Termofilik Geobacillus sp. TF14’ten saflaştırılan α-Amilaz enziminin Kitosan boncuklara kovalent immobilizasyonu
Bu çalışmada, daha önce Geobacillus sp. TF14den saflaştırılmış α-amilaz enzimi, kitosan boncuklara kovalent olarak immobilize edildi. Kitosan boncuklar, toz haldeki kitosanın % 5’lik asetik asit çözeltisinde çözülmesi ve 1 M NaOH çözeltisine damla damla eklenmesiyle elde edildi. Daha sonra boncuklar NaOH'in fazlasının giderilmesi için ard arda saf su ile yıkandı. İmmobilizasyon iki aşamada gerçekleştirildi. Öncelikle, % 2,5 Gluteraldehit çözeltisi ile reaksiyona sokularak kitosan boncuklar aktive edildi. Aktive edilmiş kitosan boncuklar immobilizasyonun tamamlanması için enzim çözeltisi ile karıştırıldı. İmmobilize edilen α-amilazın biyokimyasal karakterizasyonu da gerçekleştirildi. İmmobilize α-amilazın pH 9,00'da maksimum aktiviteye ulaştığı ve enzimin 48 saatlik bir sürede bu pH'da oldukça kararlı olduğu tespit edildi. İmmobilize enzimin optimum sıcaklık değeri 95 °C olarak belirlendi. Enzimin, bu sıcaklıkta 48 saat inkübasyon işleminden sonra başlangıçtaki aktivitesinin % 50'sini koruduğu tespit edildi. Mn2+, Co2+ ve EDTA’nın immobilize enzim aktivitesini neredeyse tamamen inhibe ettiği, diğer metal iyonlarının farklı oranlarda inhibisyona neden olduğu belirlendi. Bazı deterjanlar varlığında enzimin aktivitesini koruduğu tespit edildi. İmmobilize edilen α-amilazın nişasta esaslı birçok sanayi alanında kullanılabileceği sonucuna varılabilir.
Covalent Immobilization of α-Amylase from Thermophilic Geobacillus sp. TF14 on Chitosan Beads
In this study, α-amylase formerly purified from Geobacillus sp. TF14 strain was covalently immobilizedonto chitosan beads. Chitosan beads were prepared by dissolving chitosan powder in 5% acetic acid solutionand by addition dropwise to 1 M NaOH solution. The consisted beads were washed to remove excessiveamount of NaOH. Immobilization was carried out in two steps. Firstly, chitosan beads were activated byreacting with 2.5% Glutaraldehyde solution. Next, activated chitosan beads were mixed with enzymesolution to complete immobilization. Biochemical characterization of immobilized α-amylase was alsocarried out. It was found that immobilized α-amylase achieved maximum activity at pH 9.00 and theenzyme was quite stable at this pH over a period of 48 h. Temperature optimum of the enzyme wasdetermined as 95 °C. It was also determined that the enzyme protected 50% of its initial activity afterincubation of 48 h at this temperature. While Mn2+, Co2+ and EDTA almost completely inhibited theenzyme, other metal ions showed inhibitory effects at different ratio. In the presence of some detergentsthe enzyme conserved its initial activity. It can be concluded that the immobilized α-amylase may findapplication in many fields of starch based industries.
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