Pang Jiao, Jiang Mengtong, Liu Yuxin, Li Mingyu, Sun Jiaming, Wang Conggang, Li Xianzhen
School of Biological Engineering, Dalian Polytechnic University, Dalian 116034, Liaoning, China.
Sheng Wu Gong Cheng Xue Bao. 2022 Dec 25;38(12):4705-4718. doi: 10.13345/j.cjb.220381.
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 168 was expressed in . 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/Ca(PO) hybrid nanoflower was developed. The optimum reaction temperature of the free KatA was determined to be 35 ℃, the optimum reaction temperature of KatA/Ca(PO) hybrid nanoflowers was 30-35 ℃, and the optimum reaction pH of both was 11.0. The free KatA and KatA/Ca(PO) hybrid nanoflowers exhibited excellent stability at pH 4.0-11.0 and 25-50 ℃. The KatA/Ca(PO) 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 of the free KatA to the substrate hydrogen peroxide was (8.80±0.42) mmol/L, and the / was (13 151.53± 299.19) L/(mmol·s). The of the KatA/Ca(PO) hybrid nanoflowers was (32.75±2.96) mmol/L, and the / was (4 550.67±107.51) L/(mmol·s). Compared to the free KatA, the affinity of KatA/Ca(PO) hybrid nanoflowers to the substrate hydrogen peroxide was decreased, and the catalytic efficiency was also decreased. In summary, this study developed KatA/Ca(PO) hybrid nanoflowers using Ca as a self-assembly inducer, which enhanced the enzymatic properties and will facilitate the environmentally friendly preparation and widespread application of immobilized catalase.
过氧化氢酶广泛应用于食品、医疗和纺织工业。它具有优异的特性，包括高催化效率、高特异性和环境友好性。游离过氧化氢酶在工业中无法回收再利用，若将过氧化氢酶用作核心成分，会导致工业生物转化过程成本高昂。开发一种简单、温和、经济高效且环境友好的方法来固定化过氧化氢酶，有望提高其利用效率和酶性能。在本研究中，源自168的过氧化氢酶KatA在……中表达。经过分离和纯化后，将纯化的酶制备成酶-无机杂化纳米花形式的固定化酶，并对其酶学性质进行了研究。结果表明，纯化的KatA通过乙醇沉淀、DEAE阴离子交换色谱和疏水色谱三步法获得。然后，通过优化工艺参数，开发出了一种新型的KatA/Ca(PO)杂化纳米花。游离KatA的最佳反应温度为35℃，KatA/Ca(PO)杂化纳米花的最佳反应温度为30 - 35℃，两者的最佳反应pH均为11.0。游离KatA和KatA/Ca(PO)杂化纳米花在pH 4.0 - 11.0和25 - 50℃下表现出优异的稳定性。KatA/Ca(PO)杂化纳米花的储存稳定性比游离KatA有所提高，在4℃储存14天后仍保持82%的原始酶活性，而游离KatA仅保留50%的原始酶活性。此外，经过5次催化反应后，纳米花仍保持其初始酶活性的55%，表明它具有良好的操作稳定性。游离KatA对底物过氧化氢的Km为(8.80±0.42) mmol/L，Vmax/Km为(13 151.53±299.19) L/(mmol·s)。KatA/Ca(PO)杂化纳米花的Km为(32.75±2.96) mmol/L，Vmax/Km为(4 550.67±107.51) L/(mmol·s)。与游离KatA相比，KatA/Ca(PO)杂化纳米花对底物过氧化氢的亲和力降低，催化效率也降低。综上所述，本研究以Ca为自组装诱导剂开发了KatA/Ca(PO)杂化纳米花，增强了酶学性质，将有助于固定化过氧化氢酶的环境友好制备和广泛应用。
