Characterization, Modification, and Preliminary Application of a Novel Pectate Lyase from Paenibacillus tarimensis in...

Ramie fiber, an exceptional natural textile material, requires degumming treatment to obtain spinnable mature fibers. Pectate lyase stands as the most effective enzyme for degumming by specifically removing pectin that binds multiple gummy components. However, commercial enzyme cocktails often contain cellulase activities causing significant fiber damage. Furthermore, the high-temperature and strongly alkaline conditions inherent to ramie processing render conventional pectate lyases incompatible with this specialized industrial environment. Consequently, developing thermostable and alkali-tolerant pectate lyases tailored for ramie degumming holds critical importance. This study identified a novel alkali-thermostable pectate lyase (pel114) from Paenibacillus tarimensis and achieved its heterologous expression in Pichia pastoris. Biochemical characterization revealed that pel114 exhibits optimal activity(910 U·mg^-1) at 60°C and pH 10.0, while maintaining remarkable stability across a broad pH range (6.0-12.0). Through integrated strategies combining FireProt-predicted stability hotspots, molecular dynamics simulations of flexible regions, and consensus mutation design, we engineered the V467P/A566P double mutant, demonstrating superior thermostability, with a specific activity of 891 U·mg^-1 against polygalacturonic acid. The mutant displayed a 65°C half-life of 429.9 min-a 10-fold enhancement over the wild type (WT) (42.9 min). These findings present a promising biocatalyst with substantial potential for advancing enzymatic degumming technologies in ramie processing.