Operation Modes Can Affect the Activity of Immobilized Enzyme onto Silk Fibroin Nanofibrous Membrane

Abstract

Note ] Int. J. Indust. Entomol. Vol. 27, No. (2), pp. 322-325 (2013) 322 323 Immobilization of CT on SF nanofibrous membrane In order to immobilize CT on the SF, the amine groups of SF were activated with glutaraldehye (GA). To one piece of SF nanofibrous membrane, 10% (v/v) GA in 0.2 M sodium carbonate buffer (pH 9.2) was added to activate the SF. The reaction was continued for 1 h at 25oC. The activated SF was washed 2 times with distilled water and 3 times with 0.1 M sodium phosphate buffers (pH 7.4). The activated SF was mounted on the membrane holder and an ice-cold buffer solution of CT (1 mg/mL) in 0.1 M sodium phosphate buffer (pH 7.4) was circulated overnight at 4oC. The unbound CT was washed out 20 mL of ice-cold 0.5 M of NaCl in 0.1 M sodium phosphate buffer (pH 7.4). Further it was washed 30 mL of the same buffer but without NaCl. Quantification and Activity Test of CT The bound CT was measured by bicinchoninic acid assay methods using bovine serum albumin as standard. The amount of bound CT was calculated by subtracting the amount of remaining CT from the initial amount of CT. N-benzoyl-DLtyrosine-p-nitroanilide hydrochloride (BTPNA) was used as substrate of CT. For general purpose, the substrate solution were prepared by mixing 5 mM BTPNA in DMSO, distilled water and 0.1 M sodium phosphate buffer (pH 7.4) in a ratio of 10:15:3, respectively. In order to measure the activity of the immobilized CT, the increase of absorbance at 400 nm was measured using UV spectrometer (UVICON 923, Kontron Instruments, USA). If the value of absorbance exceeded 2 then it was diluted until it reached below 1.5. The activity of immobilized CT was defined as μmol BTPNA hydrolyzed in 1 min by 1 mg of immobilized CT applying an absorbance coefficient of e400 = 9520 M -1 cm -1 . The Michaelis-Menten parameters was calculated by the Lineweaver– Burk plot after reacting enzyme immobilized SF nanofibrous membrane with the substrate solution having different BTPNA concentration. In this case, the final concentration of BTPNA was 0.0625, 0.125, 0.25, 0.5, 1.0 and 2.0 mM. Operation modes of immobilized CT The substrate solution was reacted with enzyme immobilized SF nanofibrous membrane in two modes. For the batch reactor mode, the enzyme immobilized SF nanofibrous membrane nanofibrous membrane and operated in two different systems. First, it was operated like a batch-type reactor, where the enzyme immobilized SF nanofibrous membrane was immersed into the substrate solution. Secondly, the enzyme immobilized SF nanofibrous membrane was mounted on the membrane holder and the substrate solution was passed through the membrane. We compared the Michaelis-Menten parameters of both operations and examined the effectiveness of the membrane reactor. Materials and Methods