Molecular characterization, purification, and antioxidant activity of recombinant superoxide dismutase from the Pacific abalone Haliotis discus hannai Ino

Jingna Wu,Chunhua Fang,Hui Peng,Bei Chen,Shuji Liu,Nan Pan,Hua Hao,Zhiyu Liu,Min Xu,Kun Qiao

doi:10.1007/s11274-020-02892-5

Abstract

Superoxide dismutase (SOD) is an acidic metalloenzyme that scavenges free radicals produced by endogenous and exogenous substances. In the present study, the tissue distribution of the superoxide dismutase HdhCu/Zn-SOD was investigated in Haliotis discus hannai Ino. The expression profile after lipopolysaccharide (LPS) challenge was determined using quantitative real-time polymerase chain reaction (qPCR). To study the antioxidant activity of a recombinant HdhCu/Zn-SOD protein, the HdhCu/Zn-SOD gene was cloned into the pPIC9K vector and transformed into the Pichia pastoris GS115 strain by electroporation. After induction by methanol, the recombinant product was purified using immobilized metal affinity chromatography and confirmed using mass spectrometry. The optimal expression conditions were determined to be incubation with 0.5% methanol at pH 6.0, resulting in a stable expressed product with the molecular weight of approximately 17 kDa and 21 kDa. The enzymatic activity of HdhCu/Zn-SOD consistently increased with increasing Cu2+ concentrations and showed good thermal stability. Recombinant HdhCu/Zn-SOD showed a strong ability to scavenge superoxide anions and hydroxyl radicals and protected L929 cells against the toxicity caused by H2O2 through its in vitro antioxidant activity. The heterologous expression of HdhCu/Zn-SOD in P. pastoris and the antioxidant activity of this enzyme are reported for the first time.Graphic abstract

Highlights

Marine organisms exposed to a complex aquatic environment show increased antioxidant responses
The HdhCu/Zn-Superoxide dismutase (SOD) gene transcript was overexpressed in the digestive gland at 48 h (3.86-fold vs the control, p = 0.003) and maintained high expression until 96 h postLPS injection (2.17-fold vs the control, p = 0.033) (Fig. 2)
The mRNA expression pattern was different in hemocytes after LPS challenge, with upregulation observed at 96 h (1.87-fold vs the control, p = 0.003)

Summary

Introduction

Marine organisms exposed to a complex aquatic environment show increased antioxidant responses. Reactive oxygen species (ROS) are oxygen free radicals that are generated during aerobic cellular metabolism, such as superoxide anions (O2-), hydroxyl radicals (.OH), single oxygen (.O2), and hydrogen peroxide (H2O2) (Genestra 2007). Many types of environmental stimuli, including ultraviolet light, environmental toxins, and hyperthermia, generate high levels of ROS, resulting in cellular oxidative damage. ROS can degrade unsaturated fatty acids on the cell membrane and destroy the membrane structure (Sargis and Subbaiah 2006), leading to the cleavage of polypeptide chains (Stadtman and Levine 2003) and the formation of single-strand breaks in nuclear DNA (Melidou et al 2005). To avoid cellular oxidative damage and prevent disease and death caused by oxidative stress, marine organisms develop an effective defense system to maintain the balance of ROS. The antioxidant system of mollusks includes enzymatic antioxidants such as superoxide dismutase (SOD, EC1.15.1.1), catalase, thioredoxin peroxidase, glutathione peroxidase, and glutathione-S-transferase and nonenzymatic antioxidants such as selenium-binding protein, metallothionein, thioredoxin, ferritin, and transferrin

Methods

Results

Conclusion