多聚磷酸盐激酶双底物通道腔的理性设计及其在无细胞催化生产谷胱甘肽中的应用

Rational design of polyphosphate kinase dual-substrate channel cavity for efficient production of glutathione by cell free catalysis

摘要:

三磷酸腺苷(adenosine triphosphate,ATP)是一种重要的辅助因子，参与许多需能的生物催化反应。多聚磷酸盐激酶(polyphosphate kinases,PPK)由于其底物聚磷酸盐廉价易得，可以为消耗ATP的反应提供能量。本研究选择哈氏噬纤维菌(Cytophaga hutchinsonii)来源的ChPPK，进行了底物谱和耐受性分析，通过分子对接和定点突变，理性改造多聚磷酸盐激酶的双底物通道腔来提高PPK酶的催化活性。与野生型相比，筛选得到突变体ChPPK_K81H-K103V的相对酶活提高了326.7%，同时，双突变扩大了ChPPK的底物利用范围与耐受性，提高了该酶的耐热性与耐碱性。基于该ATP再生系统，本研究偶联谷胱甘肽双功能酶GshAB和ChPPK_K81H-K103V，破细胞后采用无细胞催化生产谷胱甘肽，加入5 mmol/L ATP后，该体系6 h可以生产(25.4±1.9) mmol/L的谷胱甘肽，比突变前的催化体系提高了41.9%。优化无细胞催化体系的缓冲液、裂解液菌体量、补料时间后，无细胞体系可产生(45.2±1.8) mmol/L谷胱甘肽，底物l-半胱氨酸的转化率达到90.4%。提高ChPPK生产ATP的能力，可有效增强底物的转化率，降低催化成本，实现了无细胞催化生产谷胱甘肽的高产量、高转化率与高经济价值的统一。本研究提供了一种绿色高效的ATP再生系统，可为消耗ATP的生物催化反应平台提供可持续动力。

关键词:多聚磷酸盐激酶;ATP再生;突变;无细胞催化;谷胱甘肽

Abstract:

ATP is an important cofactor involved in many biocatalytic reactions that require energy input. Polyphosphate kinases (PPK) can provide energy for ATP-consuming reactions due to their cheap and readily available substrate polyphosphate. We selected ChPPK from Cytophaga hutchinsonii for substrate profiling and tolerance analysis. By molecular docking and site-directed mutagenesis, we rationally engineered the dual-substrate channel cavity of polyphosphate kinase to improve the catalytic activity of PPK. Compared with the wild type, the relative enzyme activity of the screened mutant ChPPK_K81H-K103V increased by 326.7%. Meanwhile, the double mutation expanded the substrate utilization range and tolerance of ChPPK, and improved its heat and alkali resistance. Subsequently, we coupled the glutathione bifunctional enzyme GshAB and ChPPK_K81H-K103V based on this ATP regeneration system, and glutathione was produced by cell-free catalysis upon disruption of cells. This system produced (25.4±1.9) mmol/L glutathione in 6 h upon addition of 5 mmol/L ATP. Compared with the system before mutation, glutathione production was increased by 41.9%. After optimizing the buffer, bacterial mass and feeding time of this system, (45.2±1.8) mmol/L glutathione was produced in 6 h and the conversion rate of the substrate l-cysteine was 90.4%. Increasing the ability of ChPPK enzyme to produce ATP can effectively enhance the conversion rate of substrate and reduce the catalytic cost, achieving high yield, high conversion rate and high economic value for glutathione production by cell-free catalysis. This study provides a green and efficient ATP regeneration system that may further power the ATP-consuming biocatalytic reaction platform.

Key words:polyphosphate kinase;ATP regeneration;mutation;cell-free catalysis;glutathione