Protein topology engineering has emerged as a new dimension to alter protein stability and function. Inspired by the art of nature, where backbone cyclization is frequently adopted to enhance the stability of natural peptide products and thermostable enzymes; herein, we report protein topology engineering of an industrial thermolabile gamma lactamase via catenation. Two different protein catenanes were successfully constructed via SpyTag/SpyCatcher modules and two different peptide dimer domains. The designed protein catenanes were functionally synthesized in Escherichia coli. A comparison of their biochemical properties revealed that protein topology played a key role in the stability of gamma lactamase. Protein catenation enhanced both the thermo- and proteolytic stabilities of gamma lactamase. Gamma lactamase was stabilized by ∼8 °C in one of the catenated forms. Moreover, Cat1-MhIHL-V54 L and Cat2-MhIHL-V54 L displayed 1.8- and 2.4-fold higher enzyme efficiencies (Kcat/Km), respectively, than the unattenuated enzyme. Therefore, our results proved that protein catenane construction could be a general strategy to strengthen industrial biocatalysts by mechanisms distinct from those of the conventional direct evolution schemes, whereby our results offer wide applications in the fine chemical industry.

蛋白质拓扑工程已成为改变蛋白质稳定性和功能的新维度。受自然艺术的启发，经常采用骨架环化来增强天然肽产品和热稳定酶的稳定性；在此，我们报告了工业不耐热 γ 内酰胺酶通过连锁的蛋白质拓扑工程。通过 SpyTag/SpyCatcher 模块和两个不同的肽二聚体结构域成功构建了两种不同的蛋白质链。设计的蛋白质链是在大肠杆菌中功能合成的。比较它们的生化特性表明，蛋白质拓扑结构在 γ 内酰胺酶的稳定性中起着关键作用。蛋白质链增强了 γ 内酰胺酶的热稳定性和蛋白水解稳定性。γ 内酰胺酶以一种链状形式在约 8°C 下稳定。此外，Cat1-MhIHL-V54 L 和 Cat2-MhIHL-V54 L 的酶效率 (Kcat/Km) 分别比未减毒酶高 1.8 倍和 2.4 倍。因此，我们的结果证明，蛋白质链构建可能是通过与传统直接进化方案不同的机制来增强工业生物催化剂的一般策略，从而我们的结果在精细化工行业中具有广泛的应用。