Native lipase dissolved in hydrophilic green solvents: A versatile 2-phase reaction system for high yield ester synthesis

摘要

Native lipase B from Candida antarctica was evaluated for ester production in the presence of hydrophilic green solvents including DES. Ester yields of up to 98% were achieved without removing water from the system in single step transformations. Analysis of the lipid phase exhibited a low water content of 0.29mg/mL in the presence of DES in contrast to 2.5mg/mL without solvent. Thus, water adsorbing properties of the hydrophilic solvents are responsible for the equilibrium shift towards ester synthesis, which was even observed in the presence of more than 20% water in total. Non-immobilized CalB was surprisingly active in hydrophilic solvents with logP-values below -1, when 5-10% water was added to the solvent phase and initial reaction velocities were significantly higher than with other organic solvents. Optimum reaction conditions were in the range of 40-50 degrees C with a minimum content of 50 mg/kg pure CalB needed for full conversion. The solvent phase was easily separated by decantation and final product purification with established refinement procedures yielded esters of high quality. Stability testing of soluble lipase B revealed that enzyme stability in glycerol and ChCl:Gly is exceptionally high and stabilization of lipase in denaturing ChCl:U was possible in the presence of a lipid interface. The hydrophilic solvents were superior to less polar organic solvents in regards to ester yield and initial reaction velocity. A drawback of some hydrophilic solvents in lipase-catalyzed reactions is byproduct formation due to their chemical structure containing hydroxyl groups. Practical applications: The proposed biocatalytic process is suited for the preparation of green esters for cosmetic and technical applications in low-cost production equipment. As no water removal is needed to obtain high yields and no complex downstream processing is applied, the process may be conducted in simple stirred tank reactors. The recycling of the non-volatile hydrophilic solvent by example decantation can easily be done in most production units. The utilization of soluble lipases in small quantities even allows for single-use enzyme application, though the active enzyme may be recovered with the solvent and used again to minimize catalyst costs. Lipase CalB is dissolved in the hydrophilic phase consisting of a hydrogen bonding network of solvent and water molecules. Lipase catalysis takes place at the solvent/lipid interface and water is adsorbed into the solvent phase, thus shifting the equilibrium towards ester formation in the water-depleted lipid phase.