Enhancing thermal stability of glucose oxidase by fusing amphiphilic short peptide

Abstract

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.