Abstract:

This study introduced the AcGox gene from Aspergillus chevalieri into a Aspergillus niger strain via protoplast transformation technology， yielding the recombinant AcGox protein. Results indicated that recombinant AcGox exhibits optimal activity at pH 6.0 and 32 ℃， demonstrating excellent acid tolerance and good thermal stability. However， the specific activity of AcGox was only 70.81 U·mg^-1，to be a critical bottleneck limiting its application. To address this issue， this study further overexpressed genes encoding riboflavin kinase and FAD synthase in the AcGox-5 strain. RT-qPCR validation confirmed significantly elevated gene transcription levels. Ultimately， the specific activity of glucose oxidase produced by AcGox/FAD/FMN-3 strain increased to 111.55 U·mg^-1，with a 57.5% improvement. This study confirmed that cofactor FAD supply levels are a critical factor limiting the catalytic efficiency of recombinant AcGox. It provides direct experimental evidence for the "cofactor engineering" strategy in enzyme engineering and offers a scalable host modification approach for industrial glucose oxidase production， advancing the application of glucose oxidase.

Key words: Aspergillus niger, Aspergillus chevalieri, AcGox, flavin adenine dinucleotide, efficient expression

摘要：

研究通过原生质体转化技术将谢瓦曲霉（Aspergillus chevalieri）来源的葡萄糖氧化酶基因（glucose oxidase，AcGox）导入黑曲霉（A. niger）菌株，获得表达的重组AcGox蛋白。结果显示，重组AcGox蛋白的最适pH为6.0，最适温度为32 ℃，表现出优良的耐酸性和较好的热稳定性。然而，AcGox的比活性仅为70.81 U·mg^-1，成为限制其应用的关键瓶颈。为解决这一问题，研究在AcGox-5表达菌株基础上，进一步过表达编码核黄素激酶和黄素腺嘌呤二核苷酸（flavin adenine dinucleotide，FAD）合成酶基因，RT-qPCR证实2个基因的转录水平显著上调。最终，优化后的菌株AcGox/FAD/FMN-3生产的Gox比活性提升至111.55 U·mg^-1，增幅达57.5%。研究证实辅因子FAD的供给水平是限制重组AcGox催化效率的关键因素，为酶工程中“辅因子工程”策略提供了直接实验证据，并为葡萄糖氧化酶的工业化生产提供了可推广的宿主改造方案，推动了Gox的应用。

关键词: 黑曲霉, 谢瓦曲霉, 葡萄糖氧化酶, 黄素腺嘌呤二核苷酸, 高效表达