Molecular Modification Of Quinone Oxidoreductase And Its Application In Dye Decolorization

NAD（P）H:quinone oxidoreductase 1（DT-diaphorase,NQO1,QR1,EC 1.6.99.2）is a widely distributed Flavin adenine dinucleotide（FAD）dependent flavoprotein that facilitates the mandatory two electron reduction of quinone,quinoneimines,nitroaromatic compounds and azo dyes.These reductions significantly contribute to catalyze the detoxification of electrophiles,inactivation of oxidants and to scavenge superoxide directly.However,limited expression level and low enzymatic activity of naturally occurring NQO1 had made it restricted to achieve these benefits.The current study focuses on the engineering of hNQO1using directed evolution,and confirmed the activity and efficiency of improved variant towards the dye decolorization assay.The main research contents and progress are as follows:1.The hNQO1 protein producing strains was induced for protein expression and determined its expression by SDS-PAGE electrophoresis.The molecular weight was determined to be 30.8 k Da.The purification of hNQO1 protein was successfully achieved using a nickel column and protein concentration of purified enzyme was quantifying BCA assay kit.Then,the enzymatic properties and kinetics of the wild-type hNQO1 were performed.The results showed that the enzyme activity was better under acidic condition of p H 1.95 and the optimum temperature is 45?C,In addition,it was also examined that enzyme activity was increased significantly with increasing temperature from 30-45?C.In the thermal stability experiment,it is found that the hNQO1 is relatively stable at 30℃-40℃,and the stability decreases with the increase of temperature.About kinetic characterizations was described,the K_mvalue,K_catvalue,and catalytic efficiency of wild-type hNQO1 are 0.5637mmol·L^-1,0.086 s^-1,and 0.153 L·（mmol·s）^-1,respectively.2.In order to enhance the activity of hNQO1by directed evolution,the target gene of wild type hNQO1 was used as a template for the construction of ep PCR library.Various concentrations of Mn Cl₂（0.05-0.15 m M）were used in ep PCR library generation of hNQO1and used E.coli BL21-gold（DE3）cells as host for screening.Used malachite green based decolorization assay for screening of hNQO1 in the 96-well MTP.More than 2300 mutant strains were screened from the mutant library.After screening,improved variants were expressed in a shaking flask.Finally,three improved mutants（1-C7,4-C10 and 7-G12）were selected for detailed characterization.After protein purification,the activities of the improved variants of hNQO1 were 2.45 times,3.41 times and 3.48 times higher than that of the wild-type,respectively.3.Thereafter,Wild-type and improved variants of hNQO1 were expressed in Escherichia coli and compare the decolorization ability of the mutant（1-C7,4-C10,7-G12）and wild-type hNQO1 to malachite green in different glycerol concentrations（100 mmol/L,250 mmol/L,500 mmol/L,750 mmol/L,1 mol/L）.The results showed that compared to that of wild-type hNQO1,the decolorization rate of mutant 1-C7 increased by 29.9%,4-C10 up to45.3%and 7-G12 up to 30.9%as expressed in the presence of 100,250 and 500 mmol/L glycerol,respectively.It was also observed that decolorization rate decreased with the increasing concentration of glycerol.4.Furthermore,six kinds of medium small molecules（1,4-naphquinone,sodium a nthraquinone-2-Sulfonate,2-methlcyclohexa-2,5-diene-1,4-dione,1,4-dihydroxyanthracene-9,10-dion,anthraquinone,menadione）were screened by wild-type hNQO1 against the decolorization of azo dyes（congo red,and amaranth）.After screening,1,4-dihydroxyan thracene-9,10-dion was selected as a suitable mediator for this assay and collected res ults showed that whole cells of wild-type hNQO1 decolorized the Congo red up to 20.3%after repeated use for three times.While whole cells of hNQO1 decolorized am aranth up to 18.8%in reaction duration of 6 h.After this,activity assay was perform ed again to compare the ability of the modified variants and wild type hNQO1 to de colorize the azo dye,During this assay,optical density(OD₆₀₀)of whole bacterial cell s was adjusted to 2.5.Analyzed results showed that the decolorization rates of wild t ype hNQO1whole cells to Congo red and amaranth up to 29.5%and 9.8%,respective ly.While improved mutants（1-C7,4-C10,7-G12）decolorized Congo red with the rat e of 33.0%,30.9%,and 33.4%,And the decolorization rate of amaranth was 6.4%,15.1%,and 14.4%,respectively.