Abstract: SOD is an important functional protein and industrial enzyme, and its source is a hot topic in the field. Here hSOD1 and bosgrunniens SOD1 were analyzed by bioinformatics firstly, and Mt1SOD1 (E25G, P29T, E101V, C112S) and Mt2SOD1 (I18T, N20H, K31V, C112S) were obtained by site-directed mutagenesis.Condons-optimized hSOD1, Mt1SOD1, Mt2SOD1, and bosgrunniens SOD1 were totally synthesized and then cloned into pET-28a(+) to achieve recombinant plasmid for transforming into E. coli BL21(DE3). The four kinds of SOD1 were induced and expressed successfully under the conditions of 1 mmol/LIPTG, 800 μmol/L Cu2+ and 20 μmol/L Zn2+ in LB medium at 25 ℃ and 180 r/min for 16 h. Next, Ni-NTA was used to purify four recombinant SOD1s. The activity of hSOD1 obtained from 100 mL LB was 71094 U/mg, and the yield was 4.57 mg. The activity of Mt1SOD1 was 128506 U/mg, and the yield was 3.13 mg. The activity of Mt2SOD1 was 58700.1 U/mg, and the yield was 5.47 mg. The activity of bos grunniens SOD1 was 42969.5 U/mg, and the yield was 6.81 mg. The enzymatic activity of hSOD1 and Mt1SOD1 remained no change between 25 ℃ and 55 ℃, and the relative enzymatic activity of hSOD1 remained about 50% after incubation at 75 ℃ for 30 minutes, and that of Mt1SOD1 remained 30%. Under the condition of pH 3.6-10.4 for 30 min, both SOD1s can maintain more than 70% of the enzyme activity. hSOD1 and Mt1SOD1 with high activity and high stability were obtained by site-directed mutagenesis, which laid the foundation for the application of SOD1 in medical, health care, cosmetics and other fields.

Key words: Cu/Zn superoxide dismutase, Escherichia coli, site-directed mutagenesis, recombinant expression and purification, activity