Study On The Expression Of Phosphotriesterase In Escherichia Coli And Its Nano-immobilization

Phosphotriesterase（PTE,EC3.1.8.1）is considered to be an efficent biodegradant of organophosphorus pesticides.However,the low soluble expression and poor stability limit its widely application.In this study,the PTE gene（ob3）from Pseudomonas diminuta was heterologously expressed in Escherichia coli,and its enzymatic properties were determined.The fermentation conditions in 5 L fermentor were optimized and the purified PTE was immobilized by metal hybrid nanoflowers to enhance its capability in practical application.The results are as follow:（1）A low soluble expression strain E.coli BL21/p ET-20b-ob3 previously constructed in our laboratory was used as a template,ob3 gene was amplified by PCR and connected with plasmid p TIG-trx which could promote soluble expression.The recombinant strain E.coli BL21/p TIG-trx-ob3 was successfully constructed.SDS-PAGE analysis showed that the molecular weight of the expressed protein was 46 k Da,which was consistent with the theoretical value,and the enzyme activity was 6.45 U·mg^-1.The optimum reaction temperature and p H was 40℃and 10,respectively.The kinetic parameters were obtained by nonlinear curve fitting.The results showed that K_m and V_max were separately 0.116 mmol·L^-1and 24.67μmol·L^-1·min^-1,k_cat value was 224 min^-1,and k_cat/K_m was 1950 L·mmol^-1·min^-1.The fermentation conditions were optimized.It was found that IPTG 0.1 mmol·L^-1,induction for 24 h at 16℃was the most suitable condition for the expression of PTE.（2）The fermentation experiment was carried out in a 5 L fermentor.The OD₆₀₀ of the fermentor could reach 24,which was 3 times higher than that of shake flask,and the wet weight of cell was 4 times higher than that of shake flask.After purification by nickel column,the expression of the enzyme was about 110 mg·L^-1,which was 4.4 times higher than that in shake flask.（3）Four kinds of metal phosphates(Cu²⁺,Co²⁺,Mn²⁺,Ni²⁺)were employed for preparing metal-PTE hybrid nanoflowers.Under the optimum conditions,the enzyme activity recovery of Cu/Co/Mn/Ni-PTE hybrid nanoflower was 222.32%,300.74%,128.39%and123.54%,respectively.Scanning electron microscopy showed that the immobilized enzyme formed flower-like structure.The kinetic parameters showed that the catalytic efficiency(k_cat/K_m)of immobilized enzymes was 1.67,2.54,0.9 and 1.09 times higher than that of the free enzyme,respectively.In addition,compared with the free enzyme,the application range of temperature and p H of the immobilized enzyme was expanded,and its stability was improved.The relative activity of the free enzyme was about 56%after storage for ten days.Under the same conditions,the relative activity of four kinds of metal-PTE hybrid nanoflowers was 80.0%,81.3%,89.7%and 90.7%,respectively.Moreover,the relative activity of the four kinds of metal-PTE hybrid nanoflowers could serperately maintain 80.3%,77.0%,54.4%,and 44.1%relative activity after eight consecutive catalytic reactions.