Abstract

In this study, keratinase was partially purified by thermal precipitation then, entrapped and cross linking via glutaraldehyde into Na-Alginate, and examined the properties of the immobilized enzyme. The molecular weight of the enzyme was found as 63 kDa. Immobilization was achieved with 83.76% yield. Reusability of immobilized enzyme was determined as 12 times with 90% residual activity. Maximum keratinolitic activity time was measured as 40 minutes. Optimum pH for free keratinase was 8.0, for immobilized keratinase was 6.0, however, optimum temperature did not change both free and immobilized keratinase. The Michaelis–Menten constant (K_m) were found as 2.5 × 10⁻³ M and 0.011 M for free and immobilized keratinase, respectively. The maximum reaction rate (V_max) for the free and immobilized keratinase was calculated as 83.33 U mL⁻¹min⁻¹ and 12.5 10.83 U mL⁻¹min⁻¹, respectively. Furthermore, The turnover number (k_cat) and catalytic performance (k_cat/K_m) of enzymes were 58.8 min⁻¹ and 1.96 × 104 min⁻¹M⁻¹ for the free enzyme, 9.80 min⁻¹ and 8.91 × 102 min⁻¹M⁻¹ for the encapsulated enzyme, respectively. Thermodynamic parameters of free enzyme; ΔG^#: 6.62 × 104 kJ mol⁻¹; ΔG^# _E-T: −2.57 × 104 kJ mol⁻¹; ΔG^#_ES: −1.51 × 104 kJ mol⁻¹; ΔH^#: −2.60 × 103 kJ mol⁻¹; ΔS^#: −2.20 × 102 kJ mol⁻¹ K⁻¹ and the thermodynamic parameters of the encapsulated enzyme ΔG^#: 7.08 × 104 kJ mol⁻¹; ΔG^#_E-T: −1.77 × 104 kJ mol⁻¹; ΔG^#_ES: −1.17 × 104 kJ mol⁻¹; ΔH^#: −2.60 × 103 kJ mol⁻¹; ΔS^#: −2.35 × 102 kJ mol⁻¹ K⁻¹.

摘要

在本研究中，角蛋白酶通过热沉淀部分纯化，然后通过戊二醛捕获并交联成海藻酸钠，并检查固定化酶的性质。发现酶的分子量为63 kDa。以 83.76% 的产率实现了固定化。固定化酶的重复使用率为 12 次，残留活性为 90%。最大角蛋白活性时间测量为 40 分钟。游离角蛋白酶的最佳 pH 值为 8.0，固定角蛋白酶为 6.0，然而，最适温度不会改变游离角蛋白酶和固定角蛋白酶。发现游离角蛋白酶和固定角蛋白酶的 Michaelis-Menten 常数 (K _m ) 分别为 2.5 × 10 ^-3 M 和 0.011 M。最大反应速率（V _max)对于游离和固定的角蛋白酶分别计算为83.33 U mL ^-1 min ^-1和12.5 10.83 U mL ^-1 min ^-1。此外，转换数（K_猫）和催化性能（K_猫/ K_米酶）分别为58.8分钟^-1和1.96×104分钟^-1中号^-1对于游离酶，9.80分钟^-1和8.91×102分钟^-1 M ^-1分别用于封装的酶。游离酶的热力学参数；ΔG ^# : 6.62 × 104 kJ mol ^-1；ΔG ^# _ET: -2.57 × 104 kJ 摩尔^-1 ; ΔG ^# _ES : -1.51 × 104 kJ mol ^-1 ; ΔH ^# : -2.60 × 103 kJ mol ^-1 ; ΔS ^# : -2.20 × 102 kJ mol ^-1 K ^-1和封装酶的热力学参数ΔG ^# : 7.08 × 104 kJ mol ^-1；ΔG ^# _ET : -1.77 × 104 kJ mol ^-1 ; ΔG ^# _ES : -1.17 × 104 kJ mol ^-1 ; ΔH ^# : -2.60 × 103 kJ mol ^-1 ; ΔS ^#：-2.35 × 102 kJ mol ^-1 K ^-1。