Chemical Engineering Journal 146 (2009) 515–519 Contents lists available at ScienceDirect Chemical Engineering Journal journal homepage: www.elsevier.com/locate/cej Immobilization of -amylase on poly(vinylamine) and poly(vinylformamide) supports and its performance Anna Konieczna-Molenda a , Andrzej Kochanowski b , Agnieszka Walaszek b , Edgar Bortel b , Piotr Tomasik a,∗ a Department of Chemistry, Agricultural University, Balicka Str. 122, 30-149 Cracow, Poland b Faculty of Chemistry, Jagiellonian University, Ingardena Str. 3, 30-060 Cracow, Poland article info Article history: Received 28 April 2008 Received in revised form 8 October 2008 Accepted 3 November 2008 Keywords: 1,4--d-glucan-glucanhydrolase Immobilization Starch hydrolysis abstract -Amylase was immobilized on six poly(vinylamines) and three poly(vinylformamides) hydrogels poly- merized using various techniques and crosslinkers. The enzyme was covalently bound to the supports using glutaraldehyde as a spacer. The immobilization procedure was optimized involving such factors as temperature, pH, time, sequence of reactions, and kind of carrier employed. Results of the immobilization were evaluated based on analyses of the enzyme activity and stability prior and after immobilization, as well as on the immobilization yield and stability. Highly active biocomposite preparations were designed which provided their multiple application for starch hydrolysis. The selection of a carrier was essential for the activity and stability of immobilized -amylase. Poly(N-vinylformamide) crosslinked with divinyl- benzene in form of spherical beads obtained in a suspension polymerization appeared to be a superior carrier for -amylase. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Amylases play essential role as hydrolyzing enzymes widely used in food, fermentation, textile, and paper industry [1]. -Amylase (1,4--d-glucan-glucanhydrolase, EC. 3.2.1.1) is an endo-acting, widely distributed enzyme that hydrolyzes the -1,4- glycosidic bonds, by-passing -1,6-glycosidic linkages in starch and related substrates. Amylases are available from various sources, including plants, animals and microorganisms [2–5]. Their immo- bilization on water insoluble carriers seems to be the most promising way to obtain more stable products for multiple use [6–14]. There are several methods for immobilizing insoluble enzymes. For that purpose, enzymes are enveloped into a gel matrix, encap- sulated, incorporated into emulsions and membranes, bound to a support by either adsorption, coordination or covalent binding. In fact, the binding of an enzyme to a support is most common. Therefore, the selection of a suitable carrier for a given enzyme and a way of fixing enzyme to it are key problems. Glutaraldehyde ∗ Corresponding author. Tel.: +48 12 6473660; fax: +48 12 6624335. E-mail address: rrtomasi@cyf-kr.edu.pl (P. Tomasik). renders the highest enzyme stability when bound to a support pro- vided the support has amino moieties on its surface [15–18]. The amination introducing such groups to the support most commonly involved carcinogenic ethyleneimine. Use of ethylenediamine was an alternative approach. Fortunately, few years ago the precursor of polyvinylamine, PVAm, i.e. N-vinylformamide, NVF, became com- mercially available. Its polymer, poly(N-vinylformamide), PNVF, can be easily hydrolyzed to PVAm making the amination unnecessary. In this work, PVAm and PNVF have been applied as carriers for enzymes. These carriers have been produced in form of spherical beads suitable also for packing in columns where hydrodynamical factor should be considered. Effect of the immobilization depends on several parameters such as surface area, accessibility of the surface for enzymes, num- ber of activated functional groups on the support, availability for binding the functional groups on the protein, spacer used, distance between the bound enzyme and the surface of the support, and the steric orientation of the active centre. Mono- or multipoint binding of an enzyme to the support, the chemical affinity of pro- tein to the material of the support are further factors influencing immobilization [19–21]. In this paper, immobilization of highly active, thermostable - amylase on poly(vinylamine) (PVAm) and poly(N-vinylformamide) supports is described. Glutaraldehyde was used as a spacer. 1385-8947/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.cej.2008.11.009