Electro-catalytic Synthesis Of Nitro-tryptophan And The Effect Of Interface On The Product Based On P450 TxtE Enzyme

Bioelctrocatalysts is a cross-disciplinary research field,which combines enzymatic reaction and electrochemical methods.The coupling of redox enzyme and electrode realizes the electron transfer between the cofactor and the electrode.The research for electron transfer law of redox enzyme could deepen the understanding of the catalytic mechanism and also be beneficial for the desigin of efficient biocatalytic system.Because of the mild and environmental friendly experiment conditions as well as simple operations,bioelctrocatalysts has attracted much attention.It has been widely applied in the research of biosensing,biofuel batteries,and newly catalytic reaction.However,the design and development for enzymatic electrochemical synthesis are still at the preliminary stage.Aromatic nitro compounds are often used as the precurors of antibiotics,herbicides,and anticancer drugs because of the special biological properties.The methods of traditionl organic synthesis for aromatic nitro compounds are usually complex with byproducts and poor region-selectivity.Developing new strategies for obtaining nitro compounds with specific strutures are needed.Enzyme catalyzed synthesis has higher selectivity and less byproducts,but it usually has complicated mechanism,which makes it hard for engineering and regulation.Here,we took the enzyme TxtE-directed regio-selective synthesis of 4-nitro-tryptophan（4-N-W）as a model.By introducing the electrochemical reaction,we combined the features of enzymatic reaction and electrochemistry,and developed a series of relectively simple,more controllable catalysis reaction.We further analysized the related electron transfer mechanism.It will provide new idea and regulating stretagies for the bioelctrocatalysts.1.Tuning the region-selectivity in the electro-enzymatic aromatic nitration via electric field and electromediator.In the work,electrochemistry was introduced for the first time into the TxtE catalyzed tryptophan nitration.With this new reaction,we successfully synthesized the nitro-tryptophan isomers(4-N-W and 5-N-W.Here,the electrochemical power replaces the electron donor of NADPH and transferred electrons to oxidoreductase Fd via the electron mediator MV²⁺.It shortened the reaction path and improved the yield of produced nitro-tryptophan.With a series of control experiments,the possible electron transfer path was deduced to be electrode（GCE）→MV²⁺→Fd→TxtE,and the redued TxtE reacted with O₂,DEANO and L-Trp to produce nitro-tryptophan.Besides that,the catalytic performance of TxtE can be adjusted by the electric field and the electron mediator.Changing the polarity of the mediator can transform the mixed production of 4-N-W and 5-N-W to just 4-N-W.This work not only broadens the application of bioelectrocatalysis,but also provides new methods for the regulation of electroenzymic catalysis.2.Electrically driven trptophan nitration mediated with Fd-TxtE complexes on the electrode surfaceWe constructed the multilayer film of Fd-TxtE complex on the carbon glass electrode to perform the electrical driven-enzymatic catalysis of nitrotryptophan to further simplify the experimental procedure and optimize the catalytic performance of TxtE in the electric field.In this reaction,we realized the direct electron transfer between the Fd-TxtE complex and the electrode and successfully produced the nitro-tryptophan isomers.The catalytic performance of modified electrodes with different proteins was studied,and the results showed that the interaction existed between Fd and TxtE on the electrode and could improve the catalytic performance.We further studied the effects of electron mediator,reaction time,and tryptophan concentration on the electroenzyme catalytic reaction,respectively.The kinetics of the system is a first-order reaction.Comparing to the first work,this reaction system with the enzyme on the electrode showed simpler,more stable,and excellent performance.The composites modified electrode could maintain high catalytic activity within a week.This work provides a methodology for the design of efficient and sustainable bioelectrocatalytic reactors.3.Study on the effect of TxtE interface on the electron transfer during the tryptophan nitration based on the molecular self-assmblyTo deeply understand the reaction mechanism of this bioelectrocatalysis,a protein complexed monolayer was constructed on gold electrode by means of molecular self-assembly technology,and the effect of TxtE interface on charge transfer of tryptophan nitration reaction was studied.Comparing to the multilayer-modified system,the self-assembled monolayer system displayed the highest yield of nitrotryptophan production.We designed and expressed different TxtE mutants for obtaining more detailed information of the reaction.We assembled the Fd-TxtE and Fd-TxtE mutant complexes on gold electrodes through electrostatic or covalent interactions.By introducing the“solid-phase charge transfer”,we discussed the roles of protein complex orientations on the charge trange transfer of Fd-TxtE complex as well as the tryptophan nitration.Meanwhile,the electron mediator could also change the charge transfer path of the reaction,which could greatly improve the yield of nitro-L-tryptophan.These results showed that the mechanism of charge transfer was sensitive to the microenvironment in the electroenzyme catalyzed reaction,which influenced the catalytic performance of the enzyme.There are multiple strategies to adjust the enzymic microenvironment and the electroenzymic catalysis,which provide various methods for diverse bioelectrocatalytical reactions.