Toprak Kökenli Miksobakteri İzolatlarından Alfa-Amilaz Enzimi Üretimi ve Saflaştırılması

Year 2022, Volume: 20 Issue: 4, 398 - 403, 27.12.2022

Melike Baran Ekinci Aynur Gül Karahan Çakmakçı

https://doi.org/10.24323/akademik-gida.1224357

Abstract

Bu çalışmada farklı il (Antalya, Konya, Isparta) ve ilçelerden (Kulu, Şarkikaraağaç, Atabey) toplanan farklı habitatlara ait 28 toprak örneğinden 19 miksobakteri suşu izole edilmiştir. İzole edilen 19 miksobakteri arasında Myxococcus sp KK2’nin en yüksek α-amilaz aktivitesine sahip olduğu belirlenmiştir. Protein çöktürülmesi ile ilk uygulamada %40, 65 ve 80 amonyum sülfat konsantrasyonları, ikinci uygulamada ise %20, 40 ve 65 konsantrasyonları kullanılarak enzim saflığı artırılmıştır. Diyaliz ve jel filtrasyon yöntemleri kullanılarak ilk uygulamada elde edilen enzimin aktivitesi 883.27 U’ya ulaşırken enzim saflığı 28.35 kat artmıştır. İkinci uygulama ile ise enzim aktivitesi 1754.99 U olarak saptanmış ve enzim saflığı 810.24 kat artmıştır.

Keywords

Miksobakteriler, Myxococcus, Enzim saflaştırma

Supporting Institution

Süleyman Demirel Üniversitesi Bilimsel Araştırma Projeleri Koordinasyon Birimi

Project Number

209

Thanks

Bu çalışma, Prof. Dr. Aynur Gül Karahan yürütücülüğünde Süleyman Demirel Üniversitesi, Bilimsel Araştırma Projeleri Koordinasyon Birimi’nce 209’nolu “Miksobakterilerden Alfa-Amilaz Enziminin Ayırımı ve Saflaştırılması” isimli proje ile desteklenmiştir.

Alpha-Amylase Production and Purification from Myxobacteria Isolates of Soil Origin

Abstract

In this study, 19 myxobacteria strains were isolated from 28 soil samples in the different habitats. They were collected from different provinces (Antalya, Konya and Isparta) and their districts (Kulu, Şarkikaraağaç and Atabey). Among the 19 myxobacteria isolated, Myxococcus sp KK2 was determined to have the highest α-amylase activity. With protein precipitation, enzyme purity was increased by using 40, 65 and 80% ammonium sulfate concentrations in the first application, then 20, 40 and 65% concentrations in the second application. By using dialysis and gel filtration methods, the activity of the enzyme obtained in the first application reached 883.27 U while enzyme purity increased 28.35 times, and enzyme activity was 1754.99 U after the second application while enzyme purity increased 810.24 times.

Keywords

Myxobacteria, Enzyme purification, Myxococcus

Project Number

209

References

• [1] Msarah, J.M., Ibrahim, I.,Hamid, A.A., Aqma, S. (2020). Optimisation and production of alpha amylase from thermophilic Bacillus spp. and its application in food waste biodegradation. Heliyon, 6(6), e04183.
• [2] Chohan, N.A., Aruwajoye, G.S., Sewsynker-Suka, Y., Gueguim-Kana, E.B. (2020). Valorisation of potato peel wastes for bioethanol production using simultaneous saccharification and fermentation: Process optimization and kinetic assessment. Renewable Energy, 146, 1031-1040.
• [3] Gavahian, M., Chu., R., Ratchaneesiripap, P. (2021). An ultrasound-assisted extraction system to accelerate production of Mhiskey, a rice spirit-based product, inside oak barrel: Total phenolics, color, and energy consumption. Journal of Food Process Engineering, 45(6), e13861.
• [4] Xia, W., Zhang, K., Su, L., Wu, J. (2021). Microbial starch debranching enzymes: Developments and applications. Biotechnology Advances, 50, 107786.
• [5] Maniglia, C.B., Castanha, N., Le-Bail, P., Augusto, P.E.D. (2021). Starch modification through environmentally friendly alternatives: a review. Critical Reviews in Food Science and Nutrition, 61(15), 2482-2505.
• [6] Alexa, E., Negrea, M., Cocan, I. (2021). Natural improvers for bakery technology. Journal of Agroalimentary Processes and Technologies, 27(4), 392-498.
• [7] Singh, P., Kumar, S. (2019). Microbial Enzyme in Food Biotechnology. Enzymes in Food Biotechnology: Production, Applications, and Future Prospects, Chapter 2, Academic Press, United Kingdom.
• [8] Chapman, J., Ismail, A.E., Dinu, C.Z. (2018). Industrial applications of enzymes: recent advances, techniques, and outlooks. Catalysts, 8(6), 238.
• [9] Awasti, K.M., Wong, J.W.C, Kumar, S., Awasti, S.K., Wang, K., Wang, M., Ren, X., Zhao, J., Chen, H., Zhang, Z. (2018). Biodegradation of food waste using microbial cultures producing thermostable α-amylase and cellulase under different pH and temperature. Bioresource Technology (Part B), 248, 160-17.
• [10] Paul, J.S., Gupta, N., Beliya, E., Tiwari,S., Jadhav, S.K. (2021). Aspects and recent trends in microbial α-amylase: a review. Applied Biochemistry and Biotechnology, 193, 2649-2698.
• [11] Lahiri, D., Moupriya, N., Sarkar., T., Dutta, B., Ray, R.R. (2021). Antibiofilm activity of α-amylase from Bacillus subtilis and prediction of the optimized conditions for biofilm removal by Response Surface Methodology (RSM) and Artificial Neural Network (ANN). Applied Biochemistry and Biotechnology, (193), 1853-1872.
• [12] Vaikundamoorthy, R., Rajendran, R.,Selvaraju, Moorthy, K., Perumal, S. (2018). Development of thermostable amylase enzyme from Bacillus cereus for potential antibiofilm activity. Bioorganic Chemistry, 77, 494-506.
• [13] Taylor, A.J., Leach, R.M. (1995). Enzymes in the food industry. Enzymes in the Food processing (Tucker, G.A., Woods, L.F.J.,-eds). 26-41. Chapman and Hall, Glasgow.
• [14] Sarıkaya, E. (1995). -Amilaz Üreten Bazı Bacillus Suşlarının Gelişme Parametreleri, Enzim Özellik ve Üretim Koşullarının Optimizasyonu. Doktora Tezi. Ankara Üniversitesi, Fen Bilimleri Enstitüsü, Biyoloji Ana Bilim Dalı, Ankara.
• [15] Bader, C.D., Panter, F., Müller, R. (2021). In depth natural product discovery -Myxobacterial strains that provided multiple secondary metabolites. Biotechnology Advances, 39, 107480.
• [16] Buchanan, R.E., Gibbons, N.E. (1974). Bergey’s Manual of Determinative Bacteriology. Washington, USA.
• [17] Dworkin, M., (1993). Cell Surfaces and Appendages. Myxobacteria II. (Dworkin, M., Kaiser, D.,-eds) 63-84. American Society for Microbiology, Washington, USA.
• [18] Holt, J.G., Krieg, R.N. Peter, H.S., Staley, J. T., Williams, T.S. (1994). Group 16 The Fruiting, Gliding Bacteria: The Myxobacteria. Bergey’s Manuel of Determinative Bacteriology. Ed. Williams and Wilkins Baltimore, Maryland USA 515-525.
• [19] Dawid, W. (2000). Biology and global distribution of myxobacteria in soils. FEMS Microbiology Reviews, 24, 403-427.
• [20] Reichenbach, H. (1993). Biology of the Myxobacteria: Ecology and Taxonomy. Myxobacteria II. (Dworkin, M., Kaiser, D., eds) 13-63. American Society for Microbiology, Washington, USA.
• [21] Reichenbach, H., Höfle, G. (1989). The Gliding Bacteria: A Treasury of Secondary Metabolites. Bioactive Metabolites from Microorganisms. (M.E. Bushell, U. Grafe Elsevier-eds). Amsterdam, 79-98.
• [22] Madigan, MT., Martinko, J. M., Parker, J. (1997). Biology of Microorganisms. Prentice Hall International Inc., New Jersey, USA.
• [23] Shrivastava, A. Sharma, R. K. (2021). Myxobacteria and their products: current trends and future perspectives in industrial applications. Folia Microbiologica, 66, 483-507.
• [24] Far, B.E., Ahmadi, Y., Khosroushahi, A.Y., Dilmaghani, A. (2020). Microbial alpha-amylase production: progress, challenges and perspectives. Advanced Pharmaceutical Bulletin, 10(3), 350-358.
• [25] Farez-Vidal, M.E., Fernandez-Vivas, A., Arias, J.M., (1992). Production of -amylase by Myxococus coralloides D. Journal of Applied Bacteriology, 73, 148-156.
• [26] Farez-Vidal, M.E., Fernandez-Vivas A., Arias, J.M. (1995). Properties and significance of an -amylase produced by Myxococus coralloides D. Journal of Applied Bacteriology, 78, 14-19.
• [27] Gerhardt, P. (1981). Manual of Methods for General Bacteriology. American Society of Microbiology, Washington, USA.
• [28] Sharma, G., Khatri, I., Subramanian, S. (2016). Complete genome of the starch-degrading myxobacteria Sandaracinus amylolyticus DSM 53668. Genome Biology and Evolution, 8(8), 2520-2529.
• [29] Hamilton, L.M., Kelly, C.T., Fogarty, W.M. (1999). Cylodextrins and their interaction with amylolytic enzymes. Enzyme and Microbial Technology, 26, 561-567.
• [30] Mulimani, V.H., Patil, G.N., Ramalingam (2000.) -Amylase production by solid state fermentation: a new pratical approach to biotechnology courses. Biochemical Education, 28, 161-163.
• [31] Takasaki, Y. (1985). An amylase producing maltotriose from Bacillus subtilis. Agricultural and Biological Chemistry, 49(4), 1091-1097.
• [32] Kochhar, S., Dua, R.D. (1990). Alpha-amylase from Bacillus amyloliquefaciens. Biotechnology Letters, 12(5), 393-396.
• [33] Yoshigi, N., Chikano, T., Kamimura, M. (1985). Purification and properties of an amylase from Bacillus cereus NY-14. Agricultural and Biological Chemistry, 49(12), 3369-3376.
• [34] Jin, F., Li, Y., Zhong, C., Yu, H. (2001). Thermostable -amylase and - galactosidase production from the thermophilic and aerobic Bacillus sp. strain. Process Biochemistry, 36, 559-564.
• [35] Wang, S. Jeyaseelan, J., Liu, Y. Qin, W. (2016). Characterization and optimization of amylase production in WangLB, a high amylase-producing strain of Bacillus. Applied Biochemistry and Biotechnology, 180,136–151.