Journal of Molecular Catalysis B: Enzymatic 89 (2013) 52–60 Contents lists available at SciVerse ScienceDirect Journal of Molecular Catalysis B: Enzymatic jou rn al hom epa ge: www.elsevier.com/locate/molcatb A novel method for covalent immobilization of dextransucrase Mahmut Parlak a , Duran Ustek b,∗ , Aziz Tanriseven a,∗∗ a Department of Biochemistry, Gebze Institute of Technology, Kocaeli, Turkey b Department of Genetics, Institute of Experimental Medicine, Istanbul University, Turkey a r t i c l e i n f o Article history: Received 25 October 2012 Received in revised form 12 December 2012 Accepted 14 December 2012 Available online 28 December 2012 Keywords: Dextran Enzyme technology Eupergit C 250L Dextransucrase Immobilized enzyme a b s t r a c t B-512F dextransucrase is an industrial enzyme used in commercial production of dextran and prebiotics. Several researchers have studied the covalent immobilization of this enzyme with little success due to dextran masking the reactive groups on the enzyme and inactivation of the enzyme during immobiliza- tion. A novel dextransucrase was designed and produced successfully to eliminate problems faced in covalent immobilization. In production of the novel enzyme, B-512F dextransucrase was truncated at N and C terminals and fused to glutathione S-transferase. The novel dextransucrase was fully active and carried out dextran biosynthesis and acceptor reactions effectively. The novel enzyme was immobilized covalently onto Eupergit C 250L, giving rise to 100% immobilization and 83.3% activity yields. Immobi- lized enzyme was used successfully for the production of acceptor products and low molecular weight dextran. The immobilized enzyme showed no decrease in activity for 15 batch reactions and retained its initial activity at storage (4 ◦ C) for 35 days. Optimal conditions were not affected by the immobilization. The kinetic parameters for the free and immobilized enzyme were determined. The acceptor reactions using fructose, glucose, maltose, and lactose were also studied. The novel method developed could also be used in immobilization of some other biomolecules. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Leuconostoc mesenteroides NRRL B-512 FMC dextransucrase (DSR-S) synthesizes dextran using the glucosyl part of sucrose and fructose is formed as a by product. DSR-S is the sole indus- trial enzyme used in the commercial dextran production. B-512 F dextran has 95% (1→6) linear and 5% (1→3) branched link- ages. Dextrans have many uses including production of sephadex, prebiotics, and blood plasma substitute. In addition to dextran biosynthesis from sucrose, DSR-S catalyzes acceptor reactions by transferring glucosyl units from sucrose onto acceptors to produce oligosaccharides. Most of the acceptors are such carbohydrates as glucose, fructose, maltose, and isomaltose. Formation of accep- tor products results in decreases in dextran biosynthesis [1,2]. When glucose or maltose is used as an acceptor, isomaltooligosac- charides are formed. They consist of glucose units (2–20) linked with (1→6) bonds. These oligosaccharides, commercially impor- ∗ Corresponding author at: Istanbul University, Research Institute of Experimen- tal Medicine, Department of Genetics, Fatih, Istanbul, Turkey. Tel.: +90 212 414 20 00/33316; fax: +90 212 414 20 00. ∗∗ Corresponding author at: Gebze Institute of Technology, Department of Bio- chemistry, Muallimkoy, Gebze, Kocaeli, Turkey. Tel.: +90 262 605 30 51; fax: +90 262 605 31 01. E-mail addresses: dustek@istanbul.edu.tr (D. Ustek), tanriseven@gyte.edu.tr (A. Tanriseven). tant carbohydrates, are resistant to the digestive enzymes [3,4]. Isomaltooligosaccharides with monomer numbers of 2–6 are used in the bakery products, beverages, spices, seasonings, gar- ment industry, and anti-cariogenic sweeteners. In addition, these oligosaccharides acts as prebiotics which have many biological functions including the promotion of the growth of bifidobacteria in the large intestine of humans and animals [5,6]. Economical production of dextran and oligosaccharides requires the usage of immobilized dextransucrase. As can be seen in the literature that the studies on covalent immobilization of dextran- sucrase have not been very successful, Kaboli and Reilly studied the immobilization of dextransucrase using 13 different carri- ers including Enzacryl AA, Enzacryl AH and found that the best immobilization yield (19.6%) was obtained using alkylamine porous silica activated by glutaraldehyde [7]. They concluded that “dex- transucrase is an extraordinarily difficult enzyme to immobilize” and found that major difficulty is inactivation of dextransucrase during immobilization. In a study by Monsan et al., DSR-S was attached covalently onto silica support with 3.1 and 4.3% immobi- lization yields in the absence and presence of maltose, respectively [8]. Maltose is a strong acceptor which protects the active site and prevents dextran chain elongation by acceptor reactions. Alcalde et al. also immobilized DSR-S covalently onto glutaraldehyde acti- vated silica. They used native and dextran-free dextransucrase in immobilization, giving rise to 0.6 and 13% immobilization yields, respectively. This result indicates that the removal of dextran 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.molcatb.2012.12.013