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This study aimed to simultaneously enhance the enzymatic activity and thermostability of glucose oxidase (AnGOD) derived from Aspergillus niger through a semi-rational design strategy. Mutation sites were designed using the FireProt online tool and consensus sequences analysis, and the optimal mutant was screened via site-directed mutagenesis and a two-stage combinatorial mutation approach. Molecular dynamics simulations were then conducted to elucidate the mechanism of performance enhancement. Results showed that from the initial 83 designed sites, 12 single-point advantageous mutants were selected, and the optimal mutant M5 (T276F/T34V/Q90R/S53F/S74T) was obtained through two-stage combinatorial mutagenesis. Compared to the wild-type (WT), M5 exhibited a 4.2-fold increase in specific activity, with a kcat value of 755.0 s−1 and a kcat/Km value of 34.52 L·mmol−1·s−1, representing 3.67- and 2.48-fold enhancements over WT, respectively. At 65 ℃, the half-life of M5 was 30 min, representing a 30-fold increase compared to WT (1 min). Molecular dynamics simulations revealed that the average RMSD of M5 and the RMSF in non-catalytic regions were reduced by 12.9% and 12.4%, respectively, compared to the wild-type (WT). Additionally, the number of hydrogen bonds between M5 and the substrate/coenzyme increased by 10.3% and 8.8%, respectively. Notably, the S74T mutation introduced a novel hydrogen bond network, while the Q90R mutation introduced a positive charge, enhancing surface electrostatic complementarity. These synergistic effects collectively improved both the thermostability and catalytic activity of AnGOD. Through semi-rational design and combinatorial mutagenesis, this study significantly alleviated the stability-activity trade-off in AnGOD enzyme engineering, laying a foundation for the application of GOD.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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