毕赤酵母高效间接表面展示系统的构建及在固定有机磷水解酶上的应用

An efficient indirect surface display system in Pichia pastoris: construction and application in immobilization of organophosphorus hydrolase

摘要:

目的 通过毕赤酵母表面展示技术，利用SpyCatcher/SpyTag (SpyC/SpyT)生物偶联体系固定有机磷水解酶(organophosphorus hydrolase, OPH)，以解决OPH在实际应用中稳定性差和重复利用率低的问题，为有机磷农药污染的生物修复提供新方法。方法 在毕赤酵母表面展示“诱饵蛋白” SpyCatcher (SpyC)，通过增加拷贝数和优化培养条件提高展示效率。通过酶学性质分析评估固定化OPH的热稳定性、pH稳定性及重复使用性能，并考察其对基于SpyC/SpyT的特异性相互作用，将OPH-SpyTag (OPH-SpyT)高效展示于毕赤酵母表面。甲基对硫磷、乐果和毒死蜱 3种有机磷农药的水解效率。结果 毕赤酵母表面展示SpyC的效率超过(97.0±0.40)%，优化后湿细胞可结合绿色荧光蛋白(21.4±0.7) mg/g。成功将OPH展示于细胞表面，固定化OPH的热稳定性和pH稳定性显著提高，重复使用5次后仍保留50%以上的活力。在最适条件下，对100 mg/L甲基对硫磷、乐果和毒死蜱这3种有机磷农药的水解率分别达到(96.5±2.7)%、(79.5±2.3)%和(82.6±2.8)%，表明该方法对某些有机磷农药具有较高的水解效率。结论 SpyC/SpyT生物偶联体系的毕赤酵母表面展示技术可有效固定OPH，显著提升其稳定性和重复使用性能，为有机磷农药污染的生物修复提供了高效、绿色的新途径，也为毕赤酵母表面展示相关领域的研究提供了有力工具和方法。

Abstract:

Objective To develop an efficient catalyst for organophosphorus pesticide degradation by immobilizing organophosphorus hydrolase (OPH) on the surface of Pichia pastoris via the SpyCatcher/SpyTag (SpyC/SpyT) system, addressing the poor stability and low reusability of OPH in practical applications and providing a new method for the bioremediation of organophosphorus pesticide pollution.Methods The “bait protein” SpyCatcher (SpyC) was first displayed on the surface of P. pastoris, and the display efficiency was increased by increasing the copy number and optimizing the culture conditions. Then based on the specific interaction between SpyC and SpyT, OPH-SpyTag (OPH-SpyT) was efficiently displayed on the yeast surface. The thermal stability, pH stability, and reusability of immobilized OPH were evaluated, and the hydrolysis efficiency of immobilized OPH against methyl parathion, dimethoate, and chlorpyrifos was assessed.Results The display efficiency of SpyC on the P. pastoris surface reached over (97.0±0.4)%, with an optimized binding capacity of (21.4±0.7) mg green fluorescent protein for 1 g wet cells. OPH was successfully displayed on the cell surface via the SpyC/SpyT system. The immobilized OPH exhibited significantly enhanced thermal and pH stability, retaining more than 50% activity after five repeated uses. Under optimum conditions, the immobilized OPH showed the hydrolysis rates of (96.5±2.7)%, (79.5±2.3)%, and (82.6±2.8)% against 100 mg/L methyl parathion, dimethoate, and chlorpyrifos, respectively. This indicated that the method showed high hydrolysis efficiency for the organophosphorus pesticides.Conclusion The immobilization of OPH on P. pastoris surface via the SpyC/SpyT system effectively improves its stability and reusability, offering an efficient and environmentally friendly solution for the bioremediation of organophosphorus pesticide pollution. Meanwhile, this study provides a powerful tool and method for research in the field of P. pastoris surface display.