甲酸脱氢酶(TsFDH)热稳定性改造及应用

Thermal stability modification and application of formate dehydrogenase (TsFDH)

摘要:

【背景】 甲酸脱氢酶(formate dehydrogenase, FDH, EC 1.2.1.2)常用于发酵产业中的NADH再生，最近的研究表明FDH可以反向催化CO₂还原成甲酸，是一种具有前景的可以实现微生物固定CO₂的酶。【目的】 提高来自硫杆菌(Thiobacillus sp.) KNK65MA的一种FDH (TsFDH)的热稳定性。【方法】 通过PROSS网站预测，采用半理性改造方式构建了9种不同的突变体，并表征了突变体和野生型的酶学性质，同时通过突变体结构分析比酶活与热稳定性提高的原因，最后成功将TsFDH突变体应用于毕赤酵母(Komagataella phaffii) GS115-4Δ体内固定CO₂。【结果】 突变体TsFDH^A199G和TsFDH^P247K的比酶活较野生型相比分别提高了114.58%和56.17%，在45 ℃条件下孵育1 h后剩余酶活分别达到19.32%和39.47%，催化效率(k_cat/K_m)分别为0.096 L/(mmol·s)和0.042 L/(mmol·s)，T_m值分别提高0.3 ℃和0.7 ℃，这些结果说明突变体催化CO₂还原的酶活及热稳定性均有所提高。结构分析发现，突变体TsFDH^A199G和TsFDH^P247K的蛋白内部的疏水相互作用较野生型相比分别增加了2个和4个，TsFDH^P247K的离子相互作用较野生型相比增加了1个，同时TsFDH^A199G优化了活性中心的微环境。最后将TsFDH突变体转入毕赤酵母GS115-4Δ体内固定CO₂，在96 h时生物量分别提高118.04%和106.11%。【结论】 本实验成功筛选出突变体TsFDH^A199G和TsFDH^P247K，提高了TsFDH的热稳定性，填补了FDH催化CO₂还原方向热稳定性改造的空白，为酶法固定CO₂提供了2个可行的酶。

Abstract:

[Background] Formate dehydrogenase (FDH, EC 1.2.1.2) is commonly used for NADH regeneration in the fermentation industry. Recent studies have shown that FDH can reversibly catalyze CO₂ reduction to formate, serving as a promising enzyme for microbial CO₂ fixation. However, poor thermal stability is the main factor limiting the application of FDH in in vitro CO₂ fixation. [Objective] This study aimed to enhance the thermal stability of a CO₂-reducing FDH from Thiobacillus sp. KNK65MA (TsFDH) via semi-rational engineering. [Methods] Nine different variants were designed via PROSS, which predicted stabilizing mutations based on sequence conservation and structural energy minimization. The enzymatic properties of the variants and the wild type were characterized, and structural mechanisms underlying stability improvement were analyzed through homology modeling. Finally, the engineered TsFDH variants were applied to the fixation of CO₂ in vivo by Komagataella phaffii GS115-4Δ. [Results] Compared with that of the wild type, the specific activities of TsFDH^A199G and TsFDH^P247K increased by 114.58% and 56.17%, respectively. The relative enzyme activities of TsFDH^A199G and TsFDH^P247K reached 19.32% and 39.47%, respectively, after incubation at 45 ℃ for 1 h, compared with that of the wild type. TsFDH^A199G and TsFDH^P247K showed the catalytic efficiency (k_cat/K_m) of 0.096 L/(mmol·s) and 0.042 L/(mmol·s) and the T_m increases of 0.3 ℃ and 0.7 ℃, respectively. These results indicated that the enzyme activity and thermal stability of TsFDH variants catalyzing CO₂ reduction were improved. Structural analysis showed that the hydrophobic interactions within TsFDH^A199G and TsFDHP^247K increased by two and four and the ionic interactions of TsFDH^P247K increased by one, compared with those of the wild type. Meanwhile, TsFDH^A199G optimized the microenvironment of the active center. It improves the affinity between the substrate and the enzyme, and thus increased the enzymatic reaction rate. Finally, TsFDH^A199G and TsFDHP^247K were transferred to K. phaffii GS115-4Δ for CO₂ fixation, increasing the biomass at the time point of 96 h by 118.04% and 106.11%, respectively. [Conclusion] TsFDH^A199G and TsFDH^P247K were successfully screened, which successfully improved the thermal stability of TsFDH. This study fills the gap of thermal stability modification of FDH for CO₂ reduction and provides two feasible enzymes for enzymatic fixation of CO₂.