Immobilization of -Galactosidase on Fibrous Matrix by Polyethyleneimine for Production of Galacto-Oligosaccharides from Lactose Nedim Albayrak and Shang-Tian Yang* Departments of Chemical Engineering and Food Science and Technology, The Ohio State University, 140 West 19th Avenue, Columbus, Ohio 43210 The production of galacto-oligosaccharides (GOS) from lactose by Aspergillus oryzae -galactosidase immobilized on cotton cloth was studied. A novel method of enzyme immobilization involving PEI-enzyme aggregate formation and growth of aggregates on individual fibrils of cotton cloth leading to multilayer immobilization of the enzyme was developed. A large amount of enzyme was immobilized (250 mg/g support) with about 90-95% efficiency. A maximum GOS production of 25-26% (w/w) was achieved at near 50% lactose conversion from 400 g/L of lactose at pH 4.5 and 40 °C. Tri- and tetrasaccharides were the major types of GOS formed, accounting for about 70% and 25% of the total GOS produced in the reactions, respectively. Temperature and pH affected not only the reaction rate but also GOS yield to some extend. A reaction pH of 6.0 increased GOS yield by as much as 10% compared with that of pH 4.5 while decreased the reaction rate of immobilized enzyme. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme under the same reaction conditions, suggesting diffusion limitation was negligible in the packed bed reactor and the enzyme carrier. Increase in the thermal stability of PEI-immobilized enzyme was also observed. The half-life for the im- mobilized enzyme on cotton cloth was close to 1 year at 40 °C and 21 days at 50 °C. Stable, continuous operation in a plug-flow reactor was demonstrated for about 3 days without any apparent problem. A maximum GOS production of 26% (w/w) of total sugars was attained at 50% lactose conversion with a feed containing 400 g/L of lactose at pH 4.5 and 40 °C. The corresponding reactor productivity was 6 kg/L/h, which is several-hundred-fold higher than those previously reported. Introduction Biocatalyst immobilization is gaining increased atten- tion for the synthesis of industrial bioproducts ranging from neutraceuticals to chemicals. Enzyme immobiliza- tion provides many important advantages over use of enzymes in soluble form, namely, enzyme reusability, continuous operation, controlled product formation, and simplified and efficient processing. The main challenges in enzyme immobilization include not only containment of a large amount of enzyme to be immobilized while retaining most of its initial activity but also the perfor- mance of immobilized enzyme in actual production processes in industrial-type reactors. Thus, the success of immobilized enzyme is not only driven by its applica- tions but also relies on a number of factors, including enzyme, support, chemical reagent, and reactor. The enzyme support is generally considered as the most important component contributing to the performance of the immobilized biocatalyst reactor. In addition to being a very inexpensive and widely available fibrous material, cotton cloth provides a number of desirable characteris- tics, including high porosity (>95%), large specific surface area, and excellent mechanical strength. Cotton cloth has been successfully used in cell immobilization and fer- mentation studies (1-4). Cotton cloth immobilized en- zyme placed in a loose spiral shape in a plug-flow-type reactor provides good flow rates, low pressure drop, and negligible mass transfer resistance. These characteristics are also highly desirable for industrial enzyme applica- tion. Thus, cotton fabric also can be used for the develop- ment of an industrially applicable fibrous bed enzyme bioreactor where the immobilized enzyme functions as good as soluble enzyme. Although enzymes can be immobilized on cotton cloth activated with tosyl chloride, the method is somewhat tedious and involves the use of organic chemicals (5). The goal of this research was to develop the method of using polyethyleneimine (PEI) for enzyme immobilization in fibrous matrices that can be used in a fibrous-bed biocatalyst reactor. PEI, an extremely branched cationic chain polymer (6), has many applications in biochemistry because of its electrostatic interaction with negatively charged species (7). PEI has been an essential ingredient of many enzyme immobilization procedures, where it serves to coat an inert support such as porous glass microbeads (8) or charged insoluble carriers (9, 10). Cotton cloth coated with PEI has been used as a support for immobilization of several enzymes, including glucose oxidase (11), urease (12, 13), and invertase (14), and yeast cells (15). In these applications, PEI was adsorbed on the cotton cloth and then excess PEI was washed away with * To whom correspondence should be addressed. Ph: (614) 292- 6611. Fax: (614) 292-3769. E-mail: yang.15@osu.edu. 240 Biotechnol. Prog. 2002, 18, 240-251 10.1021/bp010167b CCC: $22.00 © 2002 American Chemical Society and American Institute of Chemical Engineers Published on Web 03/19/2002