Immobilization of Subtilisin Carlsberg and its use for transesterification of N-acetyl-L-phenylalanine ethyl ester in organic medium.

Abstract

In this study, inorganic-based carrier perlite (PER) and cyclodextrin-modified perlite (PER-CD) were used for Subtilisin Carlsberg (SC) immobilization. For enzyme immobilization, the supports aminated with 3-aminotriethoxysilane were first activated with glutaraldehyde (GA) and genipin (GE), and then, the immobilized enzymes (PER-SC and PER-CD-SC) were obtained. The reaction medium for SC immobilization consisted of 500mg carrier and 5ml (1mg/ml) enzyme solution. The immobilization conditions were pH 8.0, 25°C, and 2h incubation time. Free and immobilized SC were used for transesterification of N-acetyl-L-phenylalanine ethyl ester (APEE) with 1-propanol in tetrahydrofuran (THF). The transesterification activity of the enzyme and the yield of the transesterification reaction were determined by gas chromatography (GC). 50mg of immobilized or 2.5mg of free SC was added to the reaction medium, which was prepared as 1mmol APEE and 10mmol alcohol in 10mL of THF. The conditions for the transesterification reaction were 60°C and 24h of incubation. The structure and surface morphology of the prepared carriers were characterized using scanning electron microscopy (SEM) and thermogravimetric analysis (TGA). Casein substrate was used in the optimization study. The optimum temperature and pH for SC activity were found to be 50°C and pH 8.0, respectively, for free and immobilized SC. The thermal stability of immobilized SC was found to be greater than that of free SC. At the end of 4h of exposure to high temperature, the immobilized enzyme maintained its activity at approximately 50%, while the free enzyme was maintained at approximately 20%. However, modification with cyclodextrin did not alter the thermal stability. The transesterification yield was found to be approximately 55% for the free enzyme, while it was found to be approximately 68% and 77% for PER-SC and PER-CD-SC, respectively. The effect of metal ions and salts on transesterification yield was examined. The results showed that the addition of metal ions decreased the percentage of transesterification by approximately 10% compared to the control group, whereas the addition of salt significantly decreased the percentage of transesterification by 60-80% compared to the control group.