Enzyme and Microbial Technology 31 (2002) 371–383 Research papers Immobilization of Aspergillus oryzae -galactosidase on tosylated cotton cloth Nedim Albayrak, Shang-Tian Yang ∗ Departments of Chemical Engineering and Food Science and Technology, The Ohio State University, 140 West 19th Avenue, Columbus, OH 43210, USA Received 27 July 2001; received in revised form 28 January 2002; accepted 31 January 2002 Abstract Immobilization of Aspergillus oryzae -galactosidase on cotton cloth activated with p-toluenesulfonyl chloride (tosyl chloride) was studied. Enzyme immobilization on the tosylated cotton followed a nucleophilic substitution mechanism as evidenced by UV spectra. NaOH mercerization before tosylation and adding pyridine at a tosyl (g)/pyridine (ml) ratio of 0.2–0.4 increased the final activity of the immobilized enzyme by six-fold. The optimal pH for enzyme immobilization was found to be 4.5. Among different fibrous matrices tested, the knitted cotton cloth showed the highest immobilized enzyme activity. About 50 mg enzyme was immobilized onto each gram of cotton cloth with a protein coupling efficiency of ∼85% and enzyme activity yield of ∼55%. The immobilized enzyme had half-life of ∼50 days at 50 ◦ C, and more than 1 year at 40 ◦ C, an improvement of 25–28-fold as compared to free enzyme. The enzyme immobilized on cotton cloth can be used in a plug-flow reactor for continuous production of galacto-oligosaccharides (GOS) from lactose. The reactor performance was stable, and there was no loss in enzyme activity at 40 ◦ C for the 15-day period studied. The immobilized enzyme also showed same pattern and level of GOS formation from lactose as those from free enzyme reaction, indicating that the reaction kinetics was not affected by immobilization and there was no significant diffusion limitation in the immobilized enzyme reactor. The method to prepare tosylated cotton cloth for enzyme immobilization is simple, inexpensive, and scaleable for industrial applications. Thus, tosylated cotton cloth can be used as a low cost support for highly active and stable biocatalyst. © 2002 Elsevier Science Inc. All rights reserved. Keywords: -Galactosidase; Immobilized enzyme; Tosyl chloride; Cotton cloth 1. Introduction Enzyme immobilization provides easy recovery and reuse of the enzyme and many other advantages, including ease in product separation and continuous operation [1]. For successful development and application of an immobi- lized biocatalyst, the enzyme support is generally consid- ered as the most important component contributing to the performance of the reactor system. Although continuous immobilized enzyme reactors give higher productivities, minimize downtime, enzyme costs, and capital investment, large-scale applications of immobilized enzymes are rare [2] largely because the support materials used for enzyme immobilization are either too expensive or difficult to use in industrial scale. Therefore, development of new techniques for enzyme immobilization on inexpensive and industrially applicable carriers is of economical significance. Fibrous matrices with high porosity, open structures and high me- chanical strength have long been an interest in biocatalysis ∗ Corresponding author. Tel.: +1-614-292-6611; fax: +1-614-292-3769. E-mail address: yang.15@osu.edu (S.-T. Yang). due to their advantages over particulate materials, which include high specific surface area, low pressure drop, and negligible mass-transfer resistance [3,4]. Various fibrous materials, including polyethylene terephythalate (PET) fibers [5], dimethylated superfine fibers [4,6,7], cotton fab- rics [3,8], nylon fiber [9], non-woven nylon fabrics [10], silk fibers [11], silk fabrics [12–14], porous glass fiber [15], and hydrophobic hollow fiber [16,17] have been success- fully used for enzyme immobilization. However, various problems associated with these supports were also reported. One common problem in using fibrous polymeric materi- als for enzyme immobilization is the lack of active sites on the polymer. Consequently, most previous studies used glutaraldehyde as a non-specific cross-linking agent to fix the enzyme on the polymeric matrix, but the results were often unsatisfactory—with low immobilization efficiency and low final enzyme activity [13–15]. The main objective of this study was to explore the effectiveness of cotton cloth as a fibrous matrix for the development of a fibrous-bed biocatalyst reactor. Knitted cotton fabric such as terry cloth is inexpensive and widely available, and has been successfully used in several cell 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00115-1