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[Objective] To conduct site-directed mutagenesis on the Ca2+-binding sites, Leu253, and protein surface oxidation-sensitive Met residues of TSS, a collagenolytic protease derived from thermophiles, thereby screening out the variants with improved antioxidant activity, thermostability, and catalytic activity and revealing the intrinsic connection among them. [Methods] Site-directed mutagenesis was conducted on the Ca2+-binding sites (Ca1, Ca2, and Ca3) and Leu253 of TSS and the oxidation-sensitive Met residues on the protein surface. The derived variants were further evaluated for H2O2 tolerance, relative activity, and residual activity. Changes in antioxidant activity, catalytic activity, and thermostability after mutations were also studied. [Results] Mutations in amino acid residues at the three Ca2+-binding sites led to decreases in catalytic activity and/or thermostability of TSS. The Leu253 series variants constructed by simulating KP-43 did not enhance the antioxidant activity of TSS. Among the variants of oxidation-sensitive residues, M302L showed increases of 13% and 61% in the catalytic and antioxidant activities, respectively, while remaining unchanged thermostability. Ca2+ increased the catalytic activity and thermostability of TSS at high temperatures in a concentration-dependent manner. A high salinity environment with more than 2 mol/L NaCl prolonged the half-life of TSS at 75 ℃ and inhibited the maturation process of TSS proform. [Conclusion] The antioxidant and catalytic activities are positively correlated in the Leu253 and Met variants of TSS, which conforms to the “Trade-off” mechanism.
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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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