Cloning Of The Key Enzyme Genes Of 1-deoxynojirimycin Synthesis Pathway And Preparation Of Molecularly Imprinted Polymer

1-Deoxynojirimycin is an iminoglycopiperidine polyhydroxyalkaloid,which is structurally similar to glucose,can compete with glucose for binding to the active center of ?-glucosidase,and can be used as a powerful competitive inhibitor for such enzymes.1-Deoxynojirimycin can be used as a strong candidate for the treatment of many diseases,and has great medicinal and research values.However,natural strains have low yields and cannot carry out large-scale fermentation production.Implementing a directed evolution strategy for biosynthetic pathways not only results in higher biosynthetic efficiency,but also the method of which is simple and effective.And compared to Bacillus,Escherichia coli expression is more stable,research on which is more thorough,and it is easier to achieve high expression of key genes.Therefore,on the basis of previous researches,this study cloned three genes Gab T1,Yktc1,and Gut B1 from the TYB gene cluster assumed to be capable of synthesizing 1-deoxynojirimycin from Bacillus amyloliquefaciens to construct a co-expression plasmid,and the combination of existing gene functions in E.coli has enabled the construction of synthetic routes that can be expressed in E.coli.In addition,the molecular imprinting technique was used to achieve the affinity adsorption and enrichment of the product 1-deoxynojirimycin.Finally,using molecular docking research methods,1-deoxynojirimycin and acarbose with the most similar mechanism of action were compared for their ability to inhibit ?-glucosidase.Using molecular docking research methods,1-deoxynojirimycin and acarbose,which have the most similar mechanism of action,were compared for their ability to inhibit the activity of ?-glucosidase.In this study,three key genes Gab T1,Yktc1,and Gut B1 in the TYB gene cluster were first cloned from B.amyloliquefaciens.According to their functions,they are assumed to be aspartate aminotransferases,phosphatases,and alcohol dehydrogenases,respectively.The enzyme properties of the three genes were simply studied.The results showed that all the three enzymes had the enzymatic activity of putative enzymes.The key enzyme,ethanol dehydrogenase,has an optimal p H of 6 and an optimal reaction temperature of 55 ?.Second,this study further constructed the co-expression plasmid p ANY2-Gab T1-Yktc1-Gut B1-assembly of the above three genes,and made full use of the existing gene function of E.coli to complete the construction of the 1-deoxynojirimycin synthesis route,and the product was identified.At the same time,in this study,the derivatization reaction of 2,4-dinitrofluorobenzene was used to identify the product.A microplate reader was used to scan the ultraviolet absorption value.It can easily and quickly analyze the production of 1-deoxynojirimycin and its concentration,thereby significantly reducing the cost and difficulty of product identification.In addition,this study also cloned the alpha-glucosidase gene from Bacillus amyloliquefaciens and expressed it efficiently in E.coli,and verified the product of the 1-deoxynojirimycin synthesis route 1-deoxynojirimycin to alpha – Glucosidase.Enzyme activity has a significant inhibitory effect,laying a foundation for subsequent research.Third,this study uses molecular imprinting technology,using magnetic graphene as a new molecularly imprinted polymer material,alpha-glucosidase as a functional group,dopamine and glutaraldehyde as crosslinking agents,and the addition of nickel ions for the histidine tag on alpha-glucosidase was specifically adsorbed.A new molecularly imprinted polymer Fe3O4 @ GO @ ?-Glu-MIP was successfully prepared to selectively enrich the product 1-deoxynojirimycin.The preparation conditions of traditional molecularly imprinted polymers are harsh,the operation is dangerous,and some even involve a large number of toxic reagents,but the preparation of molecularly imprinted polymers in this study avoids these problems.It is worth mentioning that this study innovatively uses alpha-glucosidase as the active center,so that not only can the product 1-deoxynojirimycin be selectively enriched,but also can be immobilized.Finally,in this study,using molecular docking methods,1-deoxynojirimycin and acarbose,which have the most similar mechanism of action,were compared for their inhibitory effects on alpha-glucosidase.Through the comparison of the results,although acarbose can form hydrogen bonds with many amino acid residues,it does not bind well to the enzyme active center,so it cannot better inhibit alpha-glucosidase enzyme activity.1-Deoxynojirimycin and alpha-glucosidase can be well combined into the narrow enzyme active center,and can also form a stable hydrogen bond structure with the enzyme active center,thereby more effectively inhibiting enzyme activity.Therefore,compared with acarbose,1-deoxynojirimycin binds to alpha-glucosidase more tightly,thus providing an important reference for studying the mechanism of action of 1-deoxynojirimycin.