Recombinant Expression,Immobilization,Mycotoxin Degradation And Hydrogen Detection Of Dye-Decolorizing Peroxidase

Dye-decolorizing peroxidases（DyPs）are a novel family of peroxidases with heme as a cofactor.It has been found that DyPs not only can degrade anthraquinone dyes,but also have good degradation ability on mycotoxins.However,the poor storage stability and non-recyclability of DyPs limit their applications in industry.Magnetic chitosan（Fe₃O₄@CS）is a good immobilized enzyme carrier due to its excellent biocompatibility and low price.In this study,the optimized dye-decolorization peroxidase RhDypB gene derived from Rhodococcus jostii RHA1 was used for recombinant expression in Escherichia coli.The results showed that RhDypB could degrade aflatoxin B₁（AFB₁）and zearalenone（ZEN）simultaneously.In addition,the magnetic chitosan Fe₃O₄@CS was used as the carrier for RhDypB immobilization,which improved the storage stability and reusability of RhDypB.Moreover,a colorimetric method for hydrogen peroxide detection was established with Fe₃O₄@CS@RhDypB as the biosensor.This research provides theoretical support for the potential application of immobilized RhDypB in food processing and detection fields.The specific research contents were as follows:1.The recombinant E.coli BL21（DE3,pET28a-RhDypB）was constructed based on the gene sequence of RhDypB on NCBI.The expression of RhDypB in E.coli was achieved,and the RhDypB was successfully purified with a nickel column.The molecular weight of RhDypB was 40 k Da,Rz value was 2.4,and specific enzyme activity was 13.62 U/mg.The enzymatic properties of RhDypB were investigated and it was found that the optimum p H was 4.0,optimum temperature was 65°C,K_m was 0.86 mmol/L,k_cat was 4.02 s^-1,and k_cat/Km of 16.30mmol/L^-1 s^-1.2.It was found that RhDypB showed an effective degradation ability for AFB₁ and ZEN simultaneously.The degradation ratios of RhDypB for AFB₁ and ZEN were 85.61%and86.52%,respectively.In addition,the enzyme agents was prepared by vacuum freeze drying,and results showed that the RhDypB agents still had excellent mycotoxin degradation ability after 50 days of storage.Meanwhile,the major degradation products of AFB₁ and ZEN by RhDypB were analyzed,which were AFQ₁ and 15-OH-ZEN,respectively.Fortunately,the toxicity of both degradation products were much less than that of AFB₁ and ZEN.3.The magnetic nanoparticles Fe₃O₄ were prepared by chemical precipitation method,and then modified by chitosan.The magnetic chitosan Fe₃O₄@CS was uesd as the immobilized carrier for immobilization of RhDypB.FTIR analysis showed that the immobilized RhDypB had the characteristic peaks of Fe₃O₄,chitosan and enzyme molecules.XRD showed that the crystal structure of immobilized RhDypB cubic inverse spinel structure.SEM showed that the immobilized RhDypB had uniform particle size and distribution.Magnetization curves showed that the immobilized RhDypB was superparamagnetic.4.The optimal immobilization conditions of RhDypB were studied.The optimal immobilization parameters were as follows:crosslinking time was 80 min,crosslinking agent concentration was 2%,crosslinking p H was 6.0,crosslinking temperature was 35°C,and quantity of enzyme was 3 U.Under the optimal conditions,the protein loading of Fe₃O₄@CS@RhDypB was 24.60 mg/g,and the enzyme activity recovery was 55.21%.The enzymatic properties of immobilized RhDypB and free RhDypB were studied and results showed that the immobilized RhDypB exhibited better relative enzyme activity under different p H and temperature conditions.In addition,Fe₃O₄@CS@RhDypB could maintain 65.23%relative enzyme activity after 10 times of reuse.More importantly,the free RhDypB only has4.20%relative enzyme activity left after 9 days of storage without protective agents,while immobilized RhDypB could maintain 30.21%relative enzyme activity after 15 days of storage.5.The degradation effects of AFB₁ and ZEN were studied based on Fe₃O₄@CS@RhDypB.The results showed that the degradation ratios of AFB₁ and ZEN by Fe₃O₄@CS@RhDypB were78.12%and 83.98%,respectively.The degradation ratios of AFB₁ and ZEN were 38.50%and49.76%after reused for 5 times,and the degradation ratios of AFB₁ and ZEN were 43.11%and52.67%,respectively,after 10 days of storage.6.Based on the peroxidase properties of RhDypB,a hydrogen peroxide colorimetric detection system with ABTS as substrate was established by using Fe₃O₄@CS@RhDypB as biosensor.The results showed that the biosensor has a good detection performance for hydrogen peroxide with a detection range of 5μmol/L-50μmol/L and a detection limit of 3.3μmol/L.Compared with other hydrogen peroxide colorimetric detection methods,the reaction time of Fe₃O₄@CS@RhDypB was faster.Furthermore,Fe₃O₄@CS@RhDypB also showed excellent signal stability and selectivity.