Abstract
Previously, a new copper-zinc SOD (CuZnSOD) isolated from chestnut rose (Rosa roxburghii) with good stability was described. In this study, the biosynthetic approach was used to create recombinant CuZnSOD. RACE PCR was also used to amplify the full-length CuZnSOD gene from chestnut rose, and the ORF segment was expressed in E. coli BL21 and P. pastoris GS115. For characterization, the enzyme was isolated in two steps in E. coli and one step in P. pastoris. The biochemical properties of the two recombinant enzymes were similar, and their optimal reaction pH and temperature were 6.0 and 50°C, respectively. According to molecular dynamics simulation, the CuZnSOD showed high stability from 70 to 90°C, and eight amino acids are important for enzyme thermal stability at high temperatures. This study set the stage for industrial manufacture by filling gaps in the link between conformational changes and the thermal stability of the new CuZnSOD.
Highlights
Because of its low cost and quick production cycle, synthetic biology is already widely used in industries such as medicine, diagnostics, and food [1]
reactive oxygen species (ROS) are a class of chemicals and free radicals created by living organisms during aerobic respiration [6]
PCR was performed to get partial genes of CuZnSOD from chestnut rose using primers designed with conserved sequences (Figure 1A) of the Rosaceae family
Summary
Because of its low cost and quick production cycle, synthetic biology is already widely used in industries such as medicine, diagnostics, and food [1]. Several microorganisms were chosen as host cells for protein production due to their rapid growth and expression [1, 2]. E. coli is widely used as a host cell owing to its high genetic properties and versatile cloning tools [3]. P. pastoris is the most widely used eukaryotic expression system in industry due to its capacity to grow at extremely high cell densities on minimal medium and efficient secretory expression [5]. The body has evolved an antioxidant system to maintain the concentration of ROS, and SOD is the first line of defense against free radicals. CuZnSOD is the most important antioxidant enzyme in Production a Novel Superoxide Dismutase the SOD family, which is found mostly in eukaryotic cells’ cytoplasm, nucleus, peroxisome, and mitochondrial membrane space, as well as bacteria’s periplasmic space [10]
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