Crystal Preparation Of Oxalate Decarboxylase And Optimization Of MPEG Modified Conditions By Response Surface Methodology

Experimental of genetically engineered bacteria E.coliBL21(DE3)/pET32a/YvrK that derived from Bacillus subtilis was inducedto successfully express the objective product, oxalate decarboxylase(Oxdc). And optimization of expression conditions were studied to obtainpurified oxalate decarboxylase, which of16.8U/mL activity,1.58mg/mLprotein content, and10.63U/mg specific activity.For further explanation to catalytic mechanism and relationshipsbetween the structure and function of oxalate decarboxylase, the standingcrystallization method using saline solution as a crystallization agent wasused to made crystals of oxalate decarboxylase, successfully. And thecrystals were characterized by SDS-PAGE, field scanning electronmicroscopy and other methods. The experiment initially explored out thecrystallization conditions and preliminary learned the crystal form ofoxalate decarboxylase, which lay a foundation of further optimization forperfect single crystal of oxalate decarboxylase during integratemulti-factors.Purification of oxalate decarboxylase from crude extract was carriedout by fast protein liquid chromatography system (AKTA-FPLC), thenmPEG-aldehyde (mPEG-ALD) was used to modify the oxalatedecarboxylase. Experiments characterized the molecular weight andstructural changes of oxalate decarboxylase by SDS-PAGE and InfraredSpectroscopy (IR), comparisons of enzymatic properties such as thermalstability, thermotolerance and trypsin resistance of wild enzyme (Oxdc)and modified enzyme (mPEG-Oxdc) had been analyzed. Meanwhile,optimization experiments of single factor for PEGylation conditions werecarried out, the optimal results were as follows: mPEG-ALD/Oxdc molar ratio of50:1, pH5.0, reaction temperature of37℃, modified for12h,which could remain about67.52%of enzymatic activity and50.69%ofmodification rate. This study which improve the stability of oxalatedecarboxylase provide practical protection for its further applied research.Oxalate decarboxylase would have a higher activity, better stabilityand stronger applicability, if it is highly modified. Thus, furtheroptimization using response surface methodology (RSM) based on afour-factor-three-level Box-Behnken central composite design wasapplied to evaluate the effects of mPEG modified conditions in this work,and got the best conditions: the molar ratio of mPEG-ALD/Oxdc was47.63, reaction temperature was29.85℃, reaction time was13.1h and thereaction pH5.29. Under these conditions, the modified rate (MR) andrecovery rate of enzyme activity (RR) reached73.69%and67.58%,respectively. This experiment provide technical support for futherresearch of modified oxalate decarboxylase, as well as lay a foundationfor preparation of enzyme preparation to prevent urinary tract calculi.