Immobilization of Aspergillus oryzae β-galactosidase on tosylated cotton cloth

Immobilization of Aspergillus oryzae β-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.