Yandri Yandri , Tati Suhartati , Heri Satria , Surtini Karlinasari , Suripto D. Yuwono , Sutopo Hadi

Abstract

This study aimed to analyze the influence of chitin as immobilization agent on the stability of purified α-amylase obtained from Bacillus subtilis ITBCCB148, which was immobilized the adsorption method. A series of characterization of immobilized and purified enzymes was conducted to determine optimum temperature, pH, K_M, V_max, thermal stability, and reusability of immobilized enzyme. The enzymatic activity of free and immobilized α-amylase was determined on the basis of glucose formed using Mandels method, and protein content using the Lowry method. The experimental data demonstrated that the immobilized α-amylase can be used up to five times and has an optimum temperature of 75°C, K_M=6.04 mg.mL⁻¹ substrate, and V_max=1277.14 µmol.mL⁻¹ min⁻¹. Stability test conducted at 65°C for 80min indicated that the immobilized enzyme has 76.26% residual activity, which is equivalent to the unit activity of 582.61 U/mL. Purified enzyme had an optimum temperature of 65°C, K_M of 1.9 mg/mL substrate, and V_max of 3,508.77 µmol/mL.min^-1. Stability test performed at 65°C for 80min indicated that the residual activity of the native enzyme results in a stability test at 65°C for 80 min was 15.50% with a unit activity of 42.34 U/mL. The immobilized enzyme had t_1/2-value=346.5 min, k_i=0.002 min⁻¹, and ΔG_i was 115.51 kJ.mol⁻¹, while for the purified enzyme, tha data obtained were t½=28.88 min, ki=0.024 min⁻¹, and ΔG_i=105.14 kJ.mol⁻¹. Increased stability of the enzyme as a result of immobilization was also indicated by decreased value of ki and increased values of ΔGi and t½‎.

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