Multidimensional Computational Engineering of Pectate Lyase for Enhanced Thermostability and Moderately Improved Catalytic Efficiency

Abstract

Pectate lyases are promising industrial biocatalysts, but limited thermostability restricts their practical use. Here, we developed a multidimensional consensus computational framework integrating sequence conservation, structural dynamics, and thermodynamic prediction to identify functional mutation hotspots in PcPel1834. Iterative saturation mutagenesis yielded M5 (S212E/Q225L/F241L/L260A/G296T), which showed markedly enhanced thermostability and moderately improved apparent catalytic efficiency. M5 increased the melting temperature from 52.1 to 65.0 °C and retained nearly full activity after 24 h at 50 °C. Its specific activity and kcat/Km reached 4259 U/mg and 1427 mL/s/mg, respectively, both higher than WT. Epistatic analysis and MD simulations indicated that M5 enhanced thermostability through hydrogen-bond remodeling, backbone rigidification, and strengthened long-range communication, while distal mutations allosterically preorganized the catalytic pocket to improve substrate binding and catalytic geometry. In ramie degumming, M5 showed improved pectin removal and fiber quality. This study provides a practical strategy for engineering thermostable pectate lyases with improved catalytic performance.