Ferrocene-coupled chitosan (CS-Fc) support polymer was synthesized for the immobilization of double enzymes and single enzymes. The immobilization study was carried out after a detailed characterization study. The binding of pepsin and lipase enzymes to the polymer was supported by the shift of peaks in the FTIR spectra of free enzymes. The particle sizes of chitosan (CS) and CS-Fc were evaluated using zeta potential measurement. The optimum conditions of the enzymes in the double enzyme system did not affect each other and each showed their activity. In the presence of double enzyme immobilization, the optimum pH remained the same with free enzyme, while the optimum temperature increased differently with an increase of 10 °C. Vmax values were 0.0020 and 0.0038 for free pepsin and lipase enzyme, respectively. However, due to double enzyme immobilization, the Vmax value increased 1.75 fold for pepsin and 3.94 fold for lipase enzyme. In the double enzyme immobilization, the substrate affinity of pepsin increased by 0.4-fold, while the substrate affinity of lipase increased by about 0.3-fold. In the 5th reuse of pepsin and lipase enzymes in the presence of double enzymes (Cs-Fc-Pep) + (Cs-Fc-Lip), it was determined as 18.21% for pepsin enzyme and 90.81% for lipase enzyme.
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