School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China.
School of Biological Engineering, Dalian Polytechnic University, Dalian, 116034, China.
Protein Expr Purif. 2023 Aug;208-209:106277. doi: 10.1016/j.pep.2023.106277. Epub 2023 Apr 24.
Catalase, which catalyzes the decomposition of HO to HO and O, is widely used to reduce HO in industrial applications, such as in food processing, textile dyeing and wastewater treatment. In this study, the catalase (KatA) from Bacillus subtilis was cloned and expressed in the yeast Pichia pastoris X-33. The effect of the promoter in the expression plasmid on the activity level of the secreted KatA protein was also studied. First, the gene encoding KatA was cloned and inserted into a plasmid containing an inducible alcohol oxidase 1 promoter (pAOX1) or a constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (pGAP). The recombinant plasmids were validated by colony PCR and sequencing and then linearized and transformed into the yeast P. pastoris X-33 for expression. With the promoter pAOX1, the maximum yield of KatA in the culture medium reached 338.8 ± 9.6 U/mL in 2 days of shake flask cultivation, which was approximately 2.1-fold greater than the maximum yield obtained with the promoter pGAP. The expressed KatA was then purified from the culture medium by anion exchange chromatography, and its specific activity was determined to be 14826.58 U/mg. Finally, the purified KatA exhibited optimum activity at 25 °C and pH 11.0. Its K for hydrogen peroxide was 10.9 ± 0.5 mM, and its k/K was 5788.1 ± 25.6 s mM. Through the work presented in this article, we have therefore demonstrated efficient expression and purification of KatA in P. pastoris, which might be advantageous for scaling up the production of KatA for use in a variety of biotechnological applications.
过氧化氢酶（Catalase）能够催化 HO 分解为 HO 和 O，被广泛应用于工业领域，例如食品加工、纺织品染色和废水处理等。本研究中，我们从枯草芽孢杆菌（Bacillus subtilis）中克隆并表达了过氧化氢酶（KatA），并研究了表达载体中启动子对分泌型 KatA 蛋白活性水平的影响。首先，我们克隆了编码 KatA 的基因，并将其插入含有诱导型醇氧化酶 1 启动子（pAOX1）或组成型甘油醛-3-磷酸脱氢酶启动子（pGAP）的质粒中。通过菌落 PCR 和测序对重组质粒进行了验证，然后线性化并转化入毕赤酵母（Pichia pastoris）X-33 中进行表达。使用启动子 pAOX1，摇瓶培养 2 天，培养基中 KatA 的最大产量达到 338.8±9.6 U/mL，约是使用启动子 pGAP 时的 2.1 倍。随后，我们通过阴离子交换层析从培养基中纯化了表达的 KatA，其比活为 14826.58 U/mg。最后，纯化的 KatA 在 25°C 和 pH 11.0 时表现出最佳活性。其对过氧化氢的 K 为 10.9±0.5 mM，k/K 为 5788.1±25.6 s mM。通过本文的工作，我们在毕赤酵母中实现了 KatA 的高效表达和纯化，这可能有利于扩大 KatA 的生产规模，以应用于各种生物技术领域。
