基于融合双亲短肽提高葡萄糖氧化酶的热稳定性

Enhancing thermal stability of glucose oxidase by fusing amphiphilic short peptide

葡萄糖氧化酶(Glucose oxidase,简称GOD)可氧化葡萄糖生产葡萄糖酸及其衍生物,在新型无抗饲料添加剂的开发中具有良好的应用潜力,但其生产加工过程中尚存在热稳定差的瓶颈问题。本研究采用融合双亲短肽技术提高葡萄糖氧化酶的热稳定性,分别选取8种不同氨基酸长度、不同Linker连接的双亲短肽(Self-assembling peptides,SAPs)融合至黑曲霉来源的葡萄糖氧化酶的N端,构建了8种融合型酶SAP1-GS-GOD、SAP1-PT-GOD、SAP2-PT-GOD、SAP3-PT-GOD、SAP4-PT-GOD、SAP5-PT-GOD、SAP6-PT-GOD、SAP7-PT-GOD,并在毕赤酵母GS115中异源表达。获得的连接PT Linker的融合酶在60℃下孵育60 min后的相对酶活均高于初始酶。其中,融合酶SAP5-PT-GOD在60℃下孵育30 min的相对酶活为67%,是相同处理条件下初始酶相对酶活的10.9倍。同时,融合酶SAP1-PT-GOD、SAP2-PT-GOD、SAP3-PT-GOD、SAP5-PT-GOD的K_(cat)/K_m值较初始酶均有进一步的提高。研究表明,在葡萄糖氧化酶的N端融合以PT为Linker的目标双亲短肽能有效提高葡萄糖氧化酶的热稳定性,通过对融合酶分子内作用力进行分析,酶分子内氢键的增加对融合酶热稳定性的提高效果最为显著。上述研究获得的热稳定性提高的融合葡萄糖氧化酶及其效果机制分析对提高酶在加工和应用过程中的活性具有重要的研究意义和应用价值。

Glucose oxidase catalyzes the oxidation of β-D-glucose to gluconic acid and its derivatives, thus shows a great potential in the development of antibiotic-free feed. However, its production and processing still have the problem of poor thermal stability of enzyme activity. In this study, fusion of amphiphilic peptide technology was used to improve the stability of glucose oxidase. Herein, eight self-assembling peptides with different amino acid lengths and Linkers were fused to the N terminus of the glucose oxidase, yielding eight chimeric fusions SAP1-GS-GOD, SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD, SAP4-PT-GOD, SAP5-PT-GOD, SAP6-PT-GOD and SAP7-PT-GOD. Then, the 8 recombinant proteins were expressed in P. pastoris GS115. After separation and purification, the stability of glucose oxidase at 60 ℃was determined. The relative enzyme activities of the PT Linker-linked fusion enzyme incubated at 60 ℃ for 60 min were higher than those of the original enzyme, and the relative activity of SAP5-PT-GOD was 67% at 60 ℃ for 30 min, which was 10.9 times higher than that of the initial enzyme with the same treatment. Among them, the Kcat/Km value of SAP1-PT-GOD, SAP2-PT-GOD, SAP3-PT-GOD and SAP5-PT-GOD of the fusion enzyme was further improved than that of the initial enzyme. Through the analysis of the intramolecular force of the fusion enzyme, the increase of the thermal stability of the fusion enzyme is mainly due to the increase of the hydrogen bond. In summary, the study indicates that translational fusion of self-assembling peptides with PT Linker was able to augment the thermo-stability of glucose oxidase, which has certain potential in the production and application of glucose oxidase. The glucose oxidase with improved thermostability obtained in the above study and the related mechanism will play an important role in improving the activity of related enzymes in the proceeding of processing and application.