| In food and drug industry, safety environmental demand for products in the process of catalytic conversion is high as the conventional chemical catalyst and solvent cannot meet the current environmental needs. Development and application of new green catalysts and solvents have been receiving increasing attention. In recent years, although green solvent such as supercritical fluid, ionic liquid and deep eutectic solvent were shown to good application prospects. However, the application of un-natural ingredients and solution is not fully meeting the needs of real green, efficient catalytic reaction. Therefore, thought back to the biocatalyst, and to it’s natural medium-cytoplasm to select the reaction solvent, will be an ideal direction to solve this problem.Natural deep eutectic solvent, a novel solvent put forward based on cytoplasm components analysis in the field of biology and thinking from the concept of “deep eutectic solvent†in the field of chemistry, formed from organic small molecule metabolites such as choline derivatives, alcohols, sugars, urea and other substances. In view of this solvent may exist widely and function as medium in nature, when used as reaction medium, it is promising to solve the bottleneck that enzyme application in industry because of non-matched environment, thus provide a truly appropriate medium for biocatalytic reaction.Based on the above consideration, this paper mainly work is, for the first time to explore the solvent formed by organic small molecules introduce into biocatalytic reactions. Study on basic properties of these natural deep eutectic solvents and performance of lipases in these solvents, and preliminary assessment of conversion efficiency and possible forms of lipase catalytic transformation of oil in this novel solvent. The main research content for the following four aspects:First, instrumental analysis was to study the basic physical and chemical properties, the solvent molecular structure and discussed the formation and the interaction mechanism between components of solvent studied by a combination of 13 C NMR, 1 H NMR, FT-IR, DSC/TG. Investigation of the effect of water addition to the physicochemical and physiological properties of this solvent. The results showed that the solvent formed mixture of liquid at room temperature was mainly through hydrogen bonding; A certain amount of water, in turn, reduced the degree of internal hydrogen bond interaction in solvent and the viscosity of solvent significantly. Thus, by controlling the water content can be achieved the goal of improving the applications of this solvent. Preliminary research has proven the formation mechanism of solvent and chemical properties, and assessed the effect of water on the application of this solvent.Second, evaluated the stability and substrate selectivity of lipases in natural deep eutectic solvent. Results showed that the influence of solvents on the performance of lipase was difference, solvents containing organic acid inhibited the activity of lipase, and in solvents containing “osmolyte†such as betaine, xylitol, trehalose, lipase maintained with similar or even higher activity as that in the water. The thermal stability of lipase was observed improved obviously in these solvents. Heat preservation in solvent betaine-glycerol, choline chloride-trehalose, and betaine-xylitol in 80℃ for 2 h, enzyme activity of CALB lipase didn’t decrease obviously. The effects of natural deep eutectic solvents on fatty acids selectivity of lipase were obviously, especially for short chains of substrate. In natural deep eutectic solvents, the selectivity for C4 substrate of lipase TL100 was reduced significantly. For lipase CALA, the reactivity of C6 substrates increased after the addition of solvent.Third, examined the characteristics of esterification reaction in natural deep eutectic solvents, through the synthesis of two kinds of nutritional function product 1, 3-diacylglycerol and n-3 PUFA triglycerides verified the advantage of this solvent as biocatalytic reaction media. The substrate selectivity of lipase changed when existence of natural deep eutectic solvent in esterification reaction system. 1) the existence of choline chloride was advantageous to accumulation of diglyceride especially 1, 3-diacylglycerol. Lipase Novozym 435 catalytic esterification reactions in solvent containing choline chloride, the 1, 3- diacylglycerol content up to 42.9% at 1 h(56.7 wt%). 2) the existence of this solvent, especially the presence of urea components help for the accumulation of triglyceride in this esterification reaction. In choline chloride-urea(1:1) solvent system, lipase Novozym 435 catalytic esterification under the condition of 55℃ for 12 h, the content of n-3 PUFA triglycerides was 55.03 wt %.The last, explored a possible model of lipase catalyzed hydrolysis reaction in natural deep eutectic solvent. As one of the natural deep eutectic solvent components, choline chloride or betaine can set up a new type of aqueous two-phase system. The capacity for protein extraction and the separation mechanism of this system was evaluated, and formed a new reaction system application for oil hydrolysis. In betaine/K2HPO4 aqueous two-phase system, extraction efficiency of bovine serum albumin for more than 90%, meanwhile, provided a mild and stable environment for this protein. Examined distribution of protein and enzyme activity of 4 common commercial lipase(CALB, CALA, TL100 and AYS) in quaternary ammonium salts based aqueous two-phase system. Glycerol adsorption by those quaternary ammonium salts for the formation of solvent in hydrolysis reaction. Lipase AYS catalytic hydrolysis of olive oil, FFA content reached 91.44% at 3 h in the hydrolysis reaction system. |
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