Int. J. Chem. Technol. 2018, 2 (1), 63-67 Akkoç and Tükel 63 International Journal of Chemistry and Technology http://dergipark.gov.tr/ijct Research Article Co-ımmobilization of pullulanase, glucoamylase and glucose isomerase together with cross-linked enzyme aggregates: Fructose conversion from three enzymes and starch in one step Yakup AKKOÇ 1,* , S. Seyhan TÜKEL 2 1 Departmen of of Medical Techniques, Posof Vocational School, Ardahan University 2 Departmen of Chemistry, Faculty of Science-Literature, Cukurova University Received: 03 May 2018, Revised: 07 June 2018; Accepted: 10 June 2018 * Corresponding author’s e-mail address: yakupakkoc@gmail.com ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ABSTRACT In this study, in order to obtain fructose syrup from starch, pullulanase (PUL), glucoamylase (GA) and glucose isomerase (GI) were immobilized together with cross-linked enzyme aggregates (CLEAs), and named as 'combi-CLEAs'. The values of V max and K m from the kinetic parameters of combi-CLEAs [(GA + PUL) + GI] were found as 12 U g -1 and 0.1157 mg ml -1 in optimum conditions, respectively. After 50 uses, it was observed that the remaining activity of combi-CLEAs [GA + PUL + GI] was preserved at 46%. The thermal stability of combi-CLEAs [GA + PUL) + GI] was observed to be 44% and 30%, respectively, at 60 and 80 ° C after 24 hours. At the end of 30 days, the storage stabilities of combi-CLEAs [(GA + PUL) + GI] was determined as 56% and 13% at the temperatures of 4 and 25C, respectively. Keywords: Combi-CLEAs, fructose syrup, enzyme immobilization. Pullulunaz, glukoamilaz ve glukozizomerazın çapraz bağlı enzim agregatları ile beraber immobilizasyonu: Tek basamakta üç enzim ile nişastadan fruktoz dönüşümü ÖZ Bu çalışmada nişastadan fruktoz şurubu elde etmek için pullulunaz (PUL), glukoamilaz (GA) ve glukozizomeraz (GI) çapraz bağlı enzim agregatları (CLEAs) ile beraber immobilize edildi ve 'combi-CLEAs’ olarak isimlendirildi. Combi-CLEAs [GA + PUL) + Gİ]’nın optimum şartlardaki V max and K m değerleri sırasıyla 12 U g -1 ve 0,1157 mg ml -1 olarak bulundu. Combi-CLEAs [GA + PUL) + Gİ]’ nin 50 kullanımdan sonra kalan bağıl aktivitesinin % 46’ da koruduğu gözlendi. 24 saat sonunda, 60 ve 80 C’ de Combi-CLEAs [GA + PUL) + Gİ]’nin termal kararlılığının sırasıyla % 44 ve % 30 olduğu gözlendi. 30 günün sonunda, Combi-CLEAs [GA + PUL) + Gİ]’nin 4 ve 25 C’ deki depolama kararlılığı ise sırasıyla % 56 ve % 13 olarak belirlendi. Anahtar Kelimeler: Combi-CLEAs, fruktoz şurubu, enzim immobilizasyonu. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ 1. INTRODUCTION Pullulanase (pullulan 6-glukanohidrolaz, EC 3.2.1.41) is an enzyme which hydrolyzes the pullulan, starch, amylopectin, and β-limit dextrin, α-1,6 glycosidic bonds. 1 Whereas glucoamylase converts starch into glucose, glucose isomerase converts glucose into fructose. 2 In the food industry, in order to obtain fructose syrup, first the glucose syrup is obtained from starch, and then fructose syrup is obtained from glucose syrup. In the industry, this process is done in three stages. In the first stage, the starch is gelatinized by using alpha amylase (EC 3.2.1.1) or pullulanase (EC 3.2.1.41). In the second stage, glucose syrup is obtained by using glucoamylase (EC 3.2.1.3). In the third stage, fructose syrup is obtained from glucose by using glucose isomerase. 3 Free enzymes used commercially are for single use and have limited usage areas. With using of enzymes multiple times, the enzyme immobilization technology provides great financial advantages for the industrialists in terms of economic sense. Recently, several studies related to the immobilization of two or three enzymes by combi- CLEAs (combined cross-linked enzyme aggregates) have been conducted. 4-8 In the combi-CLEAs, the product ge-