Comparison of covalent immobilization of amylase on polystyrene pellets with pentaethylenehexamine and pentaethylene glycol spacers Feng Wang a,⇑ , Zhiguo Gu a , Zhenggang Cui a , Liming Liu b,⇑ a School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, China b State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China article info Article history: Received 15 April 2011 Received in revised form 25 July 2011 Accepted 29 July 2011 Available online 6 August 2011 Keywords: Covalent immobilization Polystyrene pellets Spacer Pentaethylenehexamine Pentaethylene glycol abstract a-Amylase from Aspergillus oryzae was covalently immobilized onto polystyrene pellets with pentaethy- lenehexamine (PS-PEHA-Ald) and pentaethylene glycol (PS-PG-Ald) carrying a terminal aldehyde group. Optimum immobilization occured at pH 8.0 and 25 °C, and at pH 7.0 and 35 °C for PS-PEHA-Ald and PS- PG-Ald, respectively. PS-PEHA-Ald immobilized enzyme retained approximately 75% of the initial activity over 45 days of storage, 70% of the initial activity after nine runs of recycling and displayed the better resistance to detrimental metal ions. PS-PG-Ald immobilized enzyme retained approximately 50% of the initial activity in 8 h at 70 °C. The catalytic efficiencies of PS-PEHA-Ald immobilized and PS-PG-Ald immobilized amylase were 1.42 and 1.29 times higher than that of native enzyme. The activation energy of the reaction mediated by the amylase was reduced by 58.1% and 57.3% when PS-PEHA-Ald and PS-PG- Ald used as support respectively. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Immobilization can greatly improve the operational properties of enzymes such as re-utilization, stability and separation from reaction products (Brady and Jordaan, 2009; Cadena et al., 2010; Paljevac et al., 2007). Various types of inorganic materials, syn- thetic polymers and macromolecules have been used as supports for immobilization of enzymes (Brady and Jordaan, 2009; Cadena et al., 2010; Ran et al., 2010). The support surface has an influence on the structure of the immobilized enzyme and thus on its ther- mal stability and catalytic activity of the immobilized enzyme. Suitable surfaces for adsorption and adhesion of enzyme create beneficial microenvironments that can improve the activity of en- zyme (Bayramoglu et al., 2011a,b; Yanagisawa et al., 2006). Direct attachment of enzymes to polymer surfaces can alter the structure of the enzyme in such a way that its reactivity is diminished. To overcome this drawback, spacer molecules have been employed to keep the enzyme molecules at a reasonable distance from the support surface to limit unfavorable interactions with the surface (Brady and Jordaan, 2009; Cao et al., 2007). Polyethylenimine (PEI), a polycationic polymer, which can establish a nondistorting and very strong ionic interaction with proteins (Mateo et al., 2000), has been used to coat the surface of support to optimize the adsorption of enzyme through the long and flexible spacer arms (Greene et al., 2005; Rocchietti et al., 2004; Yuan et al., 2009). Polyethylene glycol (PEG), a water-soluble and nontoxic long chain nonionic polymer, can provide a microen- vironment to the enzyme molecules similar to that within cells (Bayramoglu et al., 2011a,b). The addition of PEG into an enzyme solution may render the enzyme molecules more compact and ri- gid, resulting in stabilization against denaturing effects (Chae et al., 2000). Manrich et al. (2010) and Fatima and Khan (2007) used PEG to protect the enzyme during immobilization. The sur- faces coated or grafted with PEG exhibited improved hydrophilicity (Ko et al., 2001; Xu et al., 2005) and minized nonspecific adsorption to protein such as chloroperoxidase and bovine serum albumin (Bayramoglu et al., 2011a,b; Chen et al., 2008). The alcohol groups on PEG can be easily activated to allow selective and facile immo- bilization of proteins (Cha et al., 2004); however, since PEI and PEG merely adsorbed onto a surface can easily be lost along with the enzyme, covalent binding of PEI and PEG is desirable (Cadena et al., 2010; Gao et al., 2010). Polystyrene resin (PS), a close-to-spherical resin, has been employed in passive adsorption of proteins via hydrophobic 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.07.110 Abbreviations: PEI, polyethylenimine; PEG, polyethylene glycol; TAKA, a-amy- lase from Aspergillus oryzae; PS, polystyrene pellets; PEHA, pentaethylenehexamine; PG, pentaethylene glycol; Ald, aldehyde; PS-PEHA-Ald, PS pellets grafted with pentaethylenehexamine terminally activated with groups of aldehyde; PS-PG-Ald, PS pellets grafted with pentaethylene glycol terminally activated with groups of aldehyde. ⇑ Corresponding authors. Tel.: +86 510 82682316; fax: +86 510 85917763 (F. Wang). E-mail addresses: peakwf@hotmail.com, fwang@jiangnan.edu.cn (F. Wang), mingll@jiangnan.edu.cn (L.M. Liu). Bioresource Technology 102 (2011) 9374–9379 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech