Lipase-catalyzed Synthesis Of L-ascorbyl Fatty Acid Esters In2-methyltetrahydrofuran-containing Mixtures

L-Ascorbic acid (vitamin C) is one of the most widely used natural antioxidants.However, Its applications in liposoluble products such as animal oil, vegetable oil andcosmetics were limited significantly, due to its highl polarity. As compared to L-ascorbicacid, its fatty acid esters have been reported to have much higher liposolubility as well ascomparable or even improved antioxidative properties, so they have higher applicationpotentials in food, cosmetics and pharmaceutical industries. At present, L-ascorbyl fattyacid esters could be synthesized by chemical and enzymatic methods. The enzymaticapproaches have many advantages such as high efficiency, high regioselectivity, simpleprcoess, mild reaction conditions compared with the chemical routes. Therefore,undoubtedly, the enzymatic methods will become the mainstream technique for inducstrialsynthesis of L-ascorbyl fatty acid esters.Due to the strong polarity of L-ascorbic acid, the enzymatic synthesis of L-ascorbylfatty acid esters was generally conducted in polar organic solvents (acetone, t-butanol and2-methyl-2-butanol, etc). However, these solvents are prone to stripping the necessary wateron the enzyme surface and denaturing the enzyme, thus greatly limiting the industrialapplication of the enzymatic processes. Compared with the traditional organic solvents, biosolvents derived from biomass resources are renewable solvents, and can markedly reducethe dependence on fossil resources; besides, these bio solvents have betterenzyme-compatibility. In this thesis, Candida antarctica lipase B (CAL-B)-catalyzedsynthesis of a new L-ascorbyl unsaturated fatty acid ester was studied with undecylenic acidvinyl ester as the acyl donor in2-methyltetrahydrofuran (MeTHF, a novel biosolvent)-containing systems. In addition, the effect of various conditions on the enzymaticreaction was explored. Then, the enzyme performace of CAL-B in the synthesis ofL-ascorbyl fatty acid esters through transesterification between L-ascorbic acid andvegetable oil (such as olive oil) was investigated in the co-solvent mixtures of MeTHF andt-butanol.Of the immobilized lipases tested, CAL-B showed the highest activity in theregioselective undecylenoylation of L-ascorbic acid. The co-solvent mixture oft-butanol-MeTHF (1:4) was the optimal reaction medium for the enzymatic reaction, sincenot only CAL-B showed a high activity in this co-solvent mixture, but also L-ascorbic acidhad moderate solubility, thus addressing the major problem of the solvent conflict in the enzymatic acylation of polar polyhydroxylated compounds (the significant difference in thesolvent polarity for dissolving substrate and keeping satisfactory enzyme activity). Inaddition, CAL-B had much higher thermostability in the co-solvent mixture than in thepreviously widely used t-butanol,2-methyl-2-butanol and acetone. In this mixture, theapparent activation energy of this enzymatic reaction (26.6vs38.1-39.1kJ/mol) was muchlower, and the enzyme catalytic efficiency (Vmax/Km,8.4vs1.3-1.4h-1) was considerablyhigher than the corresponding values in the widely used organic solvents. The optimalsubstrate molar ratio (vinyl ester/L-ascorbic acid), molecular sieve dosage, enzyme dosageand reaction temperature are3,100mg/mL,30U/mL and50oC, respectively. Under theoptimum conditions, the initial reaction rate, maximum conversion and6-regioselectivityare41.2mM/h,88.8%and>99%, respectively, when the apparent concentration ofL-ascorbic acid was60mM. Besides, this immbolized lipase showed good operationalstability in t-butanol-MeTHF (1:4), and its relative activity is still more than80%afterreused for five times.Likewise, CAL-B also proved to be the optimum biocatalyst for the synthesis ofL-ascorbyl fatty acid esters from L-ascorbic acid and olive oil. The major products includedL-ascorbyl palmitate, oleate and linoleate, which contents were comparable to the contentsof the corresponding fatty acids in olive oil. The optimal reaction medium, enzyme dosage,substrate molar ratio (olive oil/L-ascorbic acid), L-ascorbic acid concentration, reactiontemperature for this enzymatic reaction are t-butanol-MeTHF (3:2),30U/mL,3,50mM,50oC, respectively. Under the optimum conditions, the initial reaction rate and the maximumconversion is7.5mM/h and70.7%, respectively. Additionally, the reaction conditions hadslight effect on the contents of various components in the product. CAL-B showed goodoperational stability in t-butanol-MeTHF (3:2), and the relative enzyme activity of77%remained after reused for6times. CAL-B can also catalyze the transestrification betweenL-ascorbic acid and other plant oil such as corn oil, soybean oil, rapeseed oil int-butanol-MeTHF (3:2), affording L-ascorbyl fatty acid esters with different compostions.The present study not only gained a deeper understanding of biocatalysis andbiotransformation in bio solvent-containing systems, but also provides new enzymaticapproaches to L-ascorbyl fatty acid esters with active vinyl ester and plant oil as the acyldonors, respectively.