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In this study, a novel fructosyltransferase (StFFase) was screened from Streptococcus troglodytae through gene mining. The enzyme was then engineered via computer-aided rational design to improve both its catalytic efficiency and thermostability. Based on EVCoupling predictions and experimental validation, three mutants (D104E, I232S, and S684P) with significantly enhanced enzymatic activity were selected from potential candidates. Their enzymatic activities were 2.20, 2.65, and 1.53 times that of the wild-type, respectively. Notably, the I232S mutant maintained high relative enzyme activity over a temperature range of 40~70 ℃, and its half-life at 50 ℃ was extended to 6.60 h, demonstrating superior thermostability. Kinetic analysis revealed that the I232S mutant exhibited improved affinity for the substrate sucrose, with the Km value decreasing to 171 mmol/L, and a significantly enhanced catalytic efficiency. Furthermore, a conversion rate of 42.17% for 1-kestose was achieved using this mutant under conditions of 50% sucrose concentration (w/w) and an enzyme dosage of 15 U/g sucrose. Structural simulations revealed that the mutations improved catalytic performance and thermostability by eliminating electrostatic repulsion (D104E), forming new hydrogen bonds (I232S), and stabilizing β-turn conformations (S684P). This study provides a new strategy for the rational design of fructosyltransferases and highlights the potential of the engineered enzyme for industrial fructooligosaccharide production.
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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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