小球藻病毒PBCV-1编码的Cu,Zn-SOD酶学稳定性

Enzymatic stability of Cu, Zn-SOD encoded by chlorovirus PBCV-1

将小球藻病毒模式株PBCV-1编码的Cu, Zn-SOD截短突变体tcvSOD克隆到大肠杆菌表达载体pET23a(+)后,经IPTG诱导在大肠杆菌Rosetta(DE3)菌株中表达得到了可溶性tcvSOD重组蛋白.该蛋白具有典型的SOD活性,能强烈抑制邻苯三酚的自氧化作用,最高抑制率为97%,反应体系中酶蛋白终质量浓度为0.2μg/mL时达到最大抑制,由此计算出其比活力为16 050 U/mg.tcvSOD的热稳定性和对蛋白变性剂SDS胁迫的反应与酶蛋白在反应体系中的质量浓度有关.终质量浓度为1μg/mL时tcvSOD可以耐90℃、30 min以及质量分数2%~10%的SDS处理而活性保持不变或略有丧失,终质量浓度为0.05和0.1μg/mL时则对上述处理非常敏感. 0.05、0.1、1μg/mL 3种质量浓度的tcvSOD对反应体系中pH4~12的变化,以及2~10 mol/L尿素和1~4 mol/L盐酸胍胁迫处理,酶活性保持恒定.实验结果表明tcvSOD具有较强热稳定性和pH稳定性,对常见蛋白变性剂也有良好抗性,有进一步开发利用的潜能.

A truncated copper, zinc superoxide dismutase encoded by the type species PBCV-1 of chloroviruses, tcvSOD, was cloned to an Escherichia coli expression vector pET23 a(+)and expressed as soluble recombinant protein in Rosetta(DE3)under the induction of isopropyl β-d-1-thiogalactopyranoside(IPTG). This protein could inhibit the auto-oxidation of pyrogallol strongly, and the maximal inhibition of 97% could be achieved when 0.2 μg/mL enzyme was supplemented to the reaction system. The specific activity of tcvSOD generated from above experiments was 16 050 U/mg. Thermostability and tolerance of the protein to sodium dodecyl sulfate(SDS)were related to its concentration. The activity of tcvSOD remained unchanged or lost slightly under the treatment of 90 °C for 30 min or 2%-10% of SDS when the final concentration was 1 μg/mL, but reduced much quicker when that were 0.05 and 0.1 μg/mL instead.In contrary to the thermostability and tolerance to SDS,tcvSOD was very robust to the treatments of wide ranges of pH,urea and guanidine chloride.The results shown in this paper indicated that tcvSOD has high thermostability and pH-stability,and is tolerant to several protein denaturants in wide ranges,and has the potential to be further explored for application as well.