Heterologous Expression And Dye Decolorization Analysis Of Dye-decolorizing Peroxidase From Rhodococcus Jostii

Dye decolorizing peroxidase（Dy Ps）belongs to a new superfamily of peroxidases with heme as a prosthetic group.Due to its ability to degrade anthraquinone dyes,Dy Ps has become a research hotspot in today’s green economy.The effective degradation of dyes is closely related to enzyme activity.Therefore,seeking ways to improve enzyme activity is the key to the subsequent application of Dy Ps.In this paper,the optimized bacterium Rhodococcus jostii RhDypB gene was heterologously expressed in E.coli BL21（DE3）to obtain the recombinant protein,and the corresponding basic application research was carried out.The main research content and results of this study are as follows:（1）The heterologous expression of RhDypB in Escherichia coli was realized,the effects of co-expression of the key gene hem A in heme synthesis pathway and RhDypB with single vector and dual vector,as well as the exogenous addition of heme synthesis precursor 5-aminolevulinic acid（5-ALA）on enzyme activity were analyzed.Recombinant RhDypB exist in soluble form in the supernatant buffer after bacterial cell disruption and had catalytic activity.SDS-PAGE showed that there was a clear band of interest at 40 KDa（purity～90%）,and the protein concentration could reach 100 mg·L^-1.The intracellular heme concentration and enzyme activity in the co-expression strains Eco/p E28AD,Eco/p E24AD,Eco/p CA+p E24D,and Eco/p E24D expressing only RhDypB with supplemented 5-ALA were compared,the results showed the heme concentration increased to 2.4μmol·L^-1 and 8.9μmol·L^-1 in Eco/p E24AD and Eco/p E24D+ALA compared with that of 1.53μmol·L^-1in Eco/p E24D.The binding degree（R_Z value）of RhDypB with prosthetic heme increased from 0.15 to 0.31 and0.37,respectively,and the enzyme activity increased from 1680 U·g^-1 to 3400 U·g^-1 and 3240U·g^-1,increased by 102%and 93%,respectively.The enhancement of enzyme activity in strains Eco/p E24AD and Eco/p E24D+ALA was similar,but considering the cost,the strain Eco/p E24AD was used as the object of follow-up research.（2）The effect of fermentation conditions on enzyme activity in Eco/p E24AD was analyzed.The changes of enzyme activity in various fermentation condition were detected,including adding different concentrations of glucose and ferrous chloride and controlling dissolved oxygen levels in the medium.The results showed that different concentrations of glucose inhibits enzyme activity,and a certain amount concentration of Fe²⁺and dissolved oxygen level could increase the R_Z value and activity of the enzyme.When the concentration of Fe²⁺was 40μM,the enzyme activity was two times higher than that of the control(3200U·g^-1),and the R_Z value was about 1.5 times higher than that without ferrous chloride addition.When the rotation speed was 80 r·min^-1,the highest enzyme activity was 8900 U·g^-1,and the R_Z value was 0.53.（3）Research on the decolorization of anthraquinone dye reactive blue 19 by RhDypB.The results showed that the recombinant RhDypB had a good decolorization effect on the dye.The decolorization rate of the RhDypB from Eco/p E24AD and Eco/p E24D+ALA reached 53%and51%,and the decolorization rate of Eco/p E24D was 25%.The decolorization effect of the RhDypB of Eco/p E24AD under different decolorization conditions was compared,and it was found that the dye decolorization rate was higher when the concentration of Reactive Blue 19was 60 mg·L^-1,the concentration of H₂O₂ was 6 m M,and the decolorization time was 24 h.By scanning the full wavelength of the dye before and after degradation,it was found that the peak of the dye at the maximum absorption wavelength disappeared after 24 h of degradation,indicating that the dye was degraded.Using ESI-MS to analyze the possible degradation products after enzymatic treatment of the dye,it was found that the C-N bond on the side chain of the anthraquinone ring was broken first,and then the auxochrome groups of SO3^2-and Na⁺were removed.After the oxidation continues,the anthraquinone ring was split.Phthalic acid and anthranilaldehyde were formed,and a possible degradation mechanism was proposed.