Züleyha AKPINAR, Merve KIZAKLI YILDIRIM, Hakan KARAOĞLU

Termofilik Anoxybacillus gonensis glukoz izomerazının DEAE-Sefaroz üzerine iyonik ve kovalent immobilizasyonu

Bir şekerin diğerine (glukozun fruktoza) dönüştürülmesiyle üretilen yüksek fruktozlu mısır şurubu (HFCS), pazarlama değerine sahiptir. Bu nedenle günümüze kadar farklı kaynaklardan (makro ve mikroorganizmalar) izole edilen farklı glukoz izomerazlar [(GI) (D-ksiloz ketol izomeraz, EC 5.3.1.5)] araştırılmıştır. Ayrıca endüstriyel uygulamalar için GI üretiminin maliyetinin düşürülmesi araştırılmış ve bunun için farklı teknikler uygulanmıştır. Enzim immobilizasyon yaklaşımları, enzimlerin tekrar tekrar kullanılmasına izin verdiği için öne çıkan özelliklere sahiptir. Bu çalışmada Anoxybacillus gönensis G2T (AgoGI) yabani tip enzimlerinin DEAE-sefaroz matriksi üzerinde immobilizasyonu (iyonik ve kovalent) gerçekleştirildi. Çalışmanın bir sonraki aşamasında elde edilen enzimlerin kinetik ve biyokimyasal özellikleri belirlendi. İmmobilize enzimler için optimum sıcaklık ve pH değerleri sırasıyla 85 °C ve 6.50 olarak belirlendi. DEAE-sefaroz üzerinde iyonik bağlı AgoGI'nin kinetik verileri (Vmax ve Km) 4.85±2.09 μmol/dk/mg protein ve 130,57±5,42 mM, aynı matris üzerinde kovalent immobilize AgoGI 40.51± 0.81 μmol/dk/mg protein ve 127,28±2,96 mM’dır. Sonuç olarak, DEAE-sefarozun hem kovalent hem iyonik immobilizasyon için immobilizasyon matriksi olarak kullanılması, glukoz izomerazın biyokimyasal ve kinetik parametreleri üzerinde herhangi bir olumsuz etki göstermedi. Bu nedenle, DEAE-sefaroz üzerinde immobilize edilmiş AgoGI HFCS üretimi için mükemmel ve umut verici bir araçtır.

Anahtar Kelimeler:

Anoxybacillus gönensis, DEAE-sefaroz, Glukoz izomeraz, HFCS, İmmobilizasyon

Ionic and covalent immobilization of glucose isomerase of thermophilic Anoxybacillus gonensis on DEAE-sepharose

High fructose corn syrup (HFCS), which is produced by the conversion of one sugar into another (glucose to fructose), has a marketing value. Hence, different glucose isomerases [(GI) (D-xylose ketol isomerase, EC 5.3.1.5)] isolated from different sources (macro-and microorganisms) were researched until today. In addition, the cost reduction of GI production for industrial applications has been investigated and applied with different techniques. Enzyme immobilization approaches have prominent features because they allow enzymes to be used repeatedly. In the current study, Anoxybacillus gonensis G2T glucose isomerase (AgoGI) (wild type) were immobilized with ionic and covalent binding on DEAE-sepharose matrix. Afterward, kinetic and biochemical parameters of the immobilized enzymes were evaluated. The pH and temperature parameters, in which the ionic and covalent immobilized enzymes showed the best activity, were determined as 6.50 and 85 °C, respectively. The kinetic data (Vmax and Km) of ionic bound AgoGI on DEAE-sepharose were 4.85±2.09 μmol/min/mg protein and 130,57±5,42 mM, as covalent immobilized AgoGI on the same matrix were 40.51± 0.81 μmol/min/mg protein µmol/min and 127,28±2,96 mM, respectively. Consequently, the usage of DEAE-sepharose for both covalent and ionic immobilization as immobilization matrix did not exhibit any negative effects on biochemical and kinetic parameters of glucose isomerase. Therefore, immobilized AgoGI on DEAE-sepharose was an excellent and promising tool for HFCS production.

Keywords:

Anoxybacillus gonensis, DEAE-sepharose, Glucose isomerase, HFCS, Immobilization,

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