Study On Synthesis Of Sucrose-6-acetate By Immobilized Lipase

Sucrose-6-acetate (S-6-a) is an important intermediate in the synthesis process of sucralose which is halogenated sucrose derivative. Production of S-6-a with high yield and purity using biological enzymatic synthesis has been currently attented because of its positive significance in reducing pollution caused by chemical synthesis. In this paper, immobilization of Lipozyme TL 100 L and its application to synthesis of S-6-a in nonaqueous media were studied. And also preparation of sucralose was preliminary tried combining with chemical chlorination and deacetylation from S-6-a.Firstly, adsorption of Lipozyme TL 100 L on resin was studied. The nonpolar macroporous resin of HZ-802 was the best carrier among 7 kinds of resins. The yield of S-6-a was attained more than 80% in the conditions of adsorption temperature 30℃, pH6.32, adsorption capacity between 50.47 mg/g-58.1 mg/g resin, and immobilized enzyme relative water content less than 5%. Disposing Lipozyme TL 100 L with isopropanol at the volume ratio of 0.5:1, the concentration and yield of S-6-a reached 21.42 g/L and 95.4% by the immobilized enzyme. The near-UV CD spectra of lipase indicated that the tertiary structure changed, which could lead to rigid structure enhancing or active site exposure.Secondly, preparation of cross-linked Lipozyme TL 100 L aggregates was investigated. The optimization of CLEAs preparation was using PEG400 or PEG600 as precipitants and glutaraldehyde as crosslinker; consequently, the maximum activities of transesterification were 16.17 U/g and 19.76 U/g respectively. The activity of CLEAs was obtained 34.64 U/g with additive of D-sorbierite, followed by precipitation with PEG600. The maximum yield and concentration of S-6-a were 87.46% and 49.8 g/L when CLEAs loading were 40 mg/mL, the mole ratio of vinyl acetate to sucrose was 8:1 for reacting 8 h-10 h at 30℃.Kinetics characteristics of commercial Lipozyme TLIM, immobilized Lipozyme TL 100 L by adsorption and CLEAs were studied on synthesis of S-6-a. The maximum activities of transesterification were 7.9 U/g and 18.37 U/g respectively by the former two immobilized lipase at 30℃; however, 43.22 U/g was obtained by CLEAs at 50℃. The activation energies of aforementioned biocatalysts were 39.42 kJ/mol, 32.95 kJ/mol and 29.59 kJ/mol respectively. The transesterification followed Ping-Pang Bi Bi mechanism. Operation stabilities of immobilized lipase by adsorption and CLEAs were obviously better than Lipozyme TLIM.Synthesis of S-6-a was attempted in mixed media with ionic liquid and organic solvent. The hydrophobic ionic liquids of [Bmim]PF6, [Emim]PF6 or [MoEmim]PF6 could be used as one component of the mixture. The maximum yield and concentration of S-6-a were 80.38% and 21.79 g/L by Lipozyme TLIM at the volume ratio of ionic liquid to organic solvent 4:1 and vinyl acetate versus sucrose 8:1.Synthesis of sucralose was tried from sucrose by enzyme-chemical method. After monogroup protection from sucrose to S-6-a by enzymatic transesterification, the procedures of chlorination and deacetylation were performed with chemical process. This product was separated and purified by column chromatography, then identified as sucralose by HPLC-ELSD, HPLC-MS and NMR.