Lipase-Catalyzed Synthesis Of Fructose Laurate

Sugar esters of fatty acids (sugar ester) have many applications as biocompatible and biodegradable nonionic surfactants. Application performance of sugar esters depends on the composition of amphiphilic molecules, its chemical structure, and degree of substitution. Compared to conventional methods of chemical synthesis, enzymatic preparation of sugar esters of fatty acids have many advantages, such as eco-friendly reaction conditions and high substrate selectivity and product specificity. Selective synthesis of a single product has always been one of hotspots of research in enzymatic synthesis. In this dissertation, lipase catalyzed synthesis of fructose laurate in nonaqueous media were studied. We focused on different degrees of esterification (DE, substitution degree of fructose by lauric acid) in products, and concerned selective synthesis of fructose monolaurate and fructose dilaurate. By scaning the impact of solvent media on the structure and catalytic dynamics of lipase, we could further explore the substrate specificity and selectivity of esterification, and reveal the mechanism of selective synthesis of lipase. In addition, fructose mono-ester and di-ester in two types of esterification degree products were also investigated for their application performance as surfactants.Enzymatic direct esterification of fructose with lauric acid was performed in organic solvents using two representative immobilized Candida antarctica lipase B (CALB) and Rhizomucor miehei lipase (RML). The esterification degree and conversion yield of the acylation process clearly depended on the lipase source, organic solvent property, initial water activity, temperature and substrate ratio. For all cases tested, no other than mono-and diester of fructose laurate were detected within 12h. The environmental factors affected the DE selectivities of lipase, and DE are significantly different in various reaction systems.With CALB catalyzed direct esterification of fructose with lauric acid, Significant difference of DE was observed between 2-methyl-2-butanol (2M2B) and methyl ethyl ketone (MEK), as di-ester/mono-ester molar ratio of 1.05:1 in 2M2B and 2.79:1 in MEK. CALB exerted much better selectivity for di-ester synthesis in MEK. Fourier transform infrared (FTIR) spectra showed that the secondary structure of the enzyme binding mono-ester presented distinct difference in 2M2B and MEK. Contents of β-turn and antiparallel P-sheet of CALB in 2M2B were 26.9% and 16.2%, respectively, but 19.1% and 13.2% in MEK. When mono-ester and fatty acid were directly employed for synthesis of di-ester, the maximum initial velocity of di-ester synthesis in MEK was 0.59 mmol·g (enzyme)-1·h-1, which was 2.19-fold as greater as that in 2M2B, indicating that CALB conformation in MEK was preferred for the synthesis of di-ester.ILs were selected as co-solvent with 2M2B in which CALB was assessed with low DE selectivity, and we studied the selective synthesis of fructose monolaurate. The results showed that markedly improved ratio of monolauroyl fructose was achieved in the presence of 60% [BMIM][TfO] (v/v) and 20%[BMIM][BF4] (v/v), in which the mono-acylated fructose were 85% and 78% respectively. Based on a Ping-Pong Bi-Bi kinetic model, kinetic equation was fitted, and kinetic parameters revealed that the higher affinity between fructose and acyl-enzyme intermediate was enhanced, the inhibition effect of fructose on free enzyme was weakened, and mono-acylation of fructose was dominated in the presence of IL co-solvents. The secondary structure of CALB binding substrates did change in the co-solvent system proved by Fourier transform infrared spectra. Collectively, these results demonstrated that the variance of enzyme kinetic characteristics of CALB essentially promoted the selectivity of mono-acylation in ILs/2M2B co-solvent system.We surveyed several aspects of physicochemical properties of fructose monolaurate and dilaurate as sufactants, the result showed:hydrophilic-lipophilic balance value of mono-ester is higher than di-ester, it is more suitable for use as a water-in-oil emulsifier. But the critical micelle concentration of di-ester is much lower than mono-ester, it means di-ester could more easily aggregate to form micelles in water. Emulsifying capacity of mono-ester is better than sodium dodecyl sulfonate (SDS), even surpasses di-ester. Emulsion stability of both sugar esters at low concentrations for soybean oil was stronger than SDS. Both of the esters are of low-foaming surfactants. Fructose monolaurate has stronger antibacterial activity against Staphylococcus aureus, and inhibition rate achieved 91.85% within 18 h.Taken together, thses studies proved that lipase possess DE selectivities in synthesis of fructose laurate in nonaqueous media, its mechanism is solvents changed enzyme structure, and also changed enzyme kinetic characteristics to achieve the selective synthesis of fructose monolaurate and dilaurate.