| 摘要: |
| 异源表达红灰链霉菌 (Streptomyces rubrogriseus) 的硫氧还蛋白还原酶 (thioredoxin-disulfide reductase) 基因trxB,优化诱导表达条件并对纯化后的酶学性质进行分析。先设计引物扩增得到红灰链霉菌硫氧还蛋白还原酶基因trxB,构建重组质粒pET-22b-trxB,进一步将其转化到大肠杆菌Escherichia coli BL21 (DE3) 中进行表达及诱导条件优化；最后通过镍离子亲和层析纯化硫氧还蛋白还原酶,并测定其酶学性质。克隆得到大小为969 bp的trxB,并成功构建重组表达质粒pET-22b-trxB；获得重组酶的最优诱导表达条件为IPTG浓度0.5 mM、OD600 0.8、诱导温度30℃、诱导时间4 h,纯化后的重组酶大小为38.5 kDa 。酶学性质结果表明:以羽毛粉为底物时,重组酶的最适pH为8.5,最适温度为60°C；金属离子中K+、Mg2+对酶有明显的激活作用,而Fe2+ 、Fe3+、Cu2+有明显的抑制作用；化学试剂中异丙醇、PMSF、DMSO均对重组酶活力有抑制作用,而DTT对重组酶活力有促进作用；重组酶的Km为64.04 mg/mL、Vmax为3.70 U/min。重组二硫键还原酶有较高的酶活力、良好的热稳定性,在降解角蛋白方面有一定的应用价值。 |
| 关键词: 二硫键还原酶 红灰链霉菌 异源表达 表达优化 酶学性质 |
| DOI： |
| 投稿时间：2024-10-28修订日期：2024-12-05 |
| 基金项目:国家自然科学(31660022)；广西科技重点研发计划项目(桂科AB21196019,桂科AB21220020)；大学生创新创业训练计划项目(S202210608156,S202210608164),广西海洋天然产物与组合生物合成化学重点实验室开放基金资助。 |
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| The heterologous expression of thioredoxin reductase gene from Streptomyces rubrogriseus in Escherichia coli and its enzymatic propertiesHeterologous expression of thioredoxin reductase gene from Streptomyces rubrogriseus and its enzymatic properties |
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lijiling, liurui, zhangyuxin, zhangligang, lujunming, shennaikun, zhanghongyan
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| (Guangxi Minzu University) |
| Abstract: |
| To heterologously express the thioredoxin-disulfide reductase (thioredoxin-disulfide reductase) gene trxB of Streptomyces rubrogriseus, optimize the induction expression conditions and analyze the enzymatic properties of the purified enzyme. Primers were designed to amplify the thioredoxin-disulfide reductase gene trxB of Streptomyces rubrogriseus, construct the recombinant plasmid pET-22b-trxB, and further transform it into Escherichia coli BL21 (DE3) for induction expression and optimization of induction conditions. Finally, the thioredoxin-disulfide reductase was purified by nickel ion affinity chromatography, and its enzymatic properties were determined. trxB with the size of 969 bp was cloned, and the recombinant expression plasmid pET-22b-trxB was successfully constructed. The optimal induction expression conditions for the recombinant enzyme were an IPTG concentration of 0.5 mM, an OD600 of 0.8, an induction temperature of 30°C, and an induction time of 4 h. The size of the purified recombinant enzyme was 38.5 kDa. The results of enzymatic properties indicated that when feather meal was used as the substrate, the optimal pH of the recombinant enzyme was 8.5 and the optimal temperature was 60°C. Among the metal ions, K+ and Mg2+ had significant activation effects on the enzyme, while Fe2+, Fe3+, and Cu2+ had significant inhibitory effects. Among the chemical reagents, isopropanol, PMSF, and DMSO all had inhibitory effects on the activity of the recombinant enzyme, while DTT had a promoting effect. The Km of the recombinant enzyme was 64.04 mg/mL and the Vmax was3.70 U/min. Recombinant disulfide reductase has high enzyme activity and good thermal stability, and has certain application value in the degradation of keratin. |
| Key words: disulfide bond reductase, Streptomyces rubrogriseus, heterologous expression, expression optimization, enzymatic properties |
