Optimization Of Fermentation Conditions For Recombinant Organophosphate Hydrolase MPH And The Primary Research On Enzyme Preparation

In this study, an organophosphate hydrolase MPH was expressed in Escherichia coliBL21(DE3). Experiments were designed to optimize the parameters for fermentation ofMPH. Inexpensive lactose was used as an inducer of expression of the MPD gene.Composition of the culture medium, lactose concentration, induction phase and metal ionswere tested, L₉ orthogonal test was designed to determine the optimal carbon and nitrogensource and lactose concentration. Fed-batch culture process of E.coliBL21(DE3)/pET29a-mpd were carried out to produce recombinant MPH in 7LBioengineering autocontrol fermentor. The final yield of MPH was 18.69 IU/ml.The protein was purified to homogeneity by 64.52 fold with metal affinitychromatography using Ni-NTA under non-denaturing conditions with a yield of 53.63ï¼…, itspurity was determined using SDS-PAGE. The kinetic constants, km and k_cat, for thehydrolysis of methyl parathion are 171.7μmol/L, 30.2S^-1 respectively. The purifiedrecombinant MPH displayed an optimal temperature around 15℃. The activity of thisprotein was affected by pH and the optimal pH was 7.5. Both the temperature and pHaffected the preservation of recombinant MPH, and the optimal preserved temperature andpH was 4℃and 6.5. Fe²⁺, Mn²⁺, Co²⁺, Cu²⁺ increased the activity of methyl parathionhydrolase , The metal chelating compounds EDTA·2Na and 1,10-phenanthroline almosthad no effect on the enzyme activity. The surfactants such as Tween-20, Tween-80,Triton-100 and SDS inhibited the action of recombinant MPH strongly.In addition, the preparation of the crude enzyme was also studied. With the optimizedeconomical stuffs, we cultured the E. coli BL21 (DE3)/pET29a-mpd and collected the crudeMPH. It is obviously that the protein is unstable in the Tris-HCl buffer for preservation.Poly(ethyleneglycol)6000,boracic acid,Na₂CO₃,NaCl,citrate sodium,formate sodium,glycerol and ethanol protected the protein from degradation. A kind of abluent was selected and improved for preserve the recombinant MPH, and the residual action of the proteinafter 90d's preservation was almost 100ï¼….