Fabrication Of The Molecular Tweezers Modified Support And Lipase-catalyzed Chiral Resolution

Lipase is a kind of very important biological hydrolytic enzymes.The main features of the lipase include hydrolase folding(the center is covered by a hydrophobic β-sheet from both sides by the α-helix)and the active site consists of Ser-His-Asp / Glu triplet and an oxyanion hole.In most cases,the α-helix covers the active site to form a "lid".Structural functional studies have shown that the morphology,size,and amino acid composition of internal tunnels and "lid" are crucial to the catalytic versatility of lipase.Bile salt,a biosurfactant,has amphiphilic molecules with polar and nonpolar surfaces,large volume,rigid,non-toxic and biodegradable,biocompatible.Bile salt has an interface-induced activation function to lipase,thereby promoting the chiral catalytic ability of lipase.Therefore,the combination of lipase and bile salt is expected to improve the chiral resolution of lipase in the transesterification of methyl-racemic aminoacid.In this work,firstly,six kinds of bile salts were selected to study the effects of different concentrations of bile salts on the catalytic performance of porcine pancreatic lipase(PPL).Then deoxycholic acid was used as the skeleton to graft and modify two hydroxyl groups on its structure,and a chiral asymmetric urea deoxycholic acid molecular tweezer with two "arms" and a "split hole" was prepared.UV spectrophotometric titration was used to determine the identity between molecular clamp and amino acid methyl ester.The molecular tweezers and lipase were then mixed for the determination of the transesterification of racemic amino acid methyl ester and the catalytic performance was determined by high performance liquid chromatography.The specific conclusions are as follows:1.After treatment of six kinds of bile salts,the effects of lipase activity tend to be similar: with the increase of the concentration of bile salts,the catalytic activity of lipase increase firstly and then decline.The maximum near to the critical micelle concentration(CMC)value of each bile salt.This shows that both the enantioselectivity and the catalytic activity of lipase with the bile salt are increased at the same time in a certain concentration range.2.The accuracy of the molecular tweezers structure was confirmed by NMR.The interaction between the molecular tweezers and the D/L-amino acid methyl ester was determined by UV spectral titration.The maximum absorbance of the complex showed a regular increase.According to the association constants Ka and the Gibbs free energy changes-ΔG0 to determine the interaction and chiral recognition is generated.The association constants of the molecular tweezers to L-amino acid methyl ester are higher than those of the D-amino acid methyl ester(except for valine methyl ester),indicating that the molecular tweezers bind preferentially to L-amino acid methyl ester;And the molecular tweezers have the strongest effect on the tryptophan methyl ester.This is mainly due to the fact that L-tryptophan methyl ester approaches the urea group arm in C7 and C12 positions in the cavity and that D-tryptophan methyl ester is close to the C3 position,so the binding effect of L-tryptophan methyl ester and molecular tweezers is stronger than of D-tryptophan methyl ester.3.According to the results of HPLC,it was found that the addition of lipase(PPL)in the transesterification reaction,the selectivity of transesterification was significantly increased,but the activity was decline.Adding molecular tweezers based on the presence of lipase,the selectivity was showing a slight increased,and the activity had also been improved.This shows that chiral asymmetric uridine acid molecular tweezers on the catalytic activity of lipase have an impact,but not particularly obvious.Among the six kinds of molecular tweezers,tryptophan molecular tweezers have the highest increase in activity and selectivity for enzymatic reaction when mixed with lipase.Lipase has chiral selectivity,and molecular tweezers can induce the "lid" of the lipase to open,exposing the active site,thereby increasing the catalytic activity of the lipase.4.The bile acid functionalized carrier material was prepared by graft modification of polyacrylonitrile fiber.The chiral functionalized carrier material was characterized by ATR-IR.It was proved that chiral functionalized carrier material has been successfully prepared.The catalytic activity of the immobilized lipase is 3-10 times higher than that of the free enzyme and the enantioselectivity is increased by 7-9 times.