Enzyme activity of CAT2 in prokaryotic co-expression of CAT2 and NCA1

Journal Information

J. Hefei Univ. Tech. (Nat. Sci.), Feb.2025, Vol.48 No.2: 236-240

DOI: 10.3969/j.issn.1003-5060.2025.02.014

Authors

ZHANG Ruihan $ ^{1} $, HAN Yi $ ^{2} $

(1. School of Food and Biological Engineering, Hefei University of Technology, Hefei 230601, China; 2. School of Life Sciences, Anhui Agricultural University, Hefei 230036, China)

Abstract and Keywords

Abstract: Catalase 2(CAT2) is a key enzyme that controls redox levels and regulates hydrogen peroxide signal transduction in plants. NCA1 is an important molecular chaperone of CAT2 in plants, protecting CAT2 from degradation and helping it function normally. Most of the current prokaryotic expression studies only express CAT2 protein. In order to solve this problem, prokaryotic expression vectors pMAL-p2x-CAT2 and PET28a(+)-NCA1 were constructed using CAT2 and NCA1 genes in Arabidopsis thaliana. After transferred into the same Escherichia coli competent cell BL21(DE3), co-expression of CAT2 protein in supernatant after cell destruction was successfully obtained. The study of the relevant enzymatic properties showed that the optimal temperature of CAT2 was 20 °C, and the optimal pH value was 8. The enzyme activity of CAT2 was 580 U/mg, five times that of CAT2 alone, and the protein concentration was 0.42 $ \mu $g/ $ \mu $L. NO is a signal molecule that can perform nitrosation modification on protein in plants. On the basis of successfully expressing CAT2, NO donor S-nitrosoglutathione(GSNO) was used to treat it. It was found that the enzyme activity of CAT2 was inhibited after NO treatment, and the enzyme activity was recovered after treatment with the reducing agent dithiothreitol(DTT). It is speculated that NO may regulate the enzyme activity of CAT2 through nitrosation modification, thus indirectly affecting the state of intracellular environment.

Keywords: catalase; NCA1 gene; prokaryotic expression; carbon monoxide; nitrosation

作者信息

张瑞含 $ ^{1} $，韩毅 $ ^{2} $

(1. 合肥工业大学食品与生物工程学院, 安徽合肥 230601; 2. 安徽农业大学生命科学学院, 安徽合肥 230036)

摘要和关键词

摘要: 植物过氧化氢酶2(CAT2)是控制植物体内氧化还原水平、调控过氧化氢信号传递的关键酶。在植物体内NCA1是CAT2重要的分子伴侣,可以保护CAT2不被降解并发挥其正常功能。在目前的原核表达研究中,大部分研究仅单独表达了CAT2蛋白。文章利用拟南芥CAT2和NCA1基因的全编码序列构建了原核表达载体pMAL-p2x-CAT2和PET28a(+)-NCA1,转入同一大肠杆菌感受态细胞BL21(DE3)后,共表达成功获得了存在于破细胞后上清液内的CAT2蛋白。通过对相关酶学性质的研究,发现CAT2的最适温度为20℃,最适pH值为8,酶活高达580 U/mg,是单独表达CAT2酶活的5倍,蛋白质量浓度为0.42 $ \mu $g/ $ \mu $L。NO是植物体内一种可以对蛋白进行亚硝基化修饰的信号分子,在成功表达出CAT2基础上利用NO供体亚硝基谷胱甘肽(GSNO)进行处理,发现NO处理后CAT2的酶活受到抑制,利用还原剂二硫苏糖醇(DTT)处理后可以使酶活恢复,推测NO可能通过亚硝基化修饰CAT2调控其酶活力,从而间接地影响细胞内环境的状态。

关键词: 过氧化氢酶；NCA1基因；原核表达；一氧化碳；亚硝基化

Funding

安徽省自然科学基金面上资助项目（2208085MC44）

合肥工业大学学报(自然科学版)

Navigation