谷胱甘肽双功能合成酶分子改造及应用

doi:10.11949/0438-1157.20240534

摘要/Abstract

摘要：

谷胱甘肽（GSH）是细胞内丰富的非蛋白质硫醇，其巯基作为活性基团，参与生物体内氧化还原反应，具有多种生理功能，在食品、医药及化妆品等行业得到广泛应用。谷胱甘肽双功能合成酶是合成GSH的关键酶，其催化效率的提升对GSH高效合成具有重要意义。本研究以Streptococcus thermophilus来源谷胱甘肽双功能合成酶（St-GshF）为研究对象，构建了一种基于谷胱甘肽和邻苯二甲醛反应产生荧光显色效应的高通量筛选方法，通过半理性设计对St-GshF进行分子改造，筛选获得有益突变体St-GshF（S27Q/G510P），其酶活为野生型的1.75倍。在此基础上，偶联多聚磷酸激酶，构建高效ATP循环系统并进行反应体系优化，最终谷胱甘肽的产量为17.36 g/L，产率达94.22%，为谷胱甘肽的规模化生产奠定了重要基础。

关键词: 谷胱甘肽双功能合成酶, 谷胱甘肽, 分子改造, 双酶偶联

Abstract:

Glutathione (GSH) is the most abundant non-protein thiol within cells. It has strong electron donating ability and participates in redox reactions in organisms, which endows it with multiple physiological functions and it is widely used in food, pharmaceutical and cosmetics industries. Glutathione bifunctional synthase is the key enzyme for catalyzing the production of GSH and improvement of its catalytic efficiency is of significance to GSH biosynthesis. In this study, the glutathione bifunctional synthasefrom Streptococcus thermophilus (St-GshF) was chosen for study object. A high-throughput screening method based on the fluorescence colorimetric effect generated by the reaction between GSH and ortho benzaldehyde was established. Using semi-rational design, St-GshF was engineered and the mutant St-GshF (S27Q/G510P) was obtained. Its activity was 1.75 times higher than the wild type. The polyphosphate kinase was coupled to construct the efficient glutathione biosynthesis system and after optimization of the reaction conditions, 17.36 g/L glutathione was obtained with a yield of 94.22%, laying an important foundation for the large-scale production of glutathione.

Key words: glutathione bifunctional synthetase, glutathione, molecular modification, dual enzyme coupling

中图分类号:

Q 814

宋世萍, 汤晓玲, 郑仁朝. 谷胱甘肽双功能合成酶分子改造及应用[J]. 化工学报, 2024, 75(S1): 251-258.

Shiping SONG, Xiaoling TANG, Renchao ZHENG. Molecular modification of glutathione bifunctional synthase and its application[J]. CIESC Journal, 2024, 75(S1): 251-258.

图/表 13

表1 饱和突变引物

Table 1 Primers for saturation mutation

位点	引物序列
S27MNN	5´-ACGCAGMNNTTCACGTTCGATACCGAAGTTAGC-3´
S27NNK	5´-CGTGAANNKCTGCGTGTTGACCGTCAGGGTCAG-3´
P99MNN	5´-AGACAGMNNCCACAGAACTTCGTCGGTAGCGAT-3´
P99NNK	5´-CTGTGGNNKCTGTCTATGCCGCCGCGTCTGAAA-3´
L136MNN	5´-AGCCTGMNNTTTGGTACCGTATTTTTCAGCCAG-3´
L136NNK	5´-ACCAAANNKCAGGCTATCTCTGGTATCCACTAC-3´
T505MNN	5´-CAGAGAMNNGAATTCGTCACCAGACGGAACCG-3´
T505NNK	5´-GAATTCNNKTCTCTGGAAGAAGGTCTGGCTTAC-3´
G510MNN	5´-GAAGAAMNNCTGGCTTACTACCCGCTGATCAAA-3´
G510NNK	5´-AGCCAGNNKTTCTTCCAGAGAGGTGAATTCGTC-3´

图1 邻苯二甲醛OPA与谷胱甘肽GSH激发波长扫描谱图

Fig.1 Excitation wavelength scanning spectra of o-phthalaldehyde (OPA) and glutathione (GSH)

图2 邻苯二甲醛与反应体系中主要成分发射波长扫描谱图

Fig.2 Emission wavelength scanning spectra of OPA and the main components in the reaction system

图3 邻苯二甲醛浓度对荧光响应值的影响

Fig.3 Effect of OPA concentration on fluorescence response values

图4 不同浓度GSH与邻苯二甲醛反应的荧光响应值标准曲线

Fig.4 Standard curve of fluorescence response values for different concentrations of GSH to react with OPA

图5 谷胱甘肽双功能合成酶的结构基础（箭头：β-折叠；黑色波浪线：η-螺旋；蓝色波浪线：α-螺旋）

Fig.5 Structural basis of St-GshF(arrow: β-sheet; black line: η-helix; blue line: α-helix)

表2 St-GshF及其突变体的相对酶活

Table 2 The relative enzymatic activity of St-GshF and its variants

突变体	酶活/(U/g wcw)	相对酶活/%
St-GshF	45.0	100
St-GshF(S27Q)	72.0	160.0
St-GShF(G510P)	76.2	169.3
St-GshF(S27Q/G510P)	78.75	175.1

图6 温度对St-GshF（S27Q/G510P）催化效率的影响

Fig.6 The effect of temperature on catalytic efficiency of St-GshF (S27Q/G510P)

图7 pH对St-GshF（S27Q/G510P）催化活力的影响

Fig.7 The effect of pH on catalytic efficiency of St- GshF (S27Q/G510P)

图8 金属离子对谷胱甘肽双功能合成酶St-GshF（S27Q/G510P）催化效率的影响

Fig.8 The effect of mental ions on the catalytic efficiency of St-GshF (S27Q/G510P)

图9 Mg2+浓度对St-GshF（S27Q/G510P）催化活性的影响

Fig.9 The effect of Mg2+ concentration on the catalytic efficiency of St-GshF (S27Q/G510P)

图10 不同浓度底物(L-Cys, L-Glu, Gly)对St-GshF（S27Q/G510P）催化效率的影响

Fig.10 The effect of substrate concentrations (L-Cys, L-Glu, Gly) on the catalytic efficiency of St-GshF (S27Q/G510P)

图11 谷胱甘肽双功能合成酶和多聚磷酸激酶偶联体系催化合成GSH的反应进程

Fig.11 The catalytic synthesis process of GSH by the coupling system of glutathione bifunctional synthase and polyphosphate kinase

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