Yerli Alfa-Amilaz Enzimi Üretimi İçin Yerli Fungus İzolasyonu, Makroskobik, Mikroskobik Tanımlaması, DNA Dizi Analizi ve Biyoinformatik Değerlendirmeler

Öz

Bu çalışmada ekmek yapımında ticari önemi olan alfa-amilaz enzimi üreten termofilik ve mezofilik yerli fungusların izolasyonu, makroskobik, mikroskobik ve moleküler tür teşhisleri gerçekleştirilmiştir. Mikroorganizma Kültür Koleksiyon Merkezlerine ve Mikrobiyal Gen Bankalarına genetik materyal oluşturulması ve yerli izolatlardan patente konu yerli enzim üretimi amaçlanmıştır. Afyon, Eskişehir, Uşak, Ankara termal alanlardan 23 termotolerant fungus izole edilmiştir. İzolatların makroskobik ve mikroskobik incelemeleri sonucunda yüksek amilaz enzimi üreten Aspergillus niger, Aspergillus terreus ve Trichoderma atroviride türüne ait 6 izolatın tür teşhisi yapılmıştır. İzolatların fungus spesifik primerleri kullanılarak 18S rDNA ve ITS bölgeleri PCR amplifikasyonu, moleküler tanımlaması yapılmış ve Web tabanlı BLAST analizleri ile karşılaştırılmıştır. Moleküler karakterizasyon çalışması sonucunda klasik tanımlama ile moleküler tanımlamanın birbirini desteklediği görülmüştür.

Anahtar Kelimeler

• Fungus
• izolasyon
• Internal Transcribed Spacer (ITS)
• amilaz enzimi

Isolation, Macroscopic, Microscopic Identification, DNA Sequencing and Bioinformatic Assessments of Native Fungi for the Production of Native Alpha-Amylase Enzyme

Öz

In this study, isolation of thermophilic and mesophilic native fungi producing alpha-amylase enzyme which is commercially important in bread making, macroscopic, microscopic and molecular identification were performed. Microorganism Culture Collection Centers and Microbial GenBanks to create genetic material and local isolates to produce patents of the native enzyme was aimed. 23 thermotolerant fungi were isolated from the thermal areas of Afyon, Eskişehir, Uşak and Ankara. As a result of macroscopic and microscopic investigations of isolates, 6 isolates of Aspergillus niger, Aspergillus terreus and Trichoderma atroviride species which produce high amylase enzyme species were determined. 18S rDNA and ITS regions of the isolates were compared with Web-based BLAST analysis by PCR amplification and molecular identification. As a result of molecular characterization study, it was observed that the classical identification and the molecular identification supported each other.

Anahtar Kelimeler

• Fungi
• isolation
• Internal Transcribed Spacer (ITS)
• amylase enzyme

Kaynakça

1. Barnett J and Hunter B, 1998. Illustrated Genera of Imperfect Fungi. American Phytopathological Society Press, St. Paul.
2. Cowan D, 1996. Industrial Enzyme Technology, TIBTECH, No. 14, 177-178.
3. Çetin ET, 1983. Endüstriyel Mikrobiyoloji, İstanbul Tıp Fakültesi Vakfı, Bayda Yayın, I. Baskı, 2, 145 146.
4. Haki GD and Raksit SK. 2003. Developments in industrially important thermostable enzymes: a review Bioresource Technolog, 89: 17-34.
5. Hasenekoglu I, (1991). Toprak Mikrofungusları. Atatürk Üniversitesi yayınları No. 689 Erzurum.
6. Leveque E, Janecek S, Haye B and Belarbi A, 2000. Thermophillic archaeal amylolytic enzyme. Enzyme and Microbial Technology, 26: 3-14.
7. Madigan MT, Martingo J, (2009). Brock Mikroorganizmaların Biyolojisi. Palme Yayıncılık. ISBN: 6055829629.
8. Nguyen, QD, Rezessy-Szabo J.M., Claeyssens M, Stals I, Hoschke A. 2002. Purification and characterization of amylolytic enzymes from thermophilic fungus Thermomyces lanuginosusstrain ATCC 34626. Enzyme Microb. Technol. 3:345-352.

9. Pitt JI, (1979). The Genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. Academic Press London.
10. Sahnoun M, Bejara S, Sayari A, Triki MA, Kriaa M, Kammoun R, 2012. Production, purification and haracterization of two alfa-amylase isoforms from a newly isolated Aspergillus Oryzae strain S2. Process Biochemistry 47 18–25.
11. Saleem 1, Mohsen KH, 2014. Ebrahim Science Direct Production of amylase by fungi isolated from legume seeds collectedin Almadinah Almunawwarah, Saudi ArabiaA. Journal of Taibah University for Science 8 90–97
12. Ünal A, 2015. Production of α-amylase from some thermophilic Aspergillus species and optimization of its culture medium and enzyme activity. African Journal of Biotechnology, DOI:10.5897/AJB2015.14924, 14(47): 3179-3183.
13. Verweij PE, Meis JFGM, van den Hurk P, Zoll J, Samson RA, Melchers WJG. 1995.Phylogenetic relationships of five species of Aspergillus and related taxa as deduced by comparison of sequences of small subunit ribosomal RNA. Med Mycol 33:185–90.
14. White TJ, Bmns T, Lee S, Taylor J, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ, editors. PCR meth appl. San Diego: Academic Press; p. 315–22.