糖氨基转移酶（SAT）可在特定酮糖的结构上引入氨基，生成具有生物学活性的氨基糖。这一特性已被应用于氨基糖的人工设计合成。例如，利用糖氨基转移酶WecE将井冈霉烯酮转化为高价值的α-葡萄糖苷酶抑制剂井冈霉烯胺。然而，WecE的低热稳定性及其对非天然底物催化活性不足限制了其应用。受限于广泛存在的酶稳定性-活性权衡（stability-activity trade-off）效应，同时提升酶热稳定性和催化活性具有技术挑战性。为了实现WecE热稳定性和催化活性的高效进化，我们使用进化保守性和平均突变折叠能等指标对WecE活性中心57个氨基酸残基进行评估，筛选到14个有助于同步提升热稳定性和催化活性的潜在热点，并采用组合活性中心饱和突变-迭代饱和突变（CAST-ISM）策略针对热点残基进行了正向突变筛选和迭代组合。与WecE野生型相比，第四轮迭代突变体M4（Y321F/K209F/V318R/F319V）在40 °C的半衰期提高了641.49倍，对非天然底物井冈霉烯酮的催化活性提高了31.37倍。第三轮迭代突变体M3（Y321F/K209F/V318R）在40 °C的半衰期提高了83.04倍，对井冈霉烯酮的活性提高了37.77倍。催化性能提升机制分析显示，与野生型相比，热稳定性和催化活性同时提升的突变体蛋白界面相互作用增强、氨基转移反应亲核进攻催化距离缩短。本文提供了一种热稳定性-催化活性热点评估的通用策略，为氨基糖类化合物的人工生物合成提供高稳定性和高催化活性的糖氨基转移酶。

Sugar aminotransferases (SATs) catalyze the installation of chiral amines onto specific keto sugars, producing bioactive amino sugars. Their activity has been utilized in artificial reactions, such as using the SAT WecE to transform valienone into the valuable α-glucosidase inhibitor valienamine. However, the low thermostability and limited activity on non-natural substrates have hindered their applications. Simultaneously improving stability and enzyme activity is particularly challenging owing to the acknowledged inherent trade-off between stability and activity. A customized combinatorial active-site saturation test-iterative saturation mutagenesis (CAST-ISM) strategy was used to simultaneously enhance the stability and activity of WecE toward valienone. Fourteen hotspots related to improving the stability-\activity trade-off were identified based on evolutionary conservation and the average mutation folding energy assessment of 57 residues in the active site of WecE. Positive mutagenesis and combinatorial mutations of these specific residues were accomplished via site-directed saturation mutagenesis (SSM) and iterative evolution cycles. Compared with those of the wild-type (WT) WecE, the quadruple mutant M4 (Y321F/K209F/V318R/F319V) displayed a 641.49-fold increase in half-life (t_1/2) at 40 °C and a 31.37-fold increase in activity toward the non-natural substrate valienone. The triple mutant M3 (Y321F/K209F/V318R) demonstrated an 83.04-fold increase in (t_1/2) at 40 °C and a 37.77-fold increase in activity toward valienone. The underlying mechanism was dependent on the strengthened interface interactions and shortened transamination reaction catalytic distance, compared with those of the WT, which improved the stability and activity of the obtained mutants. Thus, we accomplished a general target-oriented strategy for obtaining stable and highly active SATs for artificial amino-sugar biosynthesis applications.