SmF yöntemiyle topraktan izole edilen bacillus subtilis’den amilaz üretimi

Yıl 2019, Cilt: 12 Sayı: 3, 57 - 64, 15.12.2019

Veysi Ortakaya Sema Agüloğlu Fincan

Öz

Çalışmamızda Ergani Makam Dağı’ndan alınan toprak örneğinden Bacillus subtilis’in izolasyonu, tanımlanması ve optimizasyonu gerçekleştirilerek endüstriyel öneme sahip α-amilaz üretme yeteneği araştırıldı. Morfolojik ve biyokimyasal testler ile 16S rRNA analizi yapılarak B. subtilis olarak tanımlanan bakterinin 32. saat, 37 oC ve pH 7.0’ de maksimum üreme gösterdiği tespit edildi. Nişastalı besiyerinde bakterinin amilaz ürettiği tespit edildikten sonra enzim optimizasyonu yapıldı. Amilaz üretimi için optimum koşullar 37 oC, pH 7.0 ve 48. saat olarak belirlendi. Enzim aktivitesi üzerine metallerin etkisi incelendiğinde Cu+2’ nın enzim aktivitesini artırdığı, Zn+2, Hg+2 ve Fe+2 enzim aktivitesini kısmen inhibe ettiği, Ca+2 ise kontrole yakın bir değer gösterdiği görüldü. Karbon kaynaklarının bakteri gelişimi ve enzim üretimi üzerine inhibisyon etkisi yaptığı gözlendi.

Anahtar Kelimeler

Bacillus subtilis, tanımlama, α,, -amilaz, üretim, karakterizasyon

Amylase Production of Bacillus subtilis Isolated from Soil by SmF Method

Öz

In our study, the isolation, identification and optimization of Bacillus subtilis from soil samples were performed and the ability to produce α-amylase of industrial importance was investigated. Bacteria identified as B. subtilis by 16S rRNA analysis, morphological and biochemical tests. Maximum growth was determined at 32 hour, 37°C and pH 7.0. Enzyme optimization was performed after determination of bacterial amylase in starchy medium. Optimum conditions for amylase production were determined as 37°C, pH 7.0 and 48 h. When the effect of metals on enzyme activity was examined, it was determined that Cu2+ increased enzyme activity, Zn2+, Hg2+ and Fe2+ were partially inhibited the enzyme activity, and Ca2+ was showed close activity to the control. It was observed that carbon sources had inhibitory effect on bacterial growth and enzyme production.

Anahtar Kelimeler

Bacillus subtilis, identification, α,, -amylase, production, characterization

Kaynakça

• Saha, B.C., Jordan D.B., Bothast R.J. (2009). Enzymes, industrial (Overview), Encyclopedia of Microbiology. Academic Press, Oxford, 281-294.
• Ortakaya, V., Agüloğlu Fincan, S., Enez, B. (2017). α-Amylase from Bacillus simplex production, characterization and partial purifıcation, Fresenius Environmental Bulletin, 26, 4446-4455.
• Agüloğlu, S., Enez, B. (2014). Purification and characterization of α-amylase from thermophilic Geobacillus stearothermophilus. Starch/Stärke 66, 182–189.
• Agüloğlu Fincan, S., Enez, B. Ozdemir, S., Matman Bekler, F. (2014). Purification and characterization of thermostable α-amylase from thermophilic Anoxbacillus flavithermus. Carbohydrate Polymers, 102, 144-150.
• Ramya, L. N., Pulicherla, K. K.(2014). Molecular insight sintocold active polygalacturonase enzyme for its potential application in foodprocessing, Journal of food science and technology, 52 /9, 5484-5496.
• Nielsen, J. E., & Borchert, T. V. (2000). Protein engineering of bacterial α-amylases, Biochimica et Biophysica Acta, 1543, 253-274.
• Gupta, R., Gigra, P., Mohapatra, H., Goswami, V. K., Chauhan, B. (2003). Microbial α-amylase: biotechnological perspective. Enzyme and Microbial Technology, 38, 1599-1616.
• Wiseman, A. (1987) The Application of Enzymes in Industry, Handbook of Enzymes Biotechnology Second Edition. Chapter 3, 274-373.
• Rezzukoğlu, İ., Agüloğlu Fincan, S., Enez, B., (2018) Production and characterization of α-amylase from lactic acid bacteria isolated with whey, Biological Diversity and Conservation, 11 /2, 115-122.
• Wolfgang, A. (2007). Enzyme in industry: Productions and applications. Thirdh Completely Revised Edition. Wiley –VCH Pres., 489 pp.
• Sajedi, R.H., Naderi-Manesh, H., Khajeh, K., Ahmadvand, R., Ranjbar, B., Asoodeh, A., Moradian, F. (2005). A Ca-independent α-amylase that is active and stable at low pH from Bacillus sp. KR-8104. Enzyme and Microb. Technol., 36, 666-671.
• Bernfeld P. (1955). Enzymes carbohydrate metabolism, ln Methods in Enzymology, Academic Press, 17, 149-158.
• Lowry, O.H., Rosebrough. N.J., Farr, A.L. (1951). Protein measurement with the folin phenol reagent, Journal of Biological Chemistry, 193, 265- 275.
• Sasaki H., Kurosawa, K., Takao, S. (1986). Screening of microorganisms for raw starch saccharifying enzyme production. Agricultural Biology and Chemistry, 50, 1661-1664.
• Sarıkaya, E., Gürgün, V. (2000). Increase of the α-amylase yield by some Bacillus strains. Turkey J. Biol., 24:299-308.
• Francis, F., Sabu, A., Nampoothiri, K. M., Szakacs, G., Pandey, A. (2002). Synthesis of α-amylase by Aspergillus oryzae in solid state fermentation. Journal of Basic Microbiology, 42, 320-326.
• Ramesh, M.V., Lonsane, B.K.. (1987). Solid state fermentation for production of α- amylase by Bacillus megaterium 16M. Biotechnology Letters, 5, 323-328.
• Al-Johani N.B., Al-seeni1 M. N., Ahmed Y. M., (2017). Optimization of alkaline α-amylase production by thermophilic Bacillus subtilis, Afr J Tradit Complement Altern Med. 14/1, 288-301.
• Asgher, M., Javaid Asad, M., Rahman, S.U., Legge, R.L. (2007). A thermostable α- amylase from a moderately thermophilic Bacillus subtilis strain for starch processing. Journal of Food Engineering, 79, 950–955.
• Femi-Ola, T.O., Olowe, B.M., (2011) Characterization of alpha-amylase from Bacillus subtilis BS5 isolated from Amitermes evuncifer silvestri. Research Journal of Microbiology, 6: 140-146.
• Vidyalakshmi, R., Paranthaman and J. Indhumathi, 2009. Amylase production on submerged fermentation by Bacillus sp. World Journal of Chemistry, 4: 89-91.
• Ray R. C., Kar S., Nayak S., Swain M. R. (2008). Extracellular α-amylase production by Bacillus brevis MTCC 7521, Food Biotechnology, Vol. 22, 234–246. No. 3.
• Sankaralingam, S., Kumar, C., Shankar, T., Ramasubburayan, R., Prakash, S. 2012. Optimization of culture conditions for the production of amylase from Bacillus licheniformis on submerged fermentation. American-Eurasian J. Agric. & Environ. Sci., 12(11): 1507-1513.
• Shafiei, M., Ziaee, A.A., Amoozegar, M.A. (2010). Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting and halophilic α- amylase from a moderately halophilic bacterium Nesterenkonia sp strain F. Process Biochemistry, 45, 694-699