Multiple enzymes-induced biological cascade catalysis is indispensable in biotechnology and industrial processes. Nevertheless,the drawbacks of most natural enzymes, including poor stability and recyclability and sens...Multiple enzymes-induced biological cascade catalysis is indispensable in biotechnology and industrial processes. Nevertheless,the drawbacks of most natural enzymes, including poor stability and recyclability and sensitivity to the environment, have hindered their broader application. Here, we report a facile strategy to prepare a biomimetic cascade reaction system by combining the advantages of enzyme immobilization and biomimetic catalysis in a one-pot reaction system based on the hierarchically porous metal-organic frameworks(HP-MOFs). The hierarchically porous zirconium-porphyrin-based MOF(HPPCN-222(Fe)) synthesized by modulator-induced strategy possessed tunable hierarchical porous and peroxidase-like activity,permitting them to act as not only an efficient immobilization matrix for glucose oxidase(GOx) but also peroxidase mimics to catalyze the cascade for glucose detection. A stable, anti-interference and reusable colorimetric biosensor for glucose detection was successfully established through GOx@HP-PCN-222(Fe) on the basis of the artificial tandem catalysis. Moreover, the GOx@HP-PCN-222(Fe)-fabricated electrode was available for glucose detection by electrochemical method. This work provides a potentially universal method to design functional multi-enzymatic cascade reaction systems by integrating the merits of enzyme encapsulation and biomimetic catalysis in HP-MOFs.展开更多
Cytidine triphosphate(CTP),as a substance involved in the metabolism of phospholipids,proteins and nucleic acids,has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline.In this...Cytidine triphosphate(CTP),as a substance involved in the metabolism of phospholipids,proteins and nucleic acids,has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline.In this study,we established an in vitro six-enzyme cascade system to generate CTP.To avoid thermodynamic bottlenecks,we employed a circuitous and two-stage reaction strategy.Using cytidine as the key substrate,the final product CTP is obtained via the deamination and uridine phosphorylation pathways,relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate.Several extremophilic microbial-derived deaminases were screened and characterized,and a suitable cytidine deaminase was selected to match the first-stage reaction conditions.In addition,directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition,along with a 1.7-fold increase in activity over the wild type.After optimizing the reaction conditions,we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM,and the yield of CTP exceeded 82%within 10.0 h.展开更多
Background Dodecanedioic acid(DDA),a typical medium-chain dicarboxylic fatty acid with widespread applications,has a great synthetic value and a huge industrial market demand.Currently,a sustainable,eco-friendly and e...Background Dodecanedioic acid(DDA),a typical medium-chain dicarboxylic fatty acid with widespread applications,has a great synthetic value and a huge industrial market demand.Currently,a sustainable,eco-friendly and efficient process is desired for dodecanedioic acid production.Results Herein,a multi-enzymatic cascade was designed and constructed for the production of DDA from linoleic acid based on the lipoxygenase pathway in plants.The cascade is composed of lipoxygenase,hydroperoxide lyase,aldehyde dehydrogenase,and unidentified double-bond reductase in E.coli for the main cascade reactions,as well as NADH oxidase for cofactor recycling.The four component enzymes involved in the cascade were co-expressed in E.coli,together with the endogenous double-bond reductase of E.coli.After optimizing the reaction conditions of the rate-limiting step,43.8 g L−1 d−1 of DDA was obtained by a whole-cell one-pot process starting from renewable linoleic acid.Conclusions Through engineering of the reaction system and co-expressing the component enzymes,a sustainable and eco-friendly DDA biosynthesis route was set up in E.coli,which afforded the highest space time yield for DDA production among the current artificial multi-enzymatic routes derived from the LOX-pathway,and the productivity achieved here ranks the second highest among the current research progress in DDA biosynthesis.展开更多
基金supported by the National Natural Science Foundation of China (92061201, 21825106, 22001238)the Program for Innovative Research Team (in Science and Technology) in Universities of Henan Province (19IRTSTHN022)Zhengzhou University。
文摘Multiple enzymes-induced biological cascade catalysis is indispensable in biotechnology and industrial processes. Nevertheless,the drawbacks of most natural enzymes, including poor stability and recyclability and sensitivity to the environment, have hindered their broader application. Here, we report a facile strategy to prepare a biomimetic cascade reaction system by combining the advantages of enzyme immobilization and biomimetic catalysis in a one-pot reaction system based on the hierarchically porous metal-organic frameworks(HP-MOFs). The hierarchically porous zirconium-porphyrin-based MOF(HPPCN-222(Fe)) synthesized by modulator-induced strategy possessed tunable hierarchical porous and peroxidase-like activity,permitting them to act as not only an efficient immobilization matrix for glucose oxidase(GOx) but also peroxidase mimics to catalyze the cascade for glucose detection. A stable, anti-interference and reusable colorimetric biosensor for glucose detection was successfully established through GOx@HP-PCN-222(Fe) on the basis of the artificial tandem catalysis. Moreover, the GOx@HP-PCN-222(Fe)-fabricated electrode was available for glucose detection by electrochemical method. This work provides a potentially universal method to design functional multi-enzymatic cascade reaction systems by integrating the merits of enzyme encapsulation and biomimetic catalysis in HP-MOFs.
基金supported by the National Key Research and Development Program of China(2022YFC3401700)the National Natural Science Foundation of China(Grant No.32171478)the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(Grant No.TSBICIP-KJGG-009).
文摘Cytidine triphosphate(CTP),as a substance involved in the metabolism of phospholipids,proteins and nucleic acids,has precise drug effects and is a direct precursor for the synthesis of drugs such as citicoline.In this study,we established an in vitro six-enzyme cascade system to generate CTP.To avoid thermodynamic bottlenecks,we employed a circuitous and two-stage reaction strategy.Using cytidine as the key substrate,the final product CTP is obtained via the deamination and uridine phosphorylation pathways,relying on the irreversible reaction of cytidine triphosphate synthase to catalyze the amination of uridine triphosphate.Several extremophilic microbial-derived deaminases were screened and characterized,and a suitable cytidine deaminase was selected to match the first-stage reaction conditions.In addition,directed evolution modification of the rate-limiting enzyme CTP synthetase in the pathway yielded a variant that successfully relieved the product feedback inhibition,along with a 1.7-fold increase in activity over the wild type.After optimizing the reaction conditions,we finally carried out the catalytic reaction at an initial cytidine concentration of 20 mM,and the yield of CTP exceeded 82%within 10.0 h.
基金the financial supports from the National Key Research and Development Program of China(Nos.2019YFA0905000&2022YFC2105900)National Natural Science Foundation of China(Nos.32071475&21536004).
文摘Background Dodecanedioic acid(DDA),a typical medium-chain dicarboxylic fatty acid with widespread applications,has a great synthetic value and a huge industrial market demand.Currently,a sustainable,eco-friendly and efficient process is desired for dodecanedioic acid production.Results Herein,a multi-enzymatic cascade was designed and constructed for the production of DDA from linoleic acid based on the lipoxygenase pathway in plants.The cascade is composed of lipoxygenase,hydroperoxide lyase,aldehyde dehydrogenase,and unidentified double-bond reductase in E.coli for the main cascade reactions,as well as NADH oxidase for cofactor recycling.The four component enzymes involved in the cascade were co-expressed in E.coli,together with the endogenous double-bond reductase of E.coli.After optimizing the reaction conditions of the rate-limiting step,43.8 g L−1 d−1 of DDA was obtained by a whole-cell one-pot process starting from renewable linoleic acid.Conclusions Through engineering of the reaction system and co-expressing the component enzymes,a sustainable and eco-friendly DDA biosynthesis route was set up in E.coli,which afforded the highest space time yield for DDA production among the current artificial multi-enzymatic routes derived from the LOX-pathway,and the productivity achieved here ranks the second highest among the current research progress in DDA biosynthesis.
