Replacement of buried cysteine from zebrafish Cu/Zn superoxide dismutase and enhancement of its stability via site-directed mutagenesis.

摘要: Zebrafish Cu/Zn-superoxide dismutase (ZSOD1) has one free cysteine (Cys-7) in a first β-strand with lower thermostability. We predicted the stability would be increased single-point mutation at 70°C via I-Mutant 2.0 server, and generated mutant SOD replacement of Cys to Ala (ZSODC7A) by site-directed mutagenesis. The was expressed purified from Escherichia coli strain AD494(DE3)pLysS yield 2 mg 0.4 L culture. ZSODC7A heated 90°C. In time-dependent assay, time interval for 50% inactivation 32 min, its thermal rate constant Kd × 10−2 min−1. still activated broad pH range (2.3–12), had only moderate effect under sodium dodecyl sulfate treatment. calculated specific activity 3980 U/mg, twice that wild-type ZSOD1. addition, we soaked fish larva equal enzyme units either ZSOD1 or h, then stressed them 100 ppm paraquat induce oxidative injury. survival significant.