| The enzymatic synthesis of rare ginsenoside CK currently suffers from poor stability,long conversion cycles and non-reusability.Protecting the activity of enzyme molecules by immobilization technology and improving the stability of enzyme molecules while recovering and reusing them are potential avenues for enzyme-catalyzed technology applications.Metal organic frameworks(MOFs)are very promising platforms for enzyme immobilization because of their high specific surface area,stable backbone structure,good biocompatibility and customizable functional sites,and because their uniform pore structure can build partitioned structures to meet molecular transport and internal environmental protection.To address the problem of long conversion cycle in the conversion of ginsenoside CK by snailase,we proposed the idea of introducing β-glucosidase to rapidly convert the substrate Rb1 to Rd first,and then using snailase to catalyze the conversion of Rd to rare ginsenoside CK,so that the dual enzyme synergy may yield higher catalytic efficiency.In this work,three different enzyme immobilization strategies were used to finally prepare three different enzymes immobilized biocomposites.The biocomposites were applied to the conversion of rare ginsenoside CK from ginsenoside Rb1,and their catalytic efficiency as well as recycling performance were evaluated.The main findings of this thesis are as follows.(1)The co-precipitation method has the advantages of mild reaction conditions and simple synthesis conditions.β-glucosidase and snailase were co-immobilized in the process of Zn-BTC self-assembly by the co-precipitation method.The relative enzyme load of β-G&Sna@Zn-BTC reached 125.35 mg/g under optimal immobilization conditions.The enzymatic activities of β-G@Zn-BTC and Sna@Zn-BTC at p H 8were 28.9% and 39.2%,respectively,while β-G&Sna@Zn-BTC at p H 8 was 51.2%.The CK concentrations of the products of β-G&Sna@Zn-BTC and Sna@Zn-BTC were 0.815 and 0.499 mg/m L,respectively,after48 h of catalysis at p H 4.5 and 50°C.Meanwhile,the CK concentration of the dual enzyme co-immobilized material was 1.63 times higher than that of the single snail enzyme immobilized material.After five cycles,the enzymatic activity of the biocomposite remained 68.324±0.54% of the initial enzymatic activity of the enzyme.(2)To further improve the stability of the immobilized enzyme material and increase the reusability,the dual enzyme coimmobilization was carried out by the cross-linking strategy,and the final β-G&Sna@ZnBTC-H biocomposite was obtained with higher temperature,p H stability and organic solvent tolerance compared with the β-G&Sna@Zn-BTC obtained by the co-precipitation method.The relative enzyme loading under optimal conditions reached 123.12 mg/g,when the relative enzyme activity reached 67.3%.The immobilized enzyme material showed higher optimal catalytic temperature during the optimization of catalytic conditions,and the concentration of CK in the product was 1.227 mg/m L after 48 h of transformation at 55°C.Meanwhile,the initial enzyme activity of 82.5% was still maintained after 8 cycles due to the high stability of Zn-BTC-H prepared by hydrothermal method.(3)To further improve the efficiency of the dual enzyme cascade reaction,the immobilized material β-G@Cu-BTC-Sna@Cu-BTC was obtained by partitioned MOF self-assembly.The partitioned immobilized material maintained 86.23% of the enzyme activity after 10 cycles.The pore effect of MOF reduced the mass transfer resistance of the substrate,and the CK after 48 h of catalysis at p H 4.5 and 55°C concentration was1.29 mg/m L,which increased the conversion efficiency by 1.2-fold.The compartmentalized MOF enzyme immobilization strategy avoids the accumulation of transition products by positional control of multiple enzymes,which in turn significantly improves the catalytic efficiency. |
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