生物学杂志

生物学杂志 ›› 2024, Vol. 41 ›› Issue (1): 14-.doi: 10.3969/j.issn.2095-1736.2024.01.014

• 研究报告 • 上一篇    下一篇

来源于Pseudomonas多巴脱羧酶的异源表达、纯化及酶学性质研究

周耀林1， 孙登岳1,2， 曾志雄1， 李　霞1,2   

  1. 1. 齐鲁工业大学（山东省科学院）生物工程学部， 济南 250000；
    2. 齐鲁工业大学（山东省科学院）生物基材料与绿色造纸国家重点实验室， 济南 250000
  • 出版日期:2024-02-18 发布日期:2024-02-18
  • 通讯作者: 曾志雄，博士，教授，主要从事生物大分子结构与功能的研究，E-mail：zengzx@qlu.edu.cn；李霞，博士，研究员，主要从事多肽模拟酶设计及应用研究，E-mail：lixia5625@126.com；曾志雄和李霞为共同通信作者
  • 作者简介:周耀林，硕士研究生，主要从事多巴脱羧酶研究，E-mail：2382002192@qq.com
  • 基金资助:
    山东省自然科学基金项目（ZR2021QC038）

Heterologous expression, purification and enzymatic properties of dopa decarboxylase from Pseudomonas

ZHOU Yaolin1, SUN Dengyue1,2, ZENG Zhixiong1, LI Xia1,2   

  1. 1. Department of Bioengineering, Qilu University of Technology（Shandong Academy of Sciences）, Ji’nan 250000,
    China; 2. State Key Laboratory of Bio-based Materials and Green Paper, Qilu University of Technology
    （Shandong Academy of Sciences）, Ji’nan 250000, China
  • Online:2024-02-18 Published:2024-02-18

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摘要： 将来源于Pseudomonas的多巴脱羧酶（DOPA decarboxylase，DODC）基因序列经过PCR扩增，双酶切，连接到载体CV6-pGEX-6P-1上，经验证、测序成功构建表达载体CV6-pGEX-6P-1-DODC。转入大肠杆菌BL21（DE3）重组表达，表达条件为OD值达到0.6~0.8，异丙基β-D-1-硫代半乳糖苷（IPTG）终浓度为0.1 mmol/L，16 ℃过夜培养12~16 h。结果表明：在BL21（DE3）大肠杆菌中经诱导表达得到较高表达量的DODC融合蛋白；经过GST-亲和层析、3C蛋白酶切、离子交换层析得到纯度95%以上的DODC纯化蛋白；对DODC的酶学性质进行研究，该酶的最适反应温度为40 ℃，对温度的影响比较敏感，在20~30 ℃酶活在80%以上，超过30 ℃酶活大幅度降低，最适缓冲液为PBS缓冲液，最适反应pH值为7.5，最适底物为左旋多巴（L-DOPA），金属阳离子Ca2+对酶活力有促进作用。序列同源性分析表明，来源于Pseudomonas的DODC属于AAT-Ι超家族，并且预测出该酶的保守催化活性位点为Thr 241。

关键词: 多巴脱羧酶（DODC）, 异源表达, 蛋白纯化, 酶学性质, 底物特异性

Abstract: The dopamine decarboxylase (DODC) gene sequence from Pseudomonas was PCR amplified, double digested and ligated to the vector CV6-pGEX-6P-1, and the expression vector CV6-pGEX-6P-1-DODC was successfully constructed by validation and sequencing. Escherichia coli BL21 (DE3) was transferred for recombinant expression. The OD value was 0.6-0.8, the final concentration of isopropyl β-D-1-thiogalactoside (IPTG) was 0.1 mmol/L, and cultured at 16 ℃ overnight for 12-16 h. The results showed that DODC fusion protein with high expression was obtained in E.coli BL21(DE3) by induction expression. DODC purified protein with purity above 95% was obtained by GST-affinity chromatography, 3C protease digestion and ion exchange chromatography. The enzymatic properties of DODC were studied. The optimum reaction temperature of the enzyme was 40 ℃, which was sensitive to the effect of temperature, and the enzyme activity was more than 80% at 20-30 ℃. The enzyme activity decreased substantially above 30 ℃. The optimal buffer solution was PBS buffer solution, the optimal reaction pH was 7.5, the optimal substrate was L-DOPA. The metal cation Ca2+ promoted the enzyme activity. The sequence homology analysis showed that DODC from Pseudomonas belongs to the AAT-Ⅰ superfamily, and the conserved catalytic active site of the enzyme was predicted to be Thr 241.

Key words: DOPA decarboxylase, heterologous expression, protein purification, enzymatic properties, substrate specificity

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