Oh, T.K. 1998. Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme and Microbial Technology 22: 2-7.
Kim, Y.H., Gwon, M.N., Yang, S.Y., Park, T.K., Kim, C.G., Kim, C.W. & Song, M.D. 2002. Isolation of phytase-producing Pseudomonas sp. and optimization of its phytase production. Journal of Microbiology and Biotechnology 12: 279-285.
Kim, Y.O., Kim, H.K., Bae, K.S., Yu, J.H. & Oh, T.K. 1998. Purification and properties of a thermostable phytase from Bacillus sp. DS11. Enzyme and Microbial Technology 22: 2-7.
Konietzny, U. & Greiner, R. 2004. Bacterial phytase: potential application, in vivo function and regulation of its synthesis. Brazilian Journal of Microbiology 35: 11-18.
Kumar, V., Sinha, A.K., Makkar, H.P.S. & Becker, K. 2010. Dietary roles of phytate and phytase in human nutrition: a review. Food Chemistry 120: 945-59.
Lan, G.Q., Abdullah, N., Jalaludin, S. & Ho, Y.W. 2002. Culture conditions influencing phytase production of Mitsuokella jalaludinii, a new bacterial species from the rumen of cattle. Journal of Applied Microbiology 93: 668-674.
Lei, X.G. & Porres, J.M. 2003. Phytase enzymology, applications, and biotechnology. Biotechnology Letters 25: 1787-1794.
Lei, X.G., Weaver, J.D., Mullaney, E.J., Ullah, A.H. & Azain, M.J. 2013. Phytase, a new life for an old enzyme. Annual Review of Animal Biosciences 1: 283-309.
Neira-Vielmaa, A.A., Aguilar, C.N., Ilyina, A., Contreras-Esquivel, J.C., Carneiroda-Cunha, M.G., Michelena-Álvarez, G. & Martínez-Hernández, J.L. 2018. Purification and biochemical characterization of an Aspergillus niger phytase produced by solid-state fermentation using triticale residues as substrate. Biotechnology Reports 17: 49-54.
Ornela, P.H. O. & Guimarães, L. H. S. 2019. Purification and characterization of an alkalistable phytase produced by Rhizopus microsporus var. microsporus in submerged fermentation. Process Biochemistry 81: 70-76.
Parhamfar, M., Badoei-Dalfard, A., Khaleghi, M. & Hassanshahian, M. 2015. Purification and characterization of an acidic, thermophilic phytase from a newly isolated Geobacillus stearothermophilus strain DM12. Progress in Biological Sciences 5: 61-73.
Parhamfar, M., Badoei-Dalfard, A., Parhamfar, M. & Fahimi-Rad, S. 2016. Purification and characterization of an extracellular phosphatase enzyme from Bacillus spp. Journal of Cell and Molecular Research 8: 90-97.
Powar, V.K. & Jagannathan, V. 1982. Purification and properties of phytate specific phosphatase from Bacillus subtilis. Journal of Bacteriology 151: 1102-1108.
Puppala, K.R., Bhavsar, K., Sonalkar, V., Khire, J.M. & Dharne, M.S. 2019. Characterization of novel acidic and thermostable phytase secreting Streptomyces sp. (NCIM 5533) for plant growth promoting characteristics. Biocatalysis and Agricultural Biotechnology 18: 101-120.
Raghavendra, P. & Halami, P.M. 2009. Screening, selection and characterization of phytic acid degrading lactic acid bacteria from chicken intestine. International Journal of Food Microbiology 133: 129-134.
Ramezani-Pour, N., Badoei-Dalfard, A., Namaki-Shoushtari, A. & Karami, Z. 2015. Nitrile-metabolizing potential of Bacillus cereus strain FA12; nitrilase production, purification, and characterization. Biocatalysis and Biotransformation 33: 156-166.
Saani, D.M., Lawal, O.T. & Enujiugha, V.N. 2019. Purification and characterization of phytase from Aspergillus fumigatus isolated from African giant snail (Achatina fulica). Biocatalysis and Agricultural Biotechnology 17: 225-232.
Sajidan, A., Farouk, A., Greiner, R., Jungblut, P., Müller, E.C. & Borriss, R. 2004. Molecular and physiological characterization of a 3-phytase from the Rhizobacterium Klebsiella pneumoniae ASR1. Applied Microbiology and Biotechnology 65: 110-118.
Sambrook, J. & Russell, D. 2001. Molecular cloning: a laboratory manual, Cold Spring Harbor, New York.
Sapna, J.J. & Singh, B. 2016. Characteristics and biotechnological applications of bacterial phytases. Process Biochemistry 51: 159-169.
Selle, P.H. & Ravindran, R. 2008. Microbial phytase in poultry nutrition. Livestock Science 113: 99-122.
Shimizu, M. 1992. Purification and characterization of a phytase from Bacillus subtilis (natto) N-77. Bioscience Biotechnology and Biochemistry 56: 1266-1269.
Sievers, F., Wilm, A., Dineen, D.G., Gibson, T.J., Karplus, K., Li, W., Lopez, R., McWilliam, H., Remmert, M., Söding, J., Thompson, J.D. & Higgins, D.G. 2011. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Molecular Systems Biology 7: 539-547.
Singh, B. & Satyanarayana, T. 2009. Characterization of a HAP–phytase from a thermophilic mould Sporotrichum Thermophile. Bioresource Technology 100: 2046-2051.
Singha, B. & Satyanarayana, T. 2011. Phytases from thermophilic molds: their production, characteristics and multifarious applications. Process Biochemistry 46: 1391-1398.
Songa, H.Y. Sheikha, A. F. E. & Hu, D.M. 2019. The positive impacts of microbial phytase on its nutritional applications. Trends in Food Science and Technology 86: 553-562.
Sreedevi, S. & Reddy, B.N. 2012. Isolation, screening and optimization of phytase production from newly isolated Bacillus sp.C43. International Journal of Pharma and Bio Sciences 2: 218-231.
Sumengen, M., Dincer, S. & Kaya, A. 2012. Phytase production from Lactobacillus brevis. Turkish Journal of Biology 36: 533-541.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24: 1596-1599.
Tye, A.J., Siu, F.K., Leung, T.Y. & Lim, B.L. 2002. Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. Applied Microbiology and Biotechnology 59: 190-197.
Vasudevan, U.M., Jaiswal, A.K., Krishna, S. & Pandey, A. 2019. Thermostable phytase in feed and fuel industries. Bioresource Technology 278: 400-407.
Vasudevan, U.M., Krishna, S., Jalaja, V. & Pandey, A. 2017. Microbial phytase: impact of advances in genetic engineering in revolutionizing its properties and applications. Bioresource Technology 245: 1790-1799.
Vats, P. & Banerjee, U. 2005. Biochemical characterization of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus Niger van Tieghem. Journal of Industrial Microbiology and Biotechnology 32: 141-147.
Vohra, A. & Satyanarayana, T. 2003. Phytases: microbial sources, production, purification, and potential biotechnological applications. Critical Reviews in Biotechnology 23: 29-60.

  • تاریخ دریافت 18 خرداد 1405
  • تاریخ انتشار 18 خرداد 1405