关键词: 铜锌超氧化物歧化酶(Cu,Zn-SOD), 小球藻病毒, 热稳定性, pH稳定性, 蛋白变性剂抗性

Abstract: A truncated copper, zinc superoxide dismutase encoded by the type species PBCV-1 of chloroviruses, tcvSOD, was cloned to an Escherichia coli expression vector pET23a(+)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.- DOI:10.3969/j.issn.1000-1565.2021.03.013小球藻病毒PBCV-1编码的Cu,Zn-SOD酶学稳定性刘建荣¹,贾红玉²,陈慧慧²,刘晓飞²,康明²(1.河北大学 生物工程技术创新中心,河北 保定 071002;2.河北大学 生命科学学院,河北 保定 071002)摘 要: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0.2 μg/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1 μg/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0.1 μg/mL时则对上述处理非常敏感. 0.05、0.1、1 μg/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.关键词:铜锌超氧化物歧化酶(Cu,Zn-SOD);小球藻病毒;热稳定性;pH稳定性;蛋白变性剂抗性 中图分类号:Q502 文献标志码:A 文章编号:1000-1565(2021)03-0304-07Enzymatic stability of Cu,Zn-SOD encoded by chlorovirus PBCV-1LIU Jianrong¹, JIA Hongyu², CHEN Huihui², LIU Xiaofei², KANG Ming²(1.Innovation Center for Bioengineering and Biotechnology, Hebei University, Baoding 071002, China;2.College of Life Sciences, Hebei University, Baoding 071002,China)Abstract: A truncated copper, zinc superoxide dismutase encoded by the type species PBCV-1 of chloroviruses, tcvSOD, was cloned to an Escherichia coli expression vector pET23a(+)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.- 收稿日期:2020-09-11 基金项目:国家自然科学基金资助项目(31470192); 河北省自然科学基金资助项目(C2016201180) 第一作者:刘建荣(1963—), 女, 河北保定人, 河北大学副研究员,主要从事生化与分子生物学研究.E-mail: bioinfo5@hbu.edu.cn 通信作者:康明(1962—), 男, 河北昌黎人, 河北大学教授, 博士, 主要从事微生物代谢工程以及病毒宿主相互作用研究.E-mail: mingkang@hbu.edu.cn第3期刘建荣等:小球藻病毒PBCV-1编码的Cu,Zn-SOD酶学稳定性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.

Key words: copper,zinc-superoxide dismutase(Cu,Zn-SOD), chlorovirus, thermostability, pH-stability, tolerance to protein denaturants