化工进展 ›› 2021, Vol. 40 ›› Issue (1): 354-365.DOI: 10.16085/j.issn.1000-6613.2020-0558
杨坤1(), 卢晓雪1, 杨林松1, 赵庆欢2, 朱劼1()
收稿日期:
2020-04-09
出版日期:
2021-01-05
发布日期:
2021-01-12
通讯作者:
朱劼
作者简介:
杨坤(1995—),男,硕士研究生,研究方向为纳米催化剂。E-mail:基金资助:
Kun YANG1(), Xiaoxue LU1, Linsong YANG1, Qinghuan ZHAO2, Jie ZHU1()
Received:
2020-04-09
Online:
2021-01-05
Published:
2021-01-12
Contact:
Jie ZHU
摘要:
病毒样颗粒(VLPs)包裹纳米粒子的亚细胞结构仿生设计在催化领域潜力巨大。本文构建了一株基于毕赤酵母(Pichia pastoris)的基因工程菌,用于分泌表达重组豇豆褪绿斑驳病毒衣壳蛋白(CCMV CPs)。通过表达工艺的优化(诱导温度30℃、诱导时间96h、每24h添加终浓度体积分数1.0%的甲醇),可在发酵上清液中直接收获较高产量[(101.4±3.2)mg/L]和较高纯度(>90%)的衣壳蛋白。纯化后的CCMV CPs具有较强的体外自组装性能,能高效封装柠檬酸稳定的钌纳米颗粒(Ru-CA)(封装率约70%),制备具有核壳结构的复合纳米催化剂Ru@VLPs。与非负载型催化剂Ru-CA相比,该催化剂在硝基芳烃包括4-硝基苯酚(4-NP)和3-硝基苯磺酸钠(3-NBS)还原反应中显示出较高的催化活性,表观反应速率常数(k)分别达到约0.14min-1(4-NP还原反应)和0.16min-1(3-NBS还原反应)。经计算,它催化的4-NP还原反应活化能为32kJ/mol,略低于Ru-CA(39kJ/mol)。Ru@VLPs较高的催化性能可归因于CCMV CPs封装Ru-CA后其分散性和稳定性的提高,以及Ru纳米颗粒与衣壳蛋白功能基团(如氨基,—NH2)之间的协同作用。同时表明,Ru@VLPs亦具有较好的回收再利用性能。
中图分类号:
杨坤, 卢晓雪, 杨林松, 赵庆欢, 朱劼. 毕赤酵母分泌表达植物病毒蛋白制备自组装纳米催化剂提高催化性能[J]. 化工进展, 2021, 40(1): 354-365.
Kun YANG, Xiaoxue LU, Linsong YANG, Qinghuan ZHAO, Jie ZHU. Secretion of plant viral proteins in Pichia pastoris and self-assembled nanocatalyst for enhancing catalytic performance[J]. Chemical Industry and Engineering Progress, 2021, 40(1): 354-365.
序号 | A/℃ | B/h | C/% | CCMV蛋白含量/mg·L-1 |
---|---|---|---|---|
1 | 1(27) | 1(84) | 1(0.8) | 81.3±3.6 |
2 | 1(27) | 2(96) | 2(0.9) | 89.2±2.8 |
3 | 1(27) | 3(108) | 3(1.0) | 92.3±4.5 |
4 | 2(30) | 1(84) | 2(0.9) | 94.1±4.6 |
5 | 2(30) | 2(96) | 3(1.0) | 101.4±3.2 |
6 | 2(30) | 3(108) | 1(0.8) | 96.6±2.2 |
7 | 3(33) | 1(84h) | 3(1.0) | 83.5±4.1 |
8 | 3(33) | 2(96) | 1(0.8) | 89.6±3.4 |
9 | 3(33) | 3(108) | 2(0.9) | 87.7±3.8 |
K1 | 262.8 | 258.9 | 267.5 | |
K2 | 292.1 | 280.2 | 271.0 | |
K3 | 260.8 | 276.6 | 277.2 | |
极差 | 31.3 | 21.3 | 9.7 | |
最优方案 | A2B2C3 |
表1 CCMV蛋白表达条件优化L9(33)正交实验结果
序号 | A/℃ | B/h | C/% | CCMV蛋白含量/mg·L-1 |
---|---|---|---|---|
1 | 1(27) | 1(84) | 1(0.8) | 81.3±3.6 |
2 | 1(27) | 2(96) | 2(0.9) | 89.2±2.8 |
3 | 1(27) | 3(108) | 3(1.0) | 92.3±4.5 |
4 | 2(30) | 1(84) | 2(0.9) | 94.1±4.6 |
5 | 2(30) | 2(96) | 3(1.0) | 101.4±3.2 |
6 | 2(30) | 3(108) | 1(0.8) | 96.6±2.2 |
7 | 3(33) | 1(84h) | 3(1.0) | 83.5±4.1 |
8 | 3(33) | 2(96) | 1(0.8) | 89.6±3.4 |
9 | 3(33) | 3(108) | 2(0.9) | 87.7±3.8 |
K1 | 262.8 | 258.9 | 267.5 | |
K2 | 292.1 | 280.2 | 271.0 | |
K3 | 260.8 | 276.6 | 277.2 | |
极差 | 31.3 | 21.3 | 9.7 | |
最优方案 | A2B2C3 |
表达宿主(菌株,载体) | CCMV CPs产量 | CCMV CPs性质 | 纯化步骤 | 参考文献 |
---|---|---|---|---|
E. coli(BL21,pET23a ); E. coli(BL21(DE3), pET28a ) | 75~100mg/L | 细胞内表达 包涵体 | 细胞破裂,变性,复性,超速离心 | [ |
E. coli(Rosetta 2, pET19b) | 25.92mg/L | 细胞内表达 可溶性蛋白 | 细胞破裂,亲和色谱 | [ |
Pseudomonas fluorescens(DC487,pDOW3250) | 2.6g/L | 细胞内表达 可溶性蛋白 | 细胞破裂,PEG沉淀,蔗糖密度梯度离心 | [ |
Pichia pastoris(pPICZA) | 0.05~0.5mg/g细胞湿重; 4.8g/L | 细胞内表达 可溶性蛋白 | 细胞破裂,PEG沉淀,CsCl梯度离心 | [ |
Pichia pastoris(pPIC9K) | 约101mg/L | 分泌表达 可溶性蛋白 | 阴离子交换层析法 | 本文 |
表2 CCMV CPs在一些基因工程菌中的表达
表达宿主(菌株,载体) | CCMV CPs产量 | CCMV CPs性质 | 纯化步骤 | 参考文献 |
---|---|---|---|---|
E. coli(BL21,pET23a ); E. coli(BL21(DE3), pET28a ) | 75~100mg/L | 细胞内表达 包涵体 | 细胞破裂,变性,复性,超速离心 | [ |
E. coli(Rosetta 2, pET19b) | 25.92mg/L | 细胞内表达 可溶性蛋白 | 细胞破裂,亲和色谱 | [ |
Pseudomonas fluorescens(DC487,pDOW3250) | 2.6g/L | 细胞内表达 可溶性蛋白 | 细胞破裂,PEG沉淀,蔗糖密度梯度离心 | [ |
Pichia pastoris(pPICZA) | 0.05~0.5mg/g细胞湿重; 4.8g/L | 细胞内表达 可溶性蛋白 | 细胞破裂,PEG沉淀,CsCl梯度离心 | [ |
Pichia pastoris(pPIC9K) | 约101mg/L | 分泌表达 可溶性蛋白 | 阴离子交换层析法 | 本文 |
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