The capacities for thermal and inhibitor tolerance are critical for industrial enzymes and loss of activity is a major challenge in deploying natural enzymes for commercial applications. Protein engineering approaches， such as site-directed mutagenesis and directed evolution， have been devoted to modifying natural enzymes. Recently， a post-translation protein engineering strategy， the SpyTag/SpyCatcher system， was introduced. Here， we have generated a thermo- and ion-tolerant cyclized xylanase (C-TFX) by fusing the SpyTag and SpyCatcher peptides to its N- and C- terminus respectively. Compared with the linear enzyme， C-TFX retained greater residual activity after heating or metal ion exposure. Int... More
The capacities for thermal and inhibitor tolerance are critical for industrial enzymes and loss of activity is a major challenge in deploying natural enzymes for commercial applications. Protein engineering approaches， such as site-directed mutagenesis and directed evolution， have been devoted to modifying natural enzymes. Recently， a post-translation protein engineering strategy， the SpyTag/SpyCatcher system， was introduced. Here， we have generated a thermo- and ion-tolerant cyclized xylanase (C-TFX) by fusing the SpyTag and SpyCatcher peptides to its N- and C- terminus respectively. Compared with the linear enzyme， C-TFX retained greater residual activity after heating or metal ion exposure. Intrinsic fluorescence and circular dichroism analysis revealed that the isopeptide bond mediated by SpyTag/SpyCatcher cyclization contributed to enhanced thermo- and ion-stability， probably by stabilizing its secondary and conformational structure. In addition， the heat-challenged C-TFX was observed to degrade natural lignocellulosic substrates efficiently. The cyclized xylanase was more stable and resistent to denaturation and aggregation than the linear enzyme. The "superglue" SpyTag/SpyCatcher cyclization system enables the enzyme to maintain its structural conformation， which will be of particular interest in engineering of enzymes for industrial application such as feed additives and functional oligosaccharides production.
