Secretion of plant viral proteins in Pichia pastoris and self-assembled nanocatalyst for enhancing catalytic performance

doi:10.16085/j.issn.1000-6613.2020-0558

Abstract

Abstract:

Virus-like particles (VLPs) encapsualated metal nanoparticles (NPs) has enormous potential for application in catalysis. A highly efficient expression system was developed in this study by constructing recombinant Pichia pastoris cells for the secretion of soluble cowpea chlorotic mottle virus capsid proteins (CCMV CPs). A high yield of (101.4±3.2)mg/L CCMV CPs was harvested from the fermentation supernatant with the purity beyond 90% under the optimal induction conditions (1.0% methanol concentration at 30℃ for 96h). Resultant CPs had strong in vitro self-assembled capacity to encapsulate sodium citrate-stabilized Ru NPs (Ru-CA) to prepare hybrid nanocatalyst Ru@VLPs with core-shell structure. Compared to unsupported Ru-CA, Ru@VLPs exhibited a higher catalytic performance in the reduction of nitroarenes including 4-nitrophenol (4-NP) and 3-nitrobenzenesulfonate (3-NBS). The apparent reaction rate constant k over Ru@VLPs was calculated to be 0.14min^-1 in 4-NP reduction and 0.16min^-1 in 3-NBS one, respectively. Activation energy for 4-NP reduction over Ru@VLPs was about 32kJ/mol, also lower than that over Ru-CA (about 39kJ/mol). It was logically attributed to the enhanced stability of Ru NPs with the protection of protein cage as well as the synergistic effect between Ru NPs and some functional groups (e.g.—NH₂) on CCMV CPs. Finally, Ru@VLPs exhibited a stable and reusable capability.

Key words: Pichia pastoris, secretion, nano confinement, core-shell structure, hybrid nanocatalyst, nitroarene reduction

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。

关键词: 毕赤酵母, 分泌表达, 纳米限域, 核壳结构, 复合纳米催化剂, 硝基芳烃还原反应

CLC Number:

Q815

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.

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.

Figures/Tables 14

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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

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	[36,50]
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	[38]
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	[37]
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	[31,32]
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	[36,50]
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	[38]
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	[37]
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	[31,32]
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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