Molecular Characterisation of an Uncommon Enantioselective Lipase TALipA from Trichosporon asahii MSR54, Highlighting the Discovery of an AXSXG Signature Sequence

Abstract

A lipase-encoding gene, designated as TALipA, was effectively produced in Pichia pastoris X-33 after being cloned from Trichosporon asahii MSR54. Using affinity chromatography, the enzyme was purified with a purification fold of 1.8, and SDS-PAGE analysis confirmed it as a monomeric protein of 27 kDa. Close resemblance to bacterial and actinobacterial lipases was revealed by comparative sequence analysis, while having distinctive characteristics like a conserved AHSMG pentapeptide, where alanine replaces glycine—a rare feature among yeast lipases—and an uncommon oxyanion hole "GL." The enzyme was most active at 60 °C and pH 8.0, and it remained stable with a half-life of 68 minutes at 70 °C. TALipA displayed substrate specificity toward long-chain p-nitrophenyl esters, especially p-np palmitate, which was verified during the hydrolysis of triacylglycerides. Hydrolysis of triolein revealed regioselective behavior, while esterification of phenylethanol demonstrated solvent-dependent enantioselectivity— preferring the R-enantiomer in isopropanol and hexane and the S-enantiomer in 1,4-dioxane. The lipase was further identified as a magnesium-activated metalloenzyme, with stability in the majority of polar and non-polar solvents but activity inhibited by 10 mM EDTA. These findings suggest that TALipA represents a novel yeast lipase with significant potential as a biocatalyst for industrial applications.