The Expression,Directed Evolution Of HRP Gene And The Enzymatic Polymerization Of Aniline Or Its Derivantes

â… .Background and meaningThe polyaniline is the first conducting polymer.In the last 20 years polymerization of aniline has received a great deal of attention,because of their many possible applications in electrical,electrochemical,optical properties and their good environmental stability. Polyaniline is commonly synthesized by means of electrochemically,chemically,enzymatic polymerization of aniline or its derivatives.Presently,enzymatic synthesis of polyaniline has been explored as an more attractive alternative synthetic route to the synthesis of electronically,aqueous-solubility or optically active polyaniline.Horseradish peroxidase (EC1.11.1.7)may oxidize aniline,and it has been used extensively in the catalysis of the oxidative coupling and further polymerization of anilines.The HRP-catalyzed polymerization of monomer aniline was carried out in the presence of H₂O₂ as an oxidation substrate. Unfortunately,HRP showed lower structural stability in relative high concentration of H₂O₂. For improvement of the enzymatic,synthesis,a more attractive alternative route should enhance the tolerance of HRP to H₂O₂.As one of the random approaches for protein engineering,directed evolution techniques have proven to be an efficient route for improving the properties of enzymes even without information of the relationship between structure and function,thus yielding a number of promising results.In this process,diverse pools of the mutant library are generated in vitro via random mutagenesis and recombination of genes,followed by directed screening or selection. With the HRP as an eukaryotic protein,one of the requisites to molecular evolution is the expression in active form in a recombinant host.The expression of HRP,which is glycosylated and contains disulfide bonds,has been a major technical issue in the development of improved enzyme variants.The evolution and the expression of the HRP gene in E.coli or in S.cerevisiae,few active enzymes could be obtained or not able to secrete the functional enzyme.The Pichia pastoris host cells facilitate glycosylation and the formation of disulfide bonds,and are especially well suited for secretion of the foreign protein.The directed evolution of HRP,for specific applications,has been performed in E.coli,some of them have been expressed in Pichia pastoris,where they give rise to 5.4-folds of HRP activity toward ABTS[2,2'-azinobis(ethylbenzthiazoline-6-sulfonate)].Herein is described a vitro directed evolution of Horseradish Peroxidase in Pichia pastoris for the improvement of the tolerance of HRP to H₂O₂,aimed to the application in synthesis of PANI.Error-prone PCR and staggered extension PCR(STEP)were used to generate mutant pools.The active evolution HRP was expressed in Pichia pastoris GS115. And the enzymatic reaction kinetics,stabilities and other properties of HRP were analyzed also.â…¡.Contents and results1.The HRPC3 gene(hrp)have been cloned and analyzed from Armoracia rusticana In this process,we firstly obtained the cDNA of total Armoracia rusticana mRNA using RT-PCR technology,and then obtained the HRPC3 gene using designed primer by PCR technology.The cloned HRPC3 gene was cloned into E.coli TOP10 using pPICZα-A by chemical transformation.Comparing with the secquence of HRPC3[gi:217933]from NCBA Genebank,the homologous of cloned HRPC3 was 99.9%.2.The cloned HRPC3 geng was transformed successfully into Pichia pastoris GS115, using Pichia pastoris expression system(from Invitrogen Corporation).And the cloned HRPC3 geng was expressed successfully in Pichia pastoris GS115.Active HRP was isolated and purification obtained from culturing media,the expressed HRP was secreted out of Pichia pastoris GS115.3.Horseradish peroxidase shows a low catalyse activity toward polymerization in the presence of a relative high concentration of H₂O₂.In order to obtain mutational HRP,which were suited for using in enzymatic polymerization in the presence of H₂O₂,a directed evolution of the horseradish peroxidase were made in pichia pastoris.In the process,the error-prone PCR technology and StEP was employed separately to create mutate library.After two round of directed evolution,the selected best mutant HRP17-32,which is enhanced the tolerance to H₂O₂,showed a 14.7-fold increase in the residual activity(638.9 U/I,T₂=14.7, T₂=0.81),compared to that of wild-type enzyme.The mutate phenotype of HRP17-32 mutant,is Mut⁺,the mutant can expressed secretedly HRP17-32 under inducting by methanol, the expression level reached 0.9mg/l,and a 2.7-fold activity of HRP17-32 have been detected in culturing media,comparing with wild-type HRP recombination strain。4.The culturing condition and secreted enzyme condition of mutate HRP17-32 recombination strain have been researched,and the HRP17-32 was isolated and purified.The optimum condition of the growth and secretion HRP17-32 of recombinated Pichia pastoris GSll5 by mutate HRP17-32,is 0.75ml/L methanol,pH6.0 and culturing time for 48 hours.The total purification fold of HRP17-32 is 72.9,and the total recover yield of HRP17-32 is 62.7%.The molecular weight of HRP17-32 is ranging from 66.2 to 116.0 kDa.HRP17-32 are glycosylated in different degreen in expression.5.The 1,4-diaminobenzene(PPD)was oxidized and polymerized using HRP17-32 in a mixture of phosphate buffer and organic solvent,and the structure of poly-1,4-diaminobenzene was characterized by a Fourier transform infrared spectrum(FTIR)etc.The synthesized poly-1,4-diaminobenzene was composed of para-directed units,and molecular weight was about 1528(Mw),the polydispersity about 1.012.And the enzymatic reaction kinetics and stabilities of HRP17-32 were also analyzed.The results indicated that the Km value of HRP17-32 is 0.76 mM,the Vmax is 11.51×10^-2mmol·min^-1·mg^-1,optimum concentration of H₂O₂ is 2.5 mM,the optimum pH value is 6.0,and the optimum temperature is 45-55℃,under the reaction condition.The stability conditions of HRP17-32 was pH 5.6-7.0,reaction temperature 40℃,and 20%concentration of dioxane.The stabilities of HRP17-32 is more higher than that of wild-HRP6.HRP17-32 catalyzed polymerization of 1,4-diaminobenzene(PPD)and acrylic acid have been carried out in an aqueous buffer.And the conductivity,UV-vis spectra,FT-IR spectra,X-ray photoelectron spectroscopy(XPS),and thermo-gravimetric analysis(TGA)of the resulting polymer have been investigated.The synthesized polymer(PAnI)is a self-doped poly(1,4-diaminobenzene-acrylic acid)with 1,4-disubstituted aromatic ring,and shows electro-activity,aqueous-solubility,and higher decomposition temperature(589℃).The properties and yield of PAnI have been found to be depended on the pH of the buffer,the concentrations of H₂O₂ used,the ratio of the PPD to acrylic acid,and the time of polymerization etc. â…¢.Innovation1.Using the teachnology and methods of combinatorial chemistry,molecular biology and protein directed evolution,evolved and improved the structure and property of HRP,and application in enzymatic polymerization,has not been reported in literatures.2.Taking the enzymatic synthesis of conducting polyanilines as a specific aim,firstly evolved the HRPC3 in vitro,made the stability of HRP toward H₂O₂ improving,obtained evolved HRP17-32 and the mutant strain which can express HRP17-32,this has not been reported in literatures.3.Using the teachnology of RT-PCR,cloned HRPC3 gene(hrpC3),researching the function expression of hrpC3 in Pichia pastoris GS/115.This provided hard proof for the directed evolution of HRP in Pichia pastoris GS/115.And HRP17-32 has been expressed secretedly in Pichia pastoris GS/115 for the first time.4.The enzymatic polymerization of 1,4-diaminobenzene,and between the 1,4-diamino-benzene with acrylic acid were carried out by HRP17-32 catalyzed for the first time.And a poly-(1,4-diamino-benzene)and a poly-(1,4-diaminobenzene-acrylic acid)was obtained. And the enzymatic reaction kinetics,the stability of HRP17-32 and the factors which affect the enzymatic polymerization were analyzed too.The properties and structure of resulting polymer have been investigated by UV-vis spectra,FT-IR spectra,and so on.