The Mechanism Of The Synergistic Effects Of Natural Deep Eutectic Solvents And Enzyme And Their Applications In The Synthesis Of Lipoamino Acids

Lipoamino acids（LAA）,also known as N-acyl amino acid,have exhibited their extremely advantages in fields of food,personal care and bio-pharmacy.Moreover,Natural deep eutectic solvents（NADESs）,a new type of novel green solvent,have been proven to have extremely advantages in a variety of enzymatic synthesis systems.In this study,the activity and thermal stability of enzymes in NADESs and the mechanism of NADESs influence enzyme activity were explored.The synergistic effect of NADESs and enzyme were also studied via molecular dynamic simulation（MD）and density functional calculation（DFT）.The results are as following:（1）In this study,the potential application of NADESs used as a medium in enzymatic synthesis of lipoamino acids were investigated.The results of lipase activity and stability suggested that the activity of CALB in the B-Gly and C-Gly systems was increased by 1.68times and 1.25 times,respectively,and the thermal stability was also increased by115.48±1.36%and 108.54±1.26%,respectively.To explore the mechanism of it,the CD spectroscopy and fluorescence spectroscopy were used to study the secondary structure of CALB in different systems and found that enzyme can remain structural stability in NADESs.In addition,the results of molecular dynamic simulation（MD）showed that the increase in lipase activity can be attributed to the interaction of NADESs and substrates which may activate the substrates.（2）In the above studies,most NADESs have a high viscosity,which makes the experiment operation difficult.To reduce the viscosity of NADESs,we add water into it to form a NADESs-H₂O binary system.It was found that with the addition of water,the viscosity and polarity of NADESs can be significantly decreased.DFT results showed that there is a tendency to form hydrogen bonds between C-Gly and H₂O.The results of the excess infrared spectroscopy showed that the methyl and methylene groups of C-Gly are significantly blue-shifted,and the peak positions of the excess infrared spectrum are almost fixed,which indicated that hydrogen bonds can be formed between them.The ¹H NMR and¹H-¹H NOESY results suggested that with the addition of water,there are four types of C-Gly cluster can be formed,when 0.1<x（D₂O）<0.3,the larger clusters are decomposed into small clusters;when 0.3<x（D₂O）<0.7,the small clusters gradually decomposed into monomers;when x（D₂O）>0.7,the C-Gly structure is destroyed,and choline chloride and glycerol were released.Moreover,the addition of water can improve the activity and stability of CALB in NADESs.It can be attributed to that with the addition of water,the aggregation of NADESs decreased significantly.Therefore,more NADESs components can be diffused to the active site of CALB.（3）Then,we conducted a systematic solvents and enzymes screening processes and finally obtained a yield of up to 59.14%.Interestingly,Gemini lipoamino acids（GLAA）cannot be synthesized to any perceivable yield in NADESs without CALB,while NADESs-CALB system can obtain a high yield.The results of DFT calculation and AIM analysis showed that there is a strong non-bonding interaction between C-Gly and lauric acid molecules,and hydrogen bonds play a dominant role in it.In addition,we also found that choline cations and glycerol also have various non-bonding interactions with Cl^-,which occupy their binding sites.（4）Finally,to improve the concentration of anions,we designed a series of new NADESs by adding metal chloride hydrates,in which the yield of GLAA was increased to77.44±1.22%.MD results suggested that the molecules in new-designed NADESs have been rearranged,and forming a strong interaction between Mg-glycerol,and thus increasing the Lewis acidity of the system.In addition,the results of non-bond interaction analysis and AIM analysis suggested that more Cl^-binding sites can be exposed in the new-designed NADESs.Molecular frontier orbital analysis suggested that the electrophilicity of lauric acid was significantly improved.The IRC analysis showed that Cl^-acts as a“transporter”during the reaction processes,so that the hydrogen atoms of amino acid can be first bound by Cl^-,and then further bound by the hydroxyl groups of fatty acids,thereby reducing the energy barrier of reactions.In summary,we found that enzyme can be activated in NADESs and the hydrogen bonding interaction between Cl^-and substrates play a key role in this process.Based on this,we designed a series of new-type NADESs and the yield of GLAA was significantly improved.In this study,we aimed at laying a certain theoretical foundation for the development of a green and safe enzymatic reaction system.