Abstract
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-V54L and Cat2-MhIHL-V54L 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.
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