Lipase-Catalyzed Esterifications In Nonaqueous Reaction Systems

A new procedure for the preparation of high-purity linoleic acid from native safflower oil was developed, and the method was shown to possess industrial application potentials. The linoleic acid product with a purity of higher than 98% was obtained with overall yield of higher than 48%. The products with total conjugated linoleic acid (CLA) content of 97% and biologically active isomer content of higher than 90% were achieved by alkali-catalyzed conjugation of high-purity linoleic acid under optimized conditions. The major isomers were identified by the GC-FTIR and GC-MS analysis of the products. A solvent-free reaction system for the preparation of high-purity 1,3-diconjugated linoleoyl glycerol (1,3-dCLG) was designed. The reaction was conducted for 2-4 h using Novozym 435 as biocatalyst with N2 stirring under vacuum at 45-60 °C, and the 1,3-dCLG product of 92-98% purity was produced. In this reaction system, the evolution of esterification was monitored by the detection of the water activity over the reaction mixture. The response surface methodology (RSM) was employed to optimize the reaction conditions for the Novozym 435 catalyzed synthesis of 1,3-dCLG, and the best fitting models were established. The efficiency of the model was experimentally verified. A scale-up experiment was successfully performed based on the optimized condition, indicating the industrial potentials of the technology.Solvent-free synthesis of high-purity triconjugated linoleoyl glycerol (tCLG) employing Novozym 435 as biocatalyst was performed. And this reaction design was shown to possess industrial application values. It's estimated that 128-190 kg tCLG products of 95-98% purity with high extra value could be produced by the reaction design employing 1 kg Novozym 435. A novel hydrophobic magnetic microsphere encapsulating oleic acid-stabilized nano-sized magnetic particles was developed for lipase immobilization. Environmental scanning electron microscope, transmission electron microscope and sample vibrating magnetometer etc. were employed to characterize the carrier. The results indicated that the magnetic behavior of the microspheres could be described by Langevin equation and was proved to be superparamagnetic. The carrier has strong responsiveness and good redispersion in many media. Candida rugosa lipase (CRL) was immobilized to the porous carrier at up to 6750 IU/g carrier, remarkably higher than the previous studies. The immobilized CCL showed enhanced thermal stability and good durability in the repeated use after recovered by magnetic separations. Activity amelioration of the immobilized CRL in the hydrolysis of olive oil indicated an "interfacial activation" after immobilization.The activity of the immobilized lipase adsorbed on hydrophobic and superparamagnetic microspheres was 2 to 3-fold higher than that of the native one in catalyzing esterification in hydrophobic solvents (log P, 2-4). Exponentially increasing curves of the activity of the immobilized and native lipases against the hydrophobicity (log P) spectrums in mixed two miscible solvents were firstly observed. A solvent-free multiple-competitive reaction was designed to examine the alcohol specificity of the lipases and revealed that the alcohol specificities of the lipases had a strong chain-length dependency. Moreover, this work showed that alcohol specificity of the lipases was influenced by the nature of acyl donors, and the lipase became more preferential for longer-chain alcohol after immobilization on the hydrophobic microspheres.