Semi-rational engineering of linoleic acid isomerase improves catalytic efficiency and substrate affinity for conjugated linoleic acid biosynthesis.

Conjugated linoleic acid (CLA) is an important functional compound in the food and pharmaceutical industries, and its biosynthesis depends on linoleic acid isomerase (LAI). The natural LAI, however, suffers from low catalytic efficiency and substrate affinity, which limits CLA synthesis and industrial application. To address this, a semi-rational engineering strategy was applied to modify LAI from Propionibacterium acnes (P. acnes), constructing a soluble single-enzyme system using P229 as the parent strain. Five promising mutation sites (G36R, G7Q, Y319W, E392R, F399W) were identified and experimentally validated through overlap extension PCR. The pCold-lai-pG36R variant showed 4.56-fold higher gene expression and 1.95-fold increased enzymatic activity compared to the wild type P229 (WT). Structural analysis revealed that the G36R mutation introduced salt bridges and shorter hydrogen bonds. The purified mutant retained > 60% of its CLA productivity across 20-40 ℃ and pH 7-8, achieving optimal activity at 30 ℃ and pH 7.0 (Vmax = 21.41 μg·mL-1·h-1 and Km = 0.22 mg·mL-1). These findings highlight the potential of semi-rational engineering to enhance LAI performance, offering valuable insights for industrial applications.