过氧化氢酶-无机杂化纳米花的制备及催化特性

Preparation and catalytic properties of catalase-inorganic hybrid nanoflowers

摘要:

过氧化氢酶(catalase,CAT)是一种在食品、医疗、纺织等领域广泛应用的工业酶，具有催化效率高、专一性强、绿色环保等突出特点。工业中游离过氧化氢酶无法回收再利用，导致以其为核心的工业生物转化过程成本较高。开发一种简单、温和、低成本并且体现绿色化学理念的方法对过氧化氢酶进行固定化有望提高其利用率并且强化酶学性能，具有迫切的现实需求。本研究将源自枯草芽孢杆菌(Bacillus subtilis)168的过氧化氢酶KatA在大肠杆菌中进行重组表达，之后将分离纯化得到的纯酶以酶-无机杂化纳米花形式制备成固定化酶并进行酶学性质研究。结果显示，利用乙醇沉淀、DEAE阴离子交换层析、疏水层析3步纯化，最终获得电泳纯的重组KatA，之后通过优化制备条件获得了一种新型KatA/Ca₃(PO₄)₂杂化纳米花固定化酶。酶学性质研究结果显示，游离酶KatA的最适反应温度为35℃，KatA/Ca₃(PO₄)₂杂化纳米花的最适反应温度为30−35℃，二者最适反应pH值均为11.0。游离酶KatA和KatA/Ca₃(PO₄)₂杂化纳米花在pH4.0−11.0和25−50℃条件下均表现出较好的稳定性。KatA/Ca₃(PO₄)₂杂化纳米花显示出比游离酶KatA更好的储存稳定性，在4℃储存14d后仍保留82%的酶活力，而游离酶仅具有50%的酶活力。此外，纳米花在进行5次催化反应后仍具有55%的酶活力，表明其具有较好的操作稳定性。动力学研究结果显示，游离酶KatA对底物过氧化氢的K_m为(8.80±0.42)mmol/L，k_cat/K_m为(13151.53±299.19)L/(mmol·s)；而KatA/Ca₃(PO₄)₂杂化纳米花的K_m为(32.75±2.96)mmol/L，k_cat/K_m为(4550.67±107.51)L/(mmol·s)。与游离酶KatA相比，KatA/Ca₃(PO₄)₂杂化纳米花对底物过氧化氢的亲和力下降，同时其催化效率也有所降低。综上所述，本研究以Ca²⁺作为自组装诱导剂，成功将KatA以酶-无机杂化纳米花形式制备成固定化酶，不仅对部分酶学性能实现了强化，而且为固定化过氧化氢酶的绿色制备和规模化应用奠定了基础。

Abstract:

Catalase is widely used in the food, medical, and textile industries. It possesses exceptional properties including high catalytic efficiency, high specificity, and environmental friendliness. Free catalase cannot be recycled and reused in industry, resulting in a costly industrial biotransformation process if catalase is used as a core ingredient. Developing a simple, mild, cost-effective, and environmentally friendly approach to immobilize catalase is anticipated to improve its utilization efficiency and enzymatic performance. In this study, the catalase KatA derived from Bacillus subtilis 168 was expressed in Escherichia coli. Following separation and purification, the purified enzyme was prepared as an immobilized enzyme in the form of enzyme-inorganic hybrid nanoflowers, and the enzymatic properties were investigated. The results indicated that the purified KatA was obtained through a three-step procedure that included ethanol precipitation, DEAE anion exchange chromatography, and hydrophobic chromatography. Then, by optimizing the process parameters, a novel KatA/Ca₃(PO₄)₂ hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35℃, the optimum reaction temperature of KatA/Ca₃(PO₄)₂ hybrid nanoflowers was 30−35℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca₃(PO₄)₂ hybrid nanoflowers exhibited excellent stability at pH 4.0−11.0 and 25−50℃. The KatA/Ca₃(PO₄)₂ hybrid nanoflowers demonstrated increased storage stability than that of the free KatA, maintaining 82% of the original enzymatic activity after 14 d of storage at 4℃, whereas the free KatA has only 50% of the original enzymatic activity. In addition, after 5 catalytic reactions, the nanoflower still maintained 55% of its initial enzymatic activity, indicating that it has good operational stability. The K_m of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the k_cat/K_m was (13 151.53±299.19) L/(mmol·s). The K_m of the KatA/Ca₃(PO₄)₂ hybrid nanoflowers was (32.75±2.96) mmol/L, and the k_cat/K_m was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca₃(PO₄)₂ hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca₃(PO₄)₂ hybrid nanoflowers using Ca²⁺ as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.