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中国精品科技期刊2020
陈亚利,王倩,刘涵,等. C端非催化结构域对嗜热α-葡聚糖磷酸化酶TsGP酶学性质的影响[J]. 食品工业科技,2024,45(22):1−10. doi: 10.13386/j.issn1002-0306.2023120312.
引用本文: 陈亚利,王倩,刘涵,等. C端非催化结构域对嗜热α-葡聚糖磷酸化酶TsGP酶学性质的影响[J]. 食品工业科技,2024,45(22):1−10. doi: 10.13386/j.issn1002-0306.2023120312.
CHEN Yali, WANG Qian, LIU Han, et al. Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP[J]. Science and Technology of Food Industry, 2024, 45(22): 1−10. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023120312.
Citation: CHEN Yali, WANG Qian, LIU Han, et al. Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP[J]. Science and Technology of Food Industry, 2024, 45(22): 1−10. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2023120312.

C端非催化结构域对嗜热α-葡聚糖磷酸化酶TsGP酶学性质的影响

Effect of the C-terminal Non-catalytic Domain on the Enzymatic Properties of Thermophilic α-Glucan Phosphorylase TsGP

  • 摘要: 为获得异源表达水平与酶学性质良好的α-葡聚糖磷酸化酶并考察结构域对酶的影响,研究通过数据库挖掘和序列分析,获得了来源于超级嗜热古菌Thermococcus sp. EP1的新型α-葡聚糖磷酸化酶TsGP。利用AlphaFold2预测三维结构确定TsGP的C端为非催化结构域后,构建了C端截短的突变体ΔTsGP。在E. coli BL21(DE3)中对TsGP与ΔTsGP进行异源重组表达,并对其酶学性质进行了表征。结果表明ΔTsGP在大肠杆菌中的表达水平较TsGP提高了3.76倍。在最适反应温度为70 ℃时,ΔTsGP的比酶活为23.87 U/mg,较TsGP提高1.3倍。在50~65 ℃的工业酶催化条件下,ΔTsGP的热稳定性与TsGP相当,且酶活力更高。此外,ΔTsGP与TsGP具有相同的底物特异性,最小底物为麦芽三糖,且随着底物链长的增加,比酶活逐渐提升。以麦芽七糖为底物,ΔTsGP的kcat值为37.09 s−1,比TsGP提高了1.21倍,但底物亲和力(Km=3.30 mmol/L)降低了2.77倍。研究通过结构分析与非催化结构域截短策略成功提高了TsGP在大肠杆菌中的表达水平和工业酶催化条件下的比酶活。这为αGP酶蛋白的高效表达与应用提供了借鉴,并为进一步通过工程改造优化其性能奠定了基础。

     

    Abstract: In order to obtain α-glucan phosphorylase with good heterologous expression level and enzymatic properties and to investigate the impact of structural domains on the enzyme, a novel α-glucan phosphorylase TsGP was screened from the hyperthermophilic archaeon Thermococcus sp. EP1 through database mining and sequence analysis. After confirming that the C-terminal of TsGP was a non-catalytic domain in the three-dimensional structure predicted by AlphaFold2, a C-terminal truncated mutant ΔTsGP was constructed. Heterologous recombinant expression of TsGP and ΔTsGP was performed in E.coli BL21(DE3), and their enzymatic properties were characterized. The results showed that the expression level of ΔTsGP in E.coli was 3.76 times higher than that of TsGP. At the optimal reaction temperature 70 °C, the specific enzyme activity of ΔTsGP was 23.87 U/mg, which was 1.3 times higher than that of TsGP. Under 50~65°C which was widely applied as industrial catalytic temperature, the thermal stability of ΔTsGP was equivalent to that of TsGP, and the enzyme activity was higher. In addition, ΔTsGP and TsGP had the same substrate specificity, their smallest substrate was maltotriose, and as the substrate chain length increased, their specific enzyme activity gradually increased. Using maltoheptaose as the substrate, the kcat value of ΔTsGP was 37.09 s−1, which was 1.21 times higher than that of TsGP, but its substrate affinity (Km=3.30 mmol/L) was reduced by 2.77 times. Through structural analysis and non-catalytic domain truncation strategies, the study successfully improved the expression level of TsGP in E. coli and the specific enzyme activity under industrial enzyme catalytic conditions. The study would provide guidance for the efficient expression and application of αGPase protein and lays the foundation for further optimizing the performance of this enzyme through engineering modification.

     

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