A highly alkaline pectate lyase from Marinimicrobium sp. EAC58 and improved thermostability by rational design

Pectic oligosaccharides (POS) are novel prebiotics derived from pectin, exhibiting diverse bioactivities including antimicrobial, antitumor, antioxidant and immunomodulatory activity. As promising functional food ingredients, POS have attracted considerable attention for their health benefits. Enzymatic production via pectate lyases represents a sustainable and efficient route for POS generation. Consequently, the identification and the engineering of high-performance pectate lyases are critical for improving production efficiency and enabling the industrial application of POS.

A novel pectate lyase MarPel was successfully cloned and heterologously expressed from marine bacterium Marinimicrobium sp. EAC58. The soluble tags enhanced the expression of MarPel in Escherichia coli. The purified MarPel displayed typical pectate lyase activity and hydrolyzed pectin into unsaturated digalacturonic acid and unsaturated trigalacturonic acid. Biochemical characterization revealed that MarPel is an extremely alkaline-active enzyme with an optimal pH of 12.0 and an optimal temperature of 45 °C. To expand its industrial applicability, structure modification guided by molecular docking and molecular dynamics simulations was performed to enhance its thermostability. The optimal mutant MarPel-L262S exhibited an optimal temperature of 60 °C (15 °C higher than MarPel-WT) and a half-life (t_1/2) of 15.75 min at 60 °C, representing a 2.3-fold increase relative to the MarPel-WT.

To our knowledge, MarPel is the most alkali-tolerant pectate lyase reported to date. The L262S mutant integrates exceptional extreme-pH resilience with superior thermal stability, rendering it a promising biocatalyst candidate for the enzymatic production of POS. © 2026 Society of Chemical Industry.