Termofilik ve termotolerant filamentli fungus türlerinin endüstriyel öneme sahip enzim aktivitelerinin incelenmesi

Funguslar, temel olarak düşük maliyetli substratlarda büyüyebildikleri ve hücre dışı olarak büyük miktarda enzim salgıladıkları için endüstriyel öneme sahip birçok enzimin üretiminde yaygın olarak kullanılmaktadır. Termostabil enzimlerin endüstriyel uygulamalarda seçilmesinin en önemli sebebi ham maddelerin ön işlemleri sırasında uygulanan yüksek sıcaklıklara karşı düşük aktivite kayıplarını sağlayan stabiliteleridir. Bu çalışmada 6 termofilik ve 57 termotolerant fungus türü üzerinde endüstriyel öneme sahip olan lipaz, amilaz, selülaz, fitaz ve proteaz enzim aktiviteleri araştırılmıştır. Hücre dışı lipaz, amilaz, selülaz, fitaz, proteaz üreten fungusların taranması, farklı enzimler için özel besin ortamları kullanılarak petri kaplarında veya tüplerinde gerçekleştirilmiştir. Sonuç olarak, Humicola insolens, Lichtheimia corymbifera, Rhizomucor pussillus, Melanocarpus albomyces, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus türlerinden oluşan 63 adet kültürün 5 farklı enzimi üretme potansiyelleri belirlenmiştir ve bu kültürlerden pozitif sonuç verenler: fitaz için 18 (%28,5), proteaz için 63 (%100), amilaz için 14 (%22), lipaz için 63 (%100), selülaz için 57 (%90) olarak belirlenmiştir. Enzim tarama çalışmaları sonucunda en iyi fitaz üreticisi türler R. pusillus ve A. terreus, proteaz üreticisi tür A. fumigatus, amilaz üreticisi türler A. terreus, L. corymbifera ve R. pusillus, lipaz üreticisi tür A. fumigatus, selülaz üreticisi tür A. fumigatus olarak belirlenmiştir. Çalışmada kullanılan termofilik ve termotolerant funguslar arasından lipaz, amilaz, selülaz, proteaz ve fitaz gibi endüstriyel öneme sahip olan termostabil enzimleri üretme yeteneğine sahip olanların belirlenmesi bu fungusların ve enzimlerinin ileriki çalışmalarda değerlendirilmesine ışık tutacağı kanaatindeyiz.

Anahtar Kelimeler:

Termofilik, termotolerant, filamentli fungi, enzim

Investigation of industrially important enzyme activities of thermophilic and thermotolerant filamentous fungi

Fungi are widely used in the production of many enzymes of industrial importance, mainly because they can grow on low-cost substrates and secrete large amounts of enzymes extracellularly. The most important reason for choosing thermostable enzymes in industrial applications is their stability, which provides low activity losses against high temperatures applied during the pretreatment of raw materials. In this study, industrially important lipase, amylase, cellulase, phytase and protease enzyme activities were investigated on 6 thermophilic and 57 thermotolerant fungi species. Screening of fungi producing extracellular lipase, amylase, cellulase, phytase, protease was carried out in petri dishes or tubes using special nutrient media for different enzymes. As a result, 63 cultures consisting of Humicola insolens, Lichtheimia corymbifera, Rhizomucor pussillus, Melanocarpus albomyces, Aspergillus fumigatus, Aspergillus flavus, Aspergillus terreus species were determined to produce 5 different enzymes, and those with positive results from these cultures were determined: 18 for phytase (28,5%), 63 (100%) for protease, 14 (22%) for amylase, 63 (100%) for lipase, 57 (90%) for cellulase. As a result of enzyme screening studies, the best phytase producer species R. pusillus and A. terreus, protease producer species A. fumigatus, amylase producer species A. terreus, L. corymbifera and R. pusillus, lipase producer species A. fumigatus, cellulase producer species A. fumigatus has been determined. We believe that the determination of the thermophilic and thermotolerant fungi used in the study that are capable of producing thermostable enzymes such as lipase, amylase, cellulase, protease and phytase, which have industrial importance, will shed light on the evaluation of these fungi and their enzymes in future studies.

Keywords:

Thermophilic thermotolerant, filamentous fungi, enzyme, thermostable,

PDF

___

[1] Rajasekaran, A.K. & Maheshwari, R. (1993). Thermophilic fungi: an assesment of their potential for growth in Soil. J. Biosci. 18, 345-354.
[2] Maheshwari, R., Bharadwaj, G. & Bhat, M.K. (2000). Thermophilic fungi: their physiology and enzymes. Microbiology and Molecular Biology Reviews, 64 (3), 461-488.
[3] Mouchacca, J. (2007). Heat tolerant fungi and applied research: Addition to the previously treated group of strictly thermotolerant species. World J Microbiol Biotechnol, 23, 1755-1770.
[4] Wiseman, A. (1985), Handbook of Enzymes Biotechnology (2nd ed.) Ellis Horwood and John Wiley & Sons.
[5] Sharma, S., Vaid, S., Bhat, B., Singh, S., & Bajaj, B.K. (2019). Thermostable enzymes for industrial biotechnology. Advances in Enzyme Technology, Biomass, Biofuels, Biochemicals, 469-495. https://doi.org/10.1016/B978-0-444-64114-4.00017-0
[6] Turner, P., Mamo, G. & Karlsson, E.N. (2007). Potential and Utilization of Thermophiles and Thermostable enzymes in Biorefining. Microbial Cell Factories, 6(9), 1-23.
[7] Prabakaran, M., Thennarasu, V., Mangala, R.A., Bharathidasan, R., Chandrakala, N., & Mohan, N. (2009). Comparative studies on the enzyme activities of wild and mutant fungal strains isolated from sugarcane field. Indian Journal of Science and Technology, 2(11), 46-49.
[8] Bajaj, B.K., & Sharma, P. (2011). An alkali-thermotolerant extracellular protease from a newly isolated Streptomyces sp. DP2, New Biotechnol. 28, 725-732.
[9] Ortakaya, V., & Agüloğlu Fincan, S. (2019). Amylase production of Bacillus subtilis ısolated from soil by SmF method. Biological Diversity and Conservation, 12(3), 57-64. https://doi.org/10.5505/biodicon.2019.70288
[10] Saini, R., Saini, H.S., & Dahiya, A. (2017). Amylases: characteristics and industrial applications. J. Pharmacogn. Phytother, 6, 1865-1871.
[11] Corrêa, T.L.R., & Araújo, E.F. (2020). Fungal phytases: from genes to applications. Brazilian Journal of Microbiology, 51, 1009-1020. https://doi.org/10.1007/s42770-020-00289-y
[12] Sharma, M., & Bajaj, B.K. (2014). Cellulase production from Bacillus subtilis MS 54 and its potential for saccharification of biphasic-acid-pretreated rice straw. J. Biobased Mater. Bioenergy, 8, 449-456.
[13] Bakir, Z.B., & Metin, K. (2016). Purification and characterization of an alkali-thermostable lipase from thermophilic Anoxybacillus flavithermus HBB 134. J. Microbiol. Biotechnol. 26, 1087-1097.
[14] Choi, Y., Hyde, K.D., & Ho, W.W.H. (1999). Single spor isolation of fungi. Fungal Diversity, 3, 29-38.
[15] Gulati, H.K., Chadha, B.S., & Saini, H.S. (2007). Production, purification and characterization of thermostable phytase from thermophilic fungus Thermomyces lanuginosus TL-7. Acta Microbiologica et Immunologica Hungarica, 54 (2), 121-138.
[16] Chen, J.C. (1998). Novel screening method for extracellular phytase-producing microorganisms. Biotechnology Techniques, 12, 759-761.
[17] Balkan, B. (2008). Katı substrat fermentasyonu ile ham nişastayı parçalayan yeni bir fungal amilaz üretimi saflaştırılması ve biyokimyasal özelliklerinin belirlenmesi, Doktora tezi, Danışman Yrd.Doç.Dr. Figen Ertan, Trakya Üniversitesi, Fen Bilimleri Enstitüsü, Biyoloji AB.D.
[18] Rapp, P., & Backhaus, S. (1992). Formation of extracellular lipases by filamentous fungi, yeast and bacteria. Enzyme Microb Technol, 14, 938-943.
[19] Colen G. Junqueira R.G, & Moraes-Santos T. (2006). Isolation and screening of alkaline lipase-producing fungi from Brazilian savanna soil. World J Microbiol Biotechnol, 22, 881-885.
[20] Topal, Ş., Pembeci, C., Borcaklı, M., Batum, M., & Çeltik, Ö. (2000). Türkiye’nin tarımsal mikoflorasının endüstriyel öneme sahip bazı enzimatik aktivitelerinin ıncelenmesi-ı: amilaz, proteaz, lipaz, Turk J Biol., 24, 79–93.
[21] Tan, T.K., Yeoh, H.H., Tan, M.L., & Koh, S.K. (1987). Cellulolytic activities of some filamentous fungi. Journal of The Singapore National Academy of Science, 16, 11-16.
[22] Mitra P., Chakraverty, R., & Chandra, A. (1996). Productions of proteolytic enzymes by solid state fermentation. J Sci Ind Res, 55, 439-42.
[23] Korcan E., Özkara A., Akyıl D., Ciğerci, İ.H., & Konuk M. (2007). Farklı fungus cinslerinde endüstriyel öneme sahip bazı enzim aktivitelerinin incelenmesi. AKÜ Fen Bilimleri Dergisi, 7(1), 279-286.
[24] Tan T.K., & Leong W.F. (1986). Screening for extracellular enzymes of fungi from manufacturing wastes. Mircen journal of applied microbiology and biotechnology, 2, 445-452.
[25] Guimaraes, L.H.S., Peixoto-Nogueira, S.C., Michelin, M., Rizzatti, A.C.S., Sandrim, V.C., Zanoelo, F.F., Aquino, A.C.M.M., Junior, A.B., & Polizeli, M.L.T.M. (2006). Screening of filamentous fungi for production of enzymes of biotechnological interest. Brazilian Journal of Microbiology, 37, 474-480.
[26] Mitchell D.B., Vogel K., Weimann B.J., Pasamontes L., & van Loon A.P.G.M. (1997). The phytase subfamily of histidine acid phosphatase; isolation of genes for two novel phytases from the Aspergillus terreus and Myceliophthora thermophila. Microbiology, 143, 245-252.
[27] Singh, B., & Satyanarayana, T. (2012). Production of phytate-hydrolyzing enzymes by thermophilic moulds. African Journal of Biotechnology, 11(59), 12314-12324.
[28] Berikten, D., & Kivanc, M. (2014). Optimization of solid state fermentation for phytase production by Thermomyces lanuginosus using response surface methodology. Preparative Biochemistry and Biotechnology, 44, 834–848, https://doi.org/10.1080/10826068.2013.868357
[29] Nampoothiri K.M., Tomes G.J., Roopesh K., Szakacs G., Nagy V., Soccol C.R., & Pandey A. (2004). Thermostable phytase production by Thermoascus aurantiacus in submerged fermentation. Appl Biochem Biotechnol, 118(1-3), 205-214.
[30] Olagoke O.A. (2014). Amylase activities of some thermophilic fungi isolated from municipal solid wastes and palm-kernel stack. American Journal of Microbiology and Biotechnology, 1(2), 64-70.
[31] Idres, M.M.M., Moharram, A.M., Ahmed, M.S., Omar O., Marzouk M.E., & Yasser, M.M. (2021). α-Amylase, L-asparaginase and arginase enzymes production by fungi ısolated from rice stored under environmental condition in middle egypt. International Journal on Emerging Technologie, 12(1), 48-58.
[32] Saroj, P., Manasa P., & Narasimhulu, K. (2018) Characterization of thermophilic fungi producing extracellular lignocellulolytic enzymes for lignocellulosic hydrolysis under solid‑state fermentation. Bioresour Bioprocess, 5, 31. https://doi.org/10.1186/s40643-018-0216-6
[33] Khokhar I., Haider M.S., Mushtaq S., & Mukhtar I. (2012). Isolation and screening of highly cellulolytic filamentous fungi. J Appl Sci Environ Manag, 16, 223-226.
[34] Hamada, T.A., Abdulkreem, R.S., & Younus, H.M. (2020). Efficiency of Aspergillus Species to Produce the Lipase Enzyme from Various Types of Oil Seeds. Medico-legal Update, 20(1), 809-813.