Hybrid Nanoflower Immobilization And Recombinant Expression Of Brevibacterium Sp.COD

Cholesterol Oxidase?COD?is a multifunctional enzyme that belongs to flavo-oxidoreductase.It is an important enzyme for cholesterol degradation during biological cell metabolism and is widely used in clinical detecting,agriculture,food testing and other fields.In this study,cholesterol oxidase gene?cod?from Brevibacterium sp.was expressed in E.coli BL21,and the enzymatic properties were studied.And organic-inorganic hybrid nano-immobilization was employed,the conversion ability of COD to different substrates was investigated,and the product was identified.?1?The pET28a-cod plasmid containing the cod gene from Brevibacterium sp.was used as a PCR template to amplify the cod gene,and it was inserted into pRham^TM plasmid and further expressed in E.coli BL21.Using 7-Cl-isoxazine/FMN-affinity purification,COD was prepared with specific enzyme activity at 25.23 U·mg^-1.The optimal reaction temperature of COD was 50°C,the optimum reaction pH was 7.0,and the T_m was 60.5°C by differential scanning calorimetry?DSC?.The induction conditions of were optimized,and the concentration of the inducer was 0.25%,at 30°C for 16 h,the enzyme activity was 10.6U·mL^-1,which was 1.5 times that of the unoptimized one.?2?COD was fixed by means of nano-hybridization of organic-inorganic metals,and the non-aqueous soluble salt of Cu²⁺,Ca²⁺,Zn²⁺,Mn²⁺and Fe³⁺were selected as inorganic carrier.The immobilization conditions were optimized in terms of different enzyme concentrations and metal ion concentrations.Finally the enzyme concentration of Cu²⁺,Zn²⁺and Mn²⁺carriers was 0.15 mg·mL^-1 and the metal ion concentration was 1.6 mmol·L^-1,as the optimal condition.In addition,when 0.1 mg·mL^-1 COD was used,0.8 mmol·L^-1 Ca²⁺and 1.6mmol·L^-1 Fe³⁺were used for subsequent experiments.The immobilized nanoflowers were characterized by FE-SEM,DSC,XRD and FTIR techniques,and the enzymatic properties of the immobilized enzymes were determined.It was suggested that when Cu²⁺,Ca²⁺and Zn²⁺were inorganic carriers,the highest specific enzyme activities under optimal conditions were0.59 U·mg^-1,0.59 U·mg^-1 and 0.63 U·mg^-1,respectively.While the specific enzyme activities of Mn²⁺and Fe³⁺were 1.95 U·mg^-1,1.12 U·mg^-1,relatively,higher than those of Cu²⁺,Ca²⁺,Zn²⁺.The temperature stability,pH stability and storage stability of the immobilized enzyme were greatly improved compared with those of free enzyme.The semi-denaturation temperature of enzyme was increased from 60.5°C to 138.49°C,167.02°C,167.02°C,160.99°C and 172.85°C,respectively.?3?The substrate specificity of free enzyme and immobilized enzyme were studied by five substrates:cholesterol,stigmasterol,pregnenolone,ergosterol,?-sitosterol and dehydroepiandrosterone.The results suggested that COD,beside cholesterol,the natural product of COD,it could also converse other steroids,including pregnenolone and dehydroepiandrosterone.The converted products were separated and purified,analyzed by LC-MS,NMR,FTIR and other technical methods to determine the molecular weight and structural formula.