Heterologous Expression,Immobilization And Dye Decolorization Of Horseradish Peroxidase C1A

Horseradish peroxidase(HRP)is a kind of secretory plant peroxidase derived from horseradish.It is widely used in medical diagnosis,detection and analysis,bioremediation and other fields.However,the current industrial production method of the enzyme is mainly extracted directly from horseradish root,which is complex and expensive.In addition,the isolated and extracted natural HRP is a mixture containing more than 40 isozymes,and a large number of single component enzymes cannot be obtained,which affects the quality of the enzyme preparation.It also limits the basic research on the structure and function of HRP.Therefore,it is of great theoretical significance and industrial application value to study the preparation of monomorphic HRP with high catalytic activity by using a suitable recombinant expression system.The content of CIA isozymes in horseradish natural HRP is the most abundant and the activity is high.In this study,HRP CIA was selected as the research object to carry out heterologous expression experiment and basic application research.The main research results are as follows:(1)The heterologous recombinant expression of HRP C1Ain Escherichia coli was realized However,it could not get soluble expression and most of the recombinant protein accumulated in an insoluble non-glycosylated form in the cytoplasmic conjugate.The renaturation of HRP CIA in the form of inclusion body was studied.By exploring the renaturation conditions,an optimized refolding buffer system(20 mmol·L-1 Tris-HCl containing 0.4 mmol·L-1 GSSG,0.05 mmol·L-1 DTT,10%(v/v)glycerol,1.7 mol·L-1 urea and 2 mmol·L-1 CaC12,pH 8.5)was established.Finally,the recombinant HRP C1A with a specific enzyme activity of 2636 U·g-1 was obtained by two-step dilution ultracentrifugation.The enzymatic properties of renatured recombinant HRP CIA were analyzed.It was found that Fe3+ and Cu2+ had strong activating effect on it.(2)In order to achieve the recycling of the recombinant enzyme,the recombinant HRP CIA was immobilized in self-prepared agarose-chitosan hydrogel carrier and the immobilization efficiency was 86.9±2.5%.Its catalytic activity remained above 80%after 6 cycles.The immobilized enzyme was characterized by scanning electron microscope(SEM).The enzymatic properties of recombinant enzyme before and after immobilization were compared.It was found that the optimum pH of immobilized enzyme moved from 5.5 to 7.0 and the optimum temperature changed from 30? to 50?.The tolerance to metal ions and organic solvents was also improved,among which Co2+and dimethyl sulfoxide(DMSO)were the most obvious.(3)The catalytic activity of recombinant enzyme HRP CIA was evaluated by dye decolorization.The results showed that recombinant HRP CIA had good decolorization effect on some dyes.The decolorization rates of acid blue 129,methyl blue,methyl red and trypan blue were 76.28,75.03,72.42,and 72.78%.The decolorization effect of acid blue 129 was the most significant.(4)The decolorization solution of acid blue 129 catalyzed by HRP C1A was analyzed by ultra high performance liquid chromatography-mass spectrometry(UPLC-MS).The ion fragments showed that the C-N bond on the side chain of anthraquinone group of acid blue 129 was first broken under the catalysis of recombinant enzyme HRP CIA,and then the active hydrogen attacked the C=O bond of anthraquinone group.After multiple redox reactions,some substances formed insoluble 1-nitroanthraquinone and precipitated from the solution,while others continued to be degraded into phthalic acid and 2-amino-5-hydroxybenzenesulfonic acid catalyzed by HRP C1A.The possible degradation mechanism of acid blue 129 catalyzed by horseradish peroxidase was proposed for the first time.