Efficient monoacylglycerol synthesis by carboxylesterase EstGtA2 from Geobacillus thermodenitrificans in a solvent-free two-phase system

Abstract

The present study investigated high-yield monoacylglycerol (MAG) synthesis by bacterial lipolytic enzymes in a solvent-free two-phase system. Esterification by monoacylglycerol lipase from Bacillus sp. H-257 (H257) required a high glycerol/fatty acid molar ratio for efficient MAG synthesis. Screening of H257 homologues revealed that carboxylesterase derived from Geobacillus thermodenitrificans, EstGtA2, exhibited a higher esterification rate than H257. Moreover, neutralizing the pH of the acidic reaction solution by adding potassium hydroxide (KOH) solution further increased the esterification rate. The esterification rate by EstGtA2 reached 75% under conditions of equivalent molar amounts of glycerol and fatty acid, and the MAG rate (MAG/total glyceride) was 97%. The neutralized pH of the reaction solution likely affected the thermal stability of EstGtA2 during the esterification reaction. Screening for thermal-tolerant variants revealed that the EstGtA2S26I variant was stable at 75°C for 30min, a condition under which wild-type EstGtA2 was completely inactivated. The esterification rate by the EstGtA2S26I variant reached 90%, and the MAG rate was 96%. The addition of alkali and the use of a thermal-tolerant enzyme were important for obtaining high-yield MAG in a solvent-free two-phase system utilizing EstGtA2.