Precisely "Coating" On Porous Support Using Enzymes Depending On Bioorthogonal Chemistry And Catalytic Synthesis Of Intermediates

Chemical total synthesis has achieved great success in the synthesis of drugs and complex natural products,but many challenging issues remain,such as expensive chemical reagents,harsh reaction conditions,lengthy synthetic routes and high environmental pollution.Compared to many chemically catalysis,enzymatic reactions usually involve milder temperatures and pressures,facilitating the combination of multiple steps into a one-pot process for catalytic synthesis.In this thesis,two enzymes involved in a cascade reaction(aldo-ketoreductase and alcohol dehydrogenase)are orderly coated on the surface of the carrier to bring the enzymes into close proximity and form a substrate channel,thus enhancing the synergy and catalytic efficiency of the two enzymes.Firstly,the application of Spy Tag/Spy Catcher for enzyme linkage was explored.Tyrosine ammonia lyase(TAL)was used as the model enzyme to form cross-linked enzyme aggregates TAL-CLEs through the specific reaction of Spy Tag with Spy Catcher for self-cross-linking.The experimental results showed that the catalytic yield of the prepared enzyme aggregate TAL-CLEs reached 98.31%,4.15 times higher than that of the enzyme aggregate based on glutaraldehyde cross-linking(23.71%).After incubation at 90 ℃ for 15 min,the initial activity of 50.38% was still be maintained,and the yield of the target product to p-coumarin acid could still maintain the initial 70.13% after six times of repeated catalysis,which indicates that the use of Spy Tag and Spy Catcher as the connection mode has a mild effect on the enzyme protein,not only having a small impact on the enzyme activity but also having a high reaction rate,good specificity and stability.It is a good bio-orthogonal method that can be applied to the specific connection of enzymes.Secondly,we applied the Spy Tag/Spy Catcher system in a dual-enzyme cascade reaction to construct a dual-enzyme ordered "coating" cascade of aldo-ketoreductase AKR and alcohol dehydrogenase ADH.The fusion proteins Spy Catcher-AKR and Spy Tag-ADH were constructed by fusing Spy Catcher and Spy Tag to AKR and ADH respectively,enabling AKR and ADH to cross-link through Spy Catcher and Spy Tag.In order to orderly "coat" both enzymes on the carrier,a non-natural amino acid(4-azido-L-phenylalanine)was inserted into the enzyme protein.The enzymes were immobilised on the carrier by a click chemical reaction between the azide groups on the enzymes and the alkyne groups on the carrier,while the other enzymes were cross-linked to the immobilised enzymes by the spontaneous reaction between Spy Tag and Spy Catcher,thus achieving the orderly "coating" of enzymes.Finally,the two-enzyme ordered "coating" was applied to the catalytic synthesis of(R)-1-(2-chlorophenyl)ethanol.The experimental results showed that the catalytic yield of the ordered double enzyme coating @ADH-AKR reached 80.75%,and the ee value was higher than 99.99%.After five cycles of experiments,the catalytic reaction of the ordered double enzyme coating @ADH-AKR retained 71.01% of the initial yield,which was higher than the disordered immobilization(63.11%),indicating that the ordered coating of AKR and ADH was an efficient immobilization method,which allowing the substrate flow more efficiently between enzyme proteins,and not only did it have good repeatability,but it also had a higher catalytic efficiency.In addition,as the enzymes are ’coated’ in two layers,this means that more enzymes can be immobilised in the carrier than in a normal enzyme monolayer.This is a significant saving in resources and costs and is in line with green sustainability.In summary,an efficient method for the sequential immobilisation of multiple enzymes was identified through the sequential "coating" of the double enzymes involved in the cascade reaction by bio-orthogonalization.The application of the Spy Tag/Spy Catcher system to the self-assembly of TAL enzymes has demonstrated the efficient use of the Spy Tag/Spy Catcher as a bioorthogonal method for enzymeprotein cross-linking.On this basis,the Spy Tag/Spy Catcher was applied to the ordered "coating" of dual enzymes and used in conjunction with unnatural amino acids to construct the ordered "coating" of dual enzymes immobilised on porous carriers for the catalytic synthesis of chiral alcoholic drug intermediates.