Cascade Enzyme Reactors Constructed Via Protein Self-assembly Strategies For Pyruvate Biosynthesis

Enzymes are highly efficient and specific biocatalysts and in nature different enzymes are usually present in a cascade in the metabolic pathways of cells.The use of synthetic biology concepts to simulate the construction of artificial multi-enzyme systems is an important part of the transition from single enzyme catalysis to wholecell catalysis and occupies a special and irreplaceable position in the field of biocatalysis.To date,effective strategies that enable the assembly of multiple enzymes include protein fusion techniques,scaffold-mediated self-assembly techniques and immobilisation techniques.Immobilisation and scaffold-mediated self-assembly techniques can bring enzymes in close proximity to each other,speeding up the transfer of substances between enzymes,thereby increasing the conversion rate of cascade reactions,and are more suitable for the assembly of more than two cascade enzymes,and have great potential for the construction of artificial multi-enzyme assembly systems.Among them,the cross-linked enzyme immobilization technique has attracted much attention in the field of constructing multi-enzyme assemblies due to the advantages of low carrier cost and simple operation steps.However,the disorderly nature of the cross-linking results may cause excessive accumulation of enzymes,thereby limiting their optimal catalytic effect.The "bottom-up" protein self-assembly strategy provides an effective means of preparing stable functional materials with a defined structure.Interfacial self-assembly techniques exploit the amphiphilic nature of proteins,allowing the two-phase interface to be used as a template for the construction of assemblies with two-dimensional films or three-dimensional vesicle structures.The scaffold-mediated assembly strategy,on the other hand,allows for multi-enzyme assembly on protein scaffolds via SpyTag/SpyCatcher interactions under more benign conditions.Thus,the application of interfacial self-assembly techniques and SpyTag/SpyCatcher interactions opens up new ideas for the development of constructing artificial multi-enzyme assemblies.As an important intermediate in biological metabolism and material transformation,pyruvic acid is widely used in important industries such as chemical,pharmaceutical,cosmetic and food industries.Traditional chemical synthesis methods suffer from environmental pollution,low yields and high production costs.The enzyme-catalysed method has the advantages of simple operation,short reaction time and high product purity,and is of great value for the synthesis of pyruvic acid.Therefore,in this thesis,two methods for constructing cascade enzyme reactors using alanine aminotransferase(Ala AT5),glutamate oxidase(LGOX)and catalase(CAT)were proposed through interfacial self-assembly techniques and SpyTag/SpyCatcher interactions,and the assembly process and catalytic performance were investigated.1.Construction of a cascade enzyme reactor based on an interfacial self-assembly strategyThis chapter develops a method for the construction of multi-enzyme assemblies based on an interfacial self-assembly strategy.Ala AT5 and LGOX were obtained by in vitro expression and affinity column purification,and cascaded with free CAT in solution;the three cascade enzymes were self-assembled at the oil-water interface by electrostatic interactions with surfactants and then combined together as building blocks,and the cascade enzyme reactors with lamellar and vesicular structures were constructed by changing the assembly conditions,respectively.The cross-linking behaviour was verified by SDS-PAGE denaturing electrophoresis;the morphology of the assemblies was characterized by transmission electron microscopy,atomic force microscopy and scanning electron microscopy;and the rational distribution of the three enzymes on the assemblies was verified by laser confocal microscopy visualization.2.Investigation of the catalytic performance of a cascade enzyme reactorThis chapter investigates the catalytic performance of a previously constructed cascade enzyme reactor for the synthesis of pyruvic acid.In order to maximise the catalytic performance of the enzyme,the optimal reaction conditions were investigated and the optimal reaction temperature for the cascade enzyme was determined to be37 °C and the optimal p H to be 7.It was demonstrated that the in vitro free triple enzyme cascade system has better catalytic performance than the single enzyme system and can improve the reaction rate and product conversion.The results showed that the pyruvate yield of both assemblies was higher than that of the free enzyme system,and the vesicle structure had a better catalytic effect than that of the lamellipodia,with the conversion rates of the lamellipodia and vesicle assemblies being 1.12 and 1.41 times higher than those of the free system,respectively,after 3.5 h of reaction.The constructed cascade enzyme assembly system can effectively improve the reaction activity in the catalytic synthesis of pyruvate,and this strategy is a guideline for the construction of complex multi-enzyme assemblies.3.Construction of cascade enzyme reactors based on SpyTag/SpyCatcher interactionsThis chapter develops a method for constructing multi-enzyme assemblies based on SpyTag/SpyCatcher interactions.The protein scaffold phage P22 protein and three fusion enzymes were obtained by in vitro expression,affinity column purification and ultracentrifugation.High performance liquid chromatography and UV-Vis photometry were used to verify that the fusion enzymes were still catalytically active.The three fusion enzymes were individually assembled with the protein scaffold in buffer solution to verify the feasibility of assembly based on SpyTag/SpyCatcher interactions.Subsequently,the three fusion enzymes were assembled with the protein scaffolds and the morphology before and after assembly was characterised by transmission electron microscopy.It was demonstrated that a protein scaffold-mediated cascade enzyme reactor with a cage-like structure was successfully constructed by the self-assembly strategy and could be used for the catalytic synthesis of pyruvate.