Cloning of the glucoamylase encoding gene found in aspergillus niger genome
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Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Adana Alparslan Türkeş Bilim ve Teknoloji Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Glukoamilaz (GA), glikoz gereken her prosesin hidroliz basamağına katılan bir ekzoenzimdir. Çok önemli bir endüstriyel enzim olan glukoamilaz enzimi nişasta, glikojen ve oligosakkaritlerin indirgen olmayan uçlarından glikozidik bağlarını hidrolize ederek D-glukoz üretir. Glukoamilazı endüstri için bu kadar önemli kılan ana neden nişasta proseslerinde oynadığı roldür. Nişasta; gıda, tekstil, deterjan ilaç ve kağıt endüstrisinin ana kaynaklarından biridir. Glukoamilaz doğada; yüksek nişasta hidroliz kapasitesi sayesinde endüstride kullanılan GAI ve GAII olmak üzere iki form halinde bulunur. Birçok bakteri, maya ve fungi çeşitli formlarda glukoamilaz üretebilirler ancak özellikle fungal glukoamilaz endüstri için önem teşkil eder. Bu çalışmada; rekombinant glukoamilaz enziminin Pichia pastoris' de üretilmesi amaçlanmıştır.. Böylece endüstri açısından önemli olan glukoamilaz enzimi laboratuvar ölçeğinde, daha ekonomik ve daha kolay çoğaltılan mayada rekombinant olarak üretilebilecektir. Bu amaçla A.niger' da buunan GAI formundaki glukoamilaz enzimini koldlayan glaA genini taşıyan plasmid dizayn edilmiş ve Pichia pastoris' aktarılmıştır. glaA geninin moleküler klonlanması, dizi analizi ile ve transfeksiyon, koloni PCR ve enzim aktivitesinin belirlenmesi ile doğrulanmıştır. Buna ek olarak elde edilen recombinant enzimin farklı koşullardaki aktivitesi incelenmiştir.
Glucoamylase (GA) (1,4-?-glucosidase) is an exoamylase enzyme that is involved in the hydrolysis step of all processes in which glucose is needed. Glucoamylase which is a very important industrial enzyme produces D-glucose by hydrolyzing glucosidic linkages of starch, glycogen, and oligosaccharides from their non-reducing ends. The main reason that makes GA of such importance to the industry is its role in the starch process. Starch is one of the major components of the food, textile, laundry, pharmaceutics, and paper industries. In nature, there is two forms of glucoamylase, GAI which is commonly used in industry because of its capacity of high starch hydrolyzing and GAII. Most of the bacteria, yeasts, and fungi can produce glucoamylase in various forms, but especially the fungal glucoamylase is most often used in the industry. This study was aimed that produce recombinant glucoamylase enzyme in Pichia pastoris. In this way, an industrially important enzyme, glucoamylase, could be produced as recombinant on laboratory scale from the more economical and easier-breeding yeast strain. For this purpose plasmid that containing the glaA gene encoding the glucoamylase enzyme in GAI form found in Aspergillus niger genome was designed and transferred to Pichia pastoris. The molecular cloning of the glaA gene was confirmed by sequence analysis and transfection was verified by colony PCR and determination of enzyme activity. In addition to that, the activities of the resultant recombinant enzyme under different conditions were investigated.
Glucoamylase (GA) (1,4-?-glucosidase) is an exoamylase enzyme that is involved in the hydrolysis step of all processes in which glucose is needed. Glucoamylase which is a very important industrial enzyme produces D-glucose by hydrolyzing glucosidic linkages of starch, glycogen, and oligosaccharides from their non-reducing ends. The main reason that makes GA of such importance to the industry is its role in the starch process. Starch is one of the major components of the food, textile, laundry, pharmaceutics, and paper industries. In nature, there is two forms of glucoamylase, GAI which is commonly used in industry because of its capacity of high starch hydrolyzing and GAII. Most of the bacteria, yeasts, and fungi can produce glucoamylase in various forms, but especially the fungal glucoamylase is most often used in the industry. This study was aimed that produce recombinant glucoamylase enzyme in Pichia pastoris. In this way, an industrially important enzyme, glucoamylase, could be produced as recombinant on laboratory scale from the more economical and easier-breeding yeast strain. For this purpose plasmid that containing the glaA gene encoding the glucoamylase enzyme in GAI form found in Aspergillus niger genome was designed and transferred to Pichia pastoris. The molecular cloning of the glaA gene was confirmed by sequence analysis and transfection was verified by colony PCR and determination of enzyme activity. In addition to that, the activities of the resultant recombinant enzyme under different conditions were investigated.
Açıklama
Fen Bilimleri Enstitüsü, Nanoteknoloji ve Mühendislik Bilimleri Ana Bilim Dalı
Anahtar Kelimeler
Biyomühendislik, Bioengineering ; Biyoteknoloji
