RNL Enzymatic Henry Reaction Research And Catalytic Mechanism Analysis

The Henry reaction is an important reaction for forming C-C bond,which uses nitroalkane and aldehyde or ketone as subtracts to produce ?-nitro alcohol.Use chemical catalysts such as metal complexes to catalyze the Henry reaction has the disadvantages of harsh reaction conditions,by-products and large pollution.The enzymatic Henry reaction using the catalytic promiscuity of lipase can effectively solve above problems.At present,a few lipases have been found to have the ability to catalyze the Henry reaction,but the reactivity and selectivity are poor.And there is no report about the structural modification of the enzyme and the analysis of the catalytic mechanism.Therefore,we studied the lipase-catalyzed Henry reaction,including enzyme screening,pure enzyme expression,catalytic mechanism analysis and chemical modification.First,we screened a series of common commercial lipases by Henry model reaction to obtain RNL with good reactivity.The product yield in pure water system was 36.10%.After protein electrophoresis of RNL,we found that the purity of RNL was low.Therefore,we selected Pichia pastoris GS115 as vector to express RNL.After it,we studied the basic enzymatic properties of RNL:the optimum temperature was 40?,the optimum pH was 8.0,and the residual enzyme activity remained above 70%after being placed at 45? for 2 h.Next,we investigated the catalytic mechanism of RNL-catalyzed Henry reaction.First,by molecular docking,we found that there are five amino acid residues near the active center,may be involved in the reaction.They sre Ser81,Phe82,Ile86,Ser145,and His257.Subsequently,by molecular dynamics simulation,we explored the role of these amino acid during the reaction.We found that Ser145 and His257 in the catalytic triad can form hydrogen bonds with the substrates to transfer proton,which are key sites affecting the reactivity.Ser81,Phe82 and Ile86 can form weak bond forces with the two substrates to fix the substrates.Then,we used single point mutation to verify the simulation results,analyzing the catalytic mechanism of lipase-catalyzed Henry reactionFinally,in order to furtherly increase the reactivity of RNL we used chemical modifiers to modify the structure of RNL.The results showed that when the Arg residue on the RNL was modified with 1,2-cyclohexanedione(CHD).the product yield was increased.Then,we optimized the reaction conditions,obtaining the optimal reaction conditions:benzaldehyde 0.1 mmol;nitromethane 2.5 mmol;pH 7.5 phosphate buffer 1 mL;RNL(CHD)modified enzyme 10 mg;stirred for 24 h at 35?.The product yield was furtherly increased from 48.15%to 66.70%Subsequently,we investigated the substrates range of the reaction and found that,RNL can catalyze the reaction of various aromatic aldehydes with nitroalkanes,and all got good results.Among them,the yield of p-nitrobenzaldehyde was highest,reaching 99.43%.