Termofilik Geobacillus sp. TF14’ten saflaştırılan α-Amilaz enziminin Kitosan boncuklara kovalent immobilizasyonu

Abstract

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.

Keywords

• α-amilaz,immobilizasyon,kitosan,karakterizasyon

Covalent Immobilization of α-Amylase from Thermophilic Geobacillus sp. TF14 on Chitosan Beads

Abstract

In this study, α-amylase formerly purified from Geobacillus sp. TF14 strain was covalently immobilized onto chitosan beads. Chitosan beads were prepared by dissolving chitosan powder in 5% acetic acid solution and by addition dropwise to 1 M NaOH solution. The consisted beads were washed to remove excessive amount of NaOH. Immobilization was carried out in two steps. Firstly, chitosan beads were activated by reacting with 2.5% Glutaraldehyde solution. Next, activated chitosan beads were mixed with enzyme solution to complete immobilization. Biochemical characterization of immobilized α-amylase was also carried out. It was found that immobilized α-amylase achieved maximum activity at pH 9.00 and the enzyme was quite stable at this pH over a period of 48 h. Temperature optimum of the enzyme was determined as 95 °C. It was also determined that the enzyme protected 50% of its initial activity after incubation of 48 h at this temperature. While Mn²⁺, Co²⁺ and EDTA almost completely inhibited the enzyme, other metal ions showed inhibitory effects at different ratio. In the presence of some detergents the enzyme conserved its initial activity. It can be concluded that the immobilized α-amylase may find application in many fields of starch based industries.

Keywords

• α-Amylase,Immobilization,Chitosan,Characterization,Geobacillus sp

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