Preparation of cross-linked enzyme aggregates (CLEA) of catalase and its characterization

In this study, bovine liver catalase was immobilized as cross-linked catalase aggregates (CLEA-CAT) via precipitation with ammonium sulfate and cross-linking with glutaraldehyde. CLEA-CAT with the highest activity was obtained for the immobilization conditions as ammonium sulfate concentration % 70 (w/v), glutaraldehyde concentration 140 mM, pH 7.0 and immobilization duration 6 hours. The highest activity was obtained from CLEA-CAT prepared with 10 mg/ml CAT + 10 mg/ml Bovine serum albumin (BSA). The thermal stabilities, storage stabilities and usage in a batch type reactor of CLEA-CAT preparations were determined. Also, the images of CLEA-CAT preparations were investigated at scanning electron microscope.The conditions as pH, buffer concentration, temperature in which free catalase and CLEA-CAT preparations showed their maximum activities were determined as 7.5, 50 mM, 25°C for the free CAT and 7.5, 75 mM, 35°C for the CLEA-CAT, respectively. For free catalase at optimum defined conditions, Km, Vmax, kcat and kcat/Km values were calculated as 50 mM, 1.98x104 U/mg prot., 7.92x104 s-1 and 1.58x106 s-1M-1 and Km and Vmax values of CLEA-CAT were calculated as 43 mM and 2.0x103 U/g CLEA-CAT, respectively. It was determined that CLEA-CAT saved 30 % of its initial activity for 15 hours preincubation at 50ºC. However, free catalase saved 4 % of its initial activity at the same conditions. Free catalase was stored for 19 days at 5°C and saved 11 % of its initial activity, CLEA-CAT was stored for 2 months at 5°C and saved 10 % of its initial activity. CLEA-CAT preparations saved 29 % of their initial activities in a batch type reactor at the end of 32 usages.As a result of study, it was concluded that CLEA-CAT?s thermal and storage stabilities were higher than that of free catalase and CLEA-CAT can be used for longer at extreme conditions.Keywords: Immobilization, Cross-linked enzyme aggregates, Catalase, Glutaraldehyde.