[Screening, characterization, and application of a novel pectinase in ramie degumming]

Pectinases play a crucial role in the enzymatic degumming process of ramie. However, existing pectinases often suffer from issues such as low degumming efficiency and long processing time. Therefore, it is essential to discover novel efficient pectinases for the ramie degumming process. In this study, we performed carbohydrate-active enzymes annotation on the coding sequence (CDS) and protein sequence of Bacillus licheniformis ATCC 14580, identifying a pectate lyase BlicPL1 whose function has not yet been verified. An expression vector for this gene was constructed and expressed in Escherichia coli BL21(DE3), followed by protein purification. This study marks the first experimental confirmation that BlicPL1 functions as a pectate lyase, with a specific enzyme activity of 3 952.54 U/mg, which is higher than those of most reported pectate lyases from the polysaccharide lyase 1 (PL1) family. Enzymatic characterization revealed that the optimal pH and temperature for BlicPL1 activity were 10.0 and 60 ℃, respectively. Notably, BlicPL1 maintained high activity (over 80% relative activity) within a broad range of 50-80 °C and pH 10.0-12.0. This thermostable and alkali-tolerant profile aligns well with the high-temperature, high-pH conditions typical of ramie degumming processes. Unlike the conventional pectate lyases which are activated by Ca²⁺, BlicPL1 showcased the activity not promoted by Ca²⁺, or even inhibited by Ca²⁺, which highlighted its uniqueness in the Ca²⁺ response mechanism and suggested that BlicPL1 was a novel pectate lyase. Furthermore, BlicPL1 was employed for ramie degumming, resulting in a weight loss rate of 16.17% and a degumming rate of 44.09% for ramie fibers. Scanning electron microscopy results indicated that the treatment with BlicPL1 helped remove residual middle lamella tissue from the ramie fiber surface and reduced the adhesion of free gummy substances, resulting in smoother and more separated fibers. X-ray diffraction analysis confirmed that the characteristic cellulose peaks of the ramie fibers remained unchanged after degumming by BlicPL1, while the crystallinity index increased from 74.59% to 79.69%, indicating an increased proportion of cellulose and the successful removal of non-cellulosic components. The pectate lyase BlicPL1 identified in this study, with high activity and suitability for medium-high temperatures and alkaline environments, demonstrates significant application potential in the ramie degumming process. This work lays a foundation for advancing the industrial application of pectinases in ramie degumming.