Elif DEMİRKAN, Tuba SEVGİ, Merve BAŞKURT

Yeni İzole Edilen Bacillus sp. M-10 Suşundan α-Amilazın Üretimini Etkileyen Fiziksel Faktörlerin Optimizasyonu

Bu çalışmada Türkiye’ nin 15 farklı ilinden temin edilen toprak örneklerinden 60 bakteri izole edilmiştir. α-Amilaz pozitif olarak belirlenen en geniş açık zon çapına sahip 4 bakterinin morfolojik ve fizyolojik özellikleri araştırılmış ve hepsinin Bacillus cinsine ait olduğu saptanmıştır. 4 suşun enzim üretim kapasiteleri test edilmiştir. En yüksek α-amilaz aktivitesine sahip Bacillus suşu seçilmiş ve bu suş, Bacillus sp. M-10 olarak adlandırılmıştır. Bacillus sp. M-10’un α-amilaz üretimini etkileyen bazı fiziksel faktörlerin etkisi araştırılmıştır. Bu amaçla, sıcaklık, pH, havalandırma, inokulüm miktarı ve inokulüm yaşı gibi fiziksel parametreler denenmiştir. Bacillus sp. M-10 suşunun maksimum α-amilaz üretimi, nişastalı ortamda, 37°C sıcaklıkta, pH 7,0, havalandırma 150 rpm, inokülasyon miktarının %2.5 v/v ve inokülasyon yaşının 2 gün olduğu değerlerde optimum düzeydedir. Tarafımızdan modifiye edilen ortam, daha yüksek enzim aktivitesi elde etmek için en uygun ortam olup, enzim aktivitesi 48. saatte 30 U/ mL olarak bulunmuştur. Modifiye edilen ortamda enzim aktivitesinde yaklaşık % 42 oranında artış gözlenmiştir

Anahtar Kelimeler:

Bacillus sp., İzolasyon, Fiziksel faktörler, α-amilaz

Optimization of physical factors affecting the production of the α-amylase from a newly isolated Bacillus sp. M10 strain

In this study, sixty bacteria isolated from soil samples that come from 15 different cities of Turkey. The four isolates which had a higher ratio of clearing zone were determined to be α-amylase positive, and were investegated for morphological and physiological. All of these were defined as Bacillus. Enzyme production capacity of these 4 strain have been tested. The bacteria which has the highest α-amylase activity was selected as a Bacillus strain and these strain was named as Bacillus sp. M-10. It's investigated that physical factors which are affecting the α-amylase production of Bacillus sp. M-10. For this purpose, temperature, pH, aeration, inoculum size and inoculum age have been tested as physical parameters. In starchy medium, 37°C temperature, pH 7.0, 150 rpm for aeration, 2.5 ml inoculation size and 2 days for inoculation age were the optimum rate for maximum α-amylase production of Bacillus sp. M-10 strain. In order to enhance the production of α-amylase, medium was modified by our, and the enzyme activity was found 30 U/mL at the hour of 48. In modified environment, enzyme activity was increased about 42% rate.

Keywords:

Isolation, Bacillus sp., α-amylase, Physical factors,

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Abd-Elhalem, BT., El-Sawy, M., Gamal, RF., Abou-Taleb, KA. 2015. Production of amylases from Bacillus amyloliquefaciens under submerged fermentation using some agro-industrial byproducts. Ann. Agric. Sci., 60: 193-202.
Abou-Zeid, A. 1996. Production, purification and characterization of an extracellular alpha-amylase enzyme isolated from Aspergillus flavus. Microbios, 89: 55-66.
Abusham, RA., Rahman, RNZR., Salleh, AB., Basri, M. 2009. Optimization of physical factors affecting the production of thermo-stable organic solvent-tolerant protease from a newly isolated halo tolerant Bacillus subtilis strain Rand. Microb. Cell Fact., 8: 20-28
Ahmed, K., Munawar, S., Khan, MA. 2015. Cultural conditions for maximum alpha-amylase production by Penicillium notatum IBGE 03 using shaken flask technique of submerged fermentation. Pure Appl. Biol., 4: 306
Asgher, M., Asad, MJ., Rahman, S., Legge, R. 2007. A thermostable α-amylase from a moderately thermophilic Bacillus subtilis strain for starch processing. J. Food Eng., 79: 950-955.
Ashokkumar, B., Kayalvizhi, N., Gunasekaran, P. 2001. Optimization of media for β-fructofuranosidase production by Aspergillus niger in submerged and solid state fermentation. Process Biochem., 37: 331-338.
Dahot, M. 1986. Biosynthesis of Invertase by Penicillium expansum. J. Pure App. Sci., 5: 23-26.
Day, SA. 2015. http://alevelnotes.com/Factors-affecting-EnzymeActivity/146 Dincbas, S., Demirkan, E. 2010. Comparison of hydrolysis abilities onto soluble and commercial raw starches of immobilized and free B. amyloliquefaciens α-amylase. J. Environ. Sci., 4(11): 87-95.
Djamel, C., Ali, T., Nelly, C. 2009. Acid protease production by isolated species of Penicillium. Eur. J. Sci. Res., 25: 469-477.
Ellaiah, P., Adinarayana, K., Bhavani, Y., Padmaja, P., Srinivasulu, B. 2002. Optimization of process parameters for glucoamylase production under solid state fermentation by a newly isolated Aspergillus species. Process Biochem., 38: 615-620.
Goes, AP., Sheppard, JD. 1999. Effect of surfactants on α-amylase production in a solid substrate fermentation process. J. Chem. Technol. Biotechnol., 74: 709-712.
Gots, JS., Buchanan, R., Gibbons, N. 1975. Bergey’s Manual of Determinative Bacteriology. 9th edition, Williams & Wilkins Co., Baltimore. pp. 747-842.
Gupta, R., Beg, Q., Khan, S., Chauhan, B. 2002. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. J. Appl. Microbiol. Biotechnol., 60: 381-395.
Gupta, R., Gigras, P., Mohapatra, H., Goswami, VK., Chauhan, B. 2003. Microbial α-amylases: a biotechnological perspective. Process Biochem., 38: 1599-1616.
Gupta, R., Hiteshi, K., Rana, S. 2015. Cold active alpha amylase from a psychrophilic bacterial isolate. J. Biochem. Technol., 6: 1030-1033.
Jogezai, N., Raza, A., Abbas, F., Bajwa, M., Mohammad, D., Kakar, W., Saeed, M., Awan, A. 2011. Optimization of cultural conditions for microbial alpha amylase production. J. Microbiol. Antimicrob., 3: 221-227.
Mamma, D., Kourtoglou, E., Christakopoulos, P. 2008. Fungal multienzyme production on industrial by-products of the citrus-processing industry. Bioresour. Technol., 99: 2373-2383.
Mukhtar, H., Ikram Ul, H. 2012. Concomitant production of two proteases and alpha-amylase by a novel strain of Bacillus subtilis in a microprocessor controlled bioreactor. Braz. J. Microbiol., 43: 1072-1079.
Nascimento, WCA., Martins, MLL. 2004. Production and properties of an extracellular protease from thermophilic Bacillus sp. Braz. J. Microbiol., 35: 91-96.
Padhiar, AR., Kommu, S. 2016. Isolation, Characterization and Optimization of Bacteria producing Amylase. Int. J. Adv. Res. Biol. Sci., 3(7): 1-7.
Pandey, A., Nigam, P., Soccol, CR., Soccol, VT., Singh, D., Mohan, R. 2000. Advances in microbial amylases. Biotechnol. Appl. Biochem., 31 (2): 135-152.
Papagianni, M. 2004. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol. Adv., 22: 189-259.
Park, CW., Zipp, E. 2000. http://www.rpi.edu/dept/chem-eng/ Biotech-Environ/Projects00/temph/enzyme.html
Pazouki, M., Panda, T. 2000. Understanding the morphology of fungi. Bioprocess Eng., 22: 127-143.
Roychoudhury, S., Parulekar, SJ., Weigand, W.A. 1989. Cell growth and α-amylase production characteristics of Bacillus amyloliquefaciens. Biotech. and Bioeng. 33: 197-206.
Saleem, A., Ebrahim, MKH. 2014. Production of amylase by fungi isolated from legume seeds collected in Almadinah Almunawwarah, Saudi Arabia. J. Taibah Univ. Sci., 8: 90-97.
Saxena, L., Iyer, B.K., Ananthanarayan, L. 2007. Three phase partitioning as a novel method for purification of ragi (Eleusine coracana) bifunctional amylase/protease inhibitor. Process Biochem., 42: 491-495.
Sharma, A., Satyanarayana, T. 2011. Optimization of medium components and cultural variables for enhanced production of acidic high maltose-forming and Ca 2+-independent α-amylase by Bacillus acidicola. J. Biosci. Bioeng.,111: 550-553.
Singh, P., Gupta, P., Singh, R., Sharma, R. 2012. Factors affecting alfa Amylase Production on Submerged Fermentation by Bacillus sp. Int. J. Pha. Life Sci., 3(12): 2243-2246.
Singh, V., Sharma, R., Sharma, P. 2015. Isolation, screening and optimization of amylase producing Bacillus sp. from soil. APJHS, 2: 86-93.
Sivakumar, T., Ramasubramanian, V., Shankar, T., Vijayabaskar, P., Anandapandian, K. 2011. Screening of keratinolytic bacteria Bacillus cereus from the feather dumping soil of sivakasi. J. Basic Appl. Bio. 5: 305-314.
Sivakumar, T., Shankar, T., Vijayabaskar, P., Muthukumar, J., Nagendrakannan, E. 2012. Amylase production using Bacillus cereus isolated from a vermicompost site. Int. J. Microbiol. Res., 3: 117-123.
Sudharhsan, S., Senthilkumar, S., Ranjith, K. 2007. Physical and nutritional factors affecting the production of amylase from species of Bacillus isolated from spoiled food waste. Afr. J. Biotechnol. 6 (4): 430-435
Suribabu, K., Govardhan, TL., Hemalatha, K. 2014. Optimization of physical parameters of α-amylase producing Brevibacillus Borostelensis R1 in submerged fermentation. IJRET, 1: 517-525.
Vengadaramana, A., Balakumar, S., Arasaratnam, V. 2012. Production and optimization of α-amylase by Bacillus licheniformis ATCC 6346 in lab Bench-Scale fermenter. J. Microbiol. Biotechnol. Res., 2: 190-211.
Wanderley, KJ., Torres, FA., Moraes, LM., Ulhoa, CJ. 2004. Biochemical characterization of α-amylase from the yeast Cryptococcus flavus. FEMS Microbiol. Lett., 231:165-169.
Yoo, Y.J., Hong, J., Hatch, RT. 1987. Comparison of alphaamylase activities from different assay methods. Biotechnol. Bioeng., 30:147-151.
Zar, MS., Ul Haq, I. 2012. Optimization of the alpha amylase production from Bacillus amyloliquefaciens IIB-14 via parameter significance analysis and response surface methodology. Afr. J. Microbiol. Res., 6: 3845-3855.