Construction Of Biocatalysts Using The P450 Scaffold For The Synthesis Of Indigo From Indole

Indigo is one of the oldest pigments known to mankind and is widely used in printing and dyeing industry,pharmaceutical and food fields.The development of environmentally-friendly method for indigo production is of great significance to the sustainable development of society and the environment.In this study,we develop H2O2-dependent artificial peroxidase based on cytochrome P450BM3 monooxygenase by combination of chemical direct intervention strategy and enzyme molecular engineering technology,which could catalyze indole hydroxylation to prepare indigo.The artificial peroxidase system has simple components,does not rely on electron transfer protein and coenzyme NAD(P)H,and has excellent biocatalytic properties,providing an alternative method for green production of indigo.The specific content is as follows:Construction of mutant library of P450BM3 protein and preparation of mutant mechanism.Three mutation sites 78,87 and 268 were selected through rational design.Among them,amino acids 78 is located in the substrate channel and close to the active center,amino acids 87 is located in the active center at the end of the substrate channel,and amino acid 268 is located in the reaction active pocket I spiral link area.Ten P450BM3 mutants library of F87 A,F87G,F87A/T268 A,F87A/T268 I,F87G/T268 V,F87G/T268 A,F87G/T268 V,V78A/F87 A and V78A/F87 A were prepared by site-directed mutagenesis.The mutant library was successfully constructed through bacterial fluid sequencing.Expression and purification of P450BM3 protein.The obtained mutant library uses E.coli.as a carrier for protein expression,culture and purification.IPTG was added to induce protein expression,ultrasonic crushing,affinity chromatography and dialysis concentration to obtain the target protein.SDS-PAGE protein gel electrophoresis showed a single band with the molecular weight of 55 k Da.The UVVis spectrum of P450BM3 shows the characteristic absorption of P450 enzyme at 450 nm.Determination of catalytic capacity of P450BM3 for indole hydroxylation.The temperature,p H,H2O2 concentration and dual-function small molecule were optimized,and the enzymatic properties of P450BM3 catalyzed indole hydroxylation were determined under the optimum conditions.The results showed that the wild type P450BM3 couldn’t catalyze indole hydroxylation reaction.The benzene ring of phenylalanine(Phe)at position 87 hindered the entry of substrates.The catalytic efficiency was greatly improved after replacing Phe 87 with smaller alanine(Ala)and glycine(Gly)of P450BM3.The kcat of F87 A and F87 G mutants is 1007 min-1 and1304 min-1,respectively.In addition,the Km value of P450BM3 decreased significantly after the mutation of the hydrophilic amino acid threonine(Thr)at position 268 to the hydrophobic alanine(Ala),isoluleine(Ile)and valine(Val),indicating the construction of the hydrophobic pocket contributed to the binding of the substrate in the active site.The catalytic efficiency of F87G/T268 A mutant was up to1388 m M-1 min-1,which was 117 times that of myoglobin F43Y/H64D(kcat/Km=11.86 m M-1 min-1);32 times that of neuroglobin A15C/H64D/F49Y(kcat/Km=43.25 m M-1 min-1).In addition,the arrangement changes of amino acid at positions 78,87 and 268 can change the size and polarity of active pockets,thus affecting the selectivity and yield of substrate.The chemical selectivity of indole hydroxylation catalyzed by F87A/T268 V mutant was 80%,and the yield of indole hydroxylation catalyzed by double mutant F87G/T268 V mutant was 73%.Analysis of the catalytic mechanism.The reaction metabolites were identified by Ultraviolet Visible Spectroscopy(UV),High Performance Liquid Chromatography(HPLC),Mass Spectrometry(MASS)and Nuclear Magnetic Resonance Spectroscopy(NMR),and the catalytic mechanism of indole hydroxylation catalyzed by P450BM3 was elucidated.The docking simulation of P450BM3 protein with dual-function small molecule and indole shows that indole couldn’t enter the substrate channel of wildtype P450BM3,and the wild-type P450BM3 has no catalytic activity.The docking results of P450BM3 mutants F87A/T268 V,F87G/T268 A,F87G/T268 V and dualfunctional small molecule and indole shows that the distance between the C-3 atom of the indole and the heme iron center is the closest,which is 6.98 ? and 6.20 ? and6.84 ?,respectively.Indicating that the reaction is easy to proceed,which was consistent with the hypothesized reaction mechanism.Determination of P450BM3 for hydroxylation of indole chloride derivatives.The optimal reaction conditions of different indole chlorides were optimized respectively,and the efficiency of indole derivatives hydroxylation catalyzed by wild type P450BM3 and ten P450BM3 mutants was determined.The results showed that the wild type P450BM3 couldn’t catalyze the hydroxylation of indole chlorides,and the catalytic efficiency could be greatly improved by expanding the substrate channel with the changes of amino acids at 78 and 87 position.The conversion rate of 4-chlorinole hydroxylation catalyzed by F87 A mutant was 63%;the conversion rate of5-chloroindole hydroxylation catalyzed by F87 G mutant was 84%;the hydroxylation rate of 6-chlorindoles catalyzed by F87 G and V78A/F87 A mutants was 98%;the conversion rate of 7-chloroindole hydroxylation catalyzed by V78A/F87 G mutant was67%.