Preparation Of Metal-organic Frameworks(MOFs) Materials And Its Application In The Immobilization Of Soybean Epoxide Hydrolase

Enantiopure chiral vicinal diols have been proven to be valuable and versatile intermediates of many high-added-value chemicals such as chiral pharmaceutical, insecticide, playing an important role in the synthesis of medicines, pesticides and cosmetics. Among various biological methods, asymmetric hydrolysis of epoxides catalyzed by epoxide hydrolases(EHs) can be an economic, efficient, green method for the synthesis of enantiopure vicinal diols.Here, EHs from different resources were prepared and used for catalyzing the hydrolysis of 1, 2-epoxyoctane. As a result, EH from soybean(SEH) was found to be capble of effeciently catalyzing the asymmetric hydrolysis of 1, 2-epoxyoctane to(R)-1, 2-octanediol and consequently was selected for subsequent studies. By studying the effects of the conditions during the preparation process, the optimal preparation condition was found.It was found that the asymmetric hydrolysis of epoxide carried out in aqueous monophasic system afforded low yield and product e.e., due to poor solubility of epoxide, its pronounced non-enzymatic hydrolysis and the deactivation of free EHs. Also, it was difficult to recycle the free EHs in the reaction system. Therefore, it is of great interest to immobilize the SEH onto enzyme carriers. As the enzyme carriers, metal organic frameworks(MOFs) have many unparallel advantages. MOFs such as MOF-199, ZIF-8, Ui O-66-NH2 and nano-Ui O-66-NH2 were used to immobilize SEH via precipated-cross-linking process, respectively. The results showed that nano-Ui O-66-NH2-immobilized SEH exhibited best immoblization efficiency. The nano-Ui O-66-NH2 was also used to immobilize SEH via different methods such as precipated-cross-linking, activated-cross-linking and adsorption. The results indicated precipated-cross-linking was the most effective way for nano-Ui O-66-NH2 immobilizing SEH. The nano-Ui O-66-NH2 with abundant amount of-NH2 group was prepared under stirring and the size of Ui O-66-NH2 reached the nano-scale level. The effects of immobilization condition of the nano-Ui O-66-NH2 immobilized SHE, such as cross-linking agent concentration, cross linking time, the mass ratio of the carrier and SEH were studied. The results revealed that the highest relative activity recovery was obtained under the glutaraldehyde concentration of 130 mmol/L, cross-linking time of 2 h and the mass ratio of carrier and SEH of 10:1. Under the optimum conditions, the relative activity recovery and enzyme loading were 88.04% and 87.3 mg enzyme/carrier g, relatively.The structural changes, the integrity of the crystalline and the apparence of Ui O-66-NH2 were characterized by FTIR, XRD and SEM. The SEM of the immobilized SEH showed the size of the catalyst was about 600 nm, which indicated the catalyst was at nano-scale. When 1, 2-epoxyoctane was used as the substrate, the optimum p H of SEH@Ui O-66-NH2 and free SEH were 7.0 and 6.5, respectively; the optimum temperatures of SEH@Ui O-66-NH2 and free SEH were 40℃ and 35℃. In addition, the SEH@Ui O-66-NH2 was superior to its free counterpart in terms of thermal stability, p H stability and organic solvent tolerance. The stability of Ui O-66-NH2 and SEH@Ui O-66-NH2 were also studied. After incubation in PBS(7.0,50 mmol/L)at 40℃ for 2 h, the XRD showed they still retained the crystal integrity which indicated the good water stability of Ui O-66-NH2 and SEH@Ui O-66-NH2.A comparative study was made of the biocatalytic asymmetric hydrolysis of 1, 2-epoxyoctane with free SEH and SEH@Ui O-66-NH2 in the aqueous system. The yield and e.e. value of the product catalyzed by SEH@Ui O-66-NH2 were 41.39% and 81.21%, respectively, which was a little higher than that of the free SEH(yield 41.54%, e.e. value respectively.In the present study, we reported, for the first time, the immobilization of SEH onto the surface of MOFs Ui O-66-NH2 to obtain the nano-catalyst and the characterization of the catalyst. The nano-catalyst was used for catalyzing the asymmetric hydrolysis of 1, 2-epoxyoctane to(R)-1, 2-octanediol in DES-containing system. This study rationally designed MOFs and used them to immobilize SEH, which would increase the the researchers’ understanding of the intereaction of enzymes and carriers. This will undoubtly enable the researchers to design the MOFs as the enzymes carriers more rationally and scientificly according to the practical requirement. Furthermore, this study also provides an efficient way to produce enantiopure ortho-diols.