Studies On Oxalate Decarboxylase Modified With The Adsorption Molecule On Calcium Oxalate Crystals

Oxalate decarboxylase（Oxdc, EC 4.1.1.2） is a kind of homogenous polymerase with the manganese ion and belongs to the protein cupin superfamily. In plants and microorganisms, Oxdc is one of the main enzyme which can catalyze the decomposition of oxalate to formic acid and CO₂ without cofactors.Based on the characterisitics of highly substrate specificity and high-efficiency of enzymecatalyzed reaction, it has been applied widely in agriculture, food, industrial process, medical, biological monitoring and other fields; especially in the field of calcium oxalate stone, it has been widely concerned. But in practical application, the concentration of the enzyme agent obtained from appropriate administration route will be rapidly diluted in urinary system under the state. Therefore, the aim of this paper is to improve the application performance of Oxdc by chemicial modification, in order to provide the theoretical reference for developing the enzymic preparations to prevent and cure urinary calculi. The main contents and results of this research are as follows:1. Under in vitro experimental conditions, the factors that affect the redissolution of calcium oxalate crystals catalyzed by Oxdc were studied. The results indicated that when 4 U of Oxdc was added to the 10-mL reaction system which was subsequently allowed to react at pH 3.0 and 37℃ for a week, the re-dissolution rate of calcium oxalate crystals was approximately 5%. The experimental results showed that the degradation product formate produced an inhibitory effect on the redissolution of calcium oxalate crystals catalyzed by Oxdc.2. This experiment was preliminarily screened the molecule adsorbed on calcium oxalate crystals, and the adsorption of ethylenediaminetetraacetic acid（EDTA） onto calcium oxalate monohydrate crystals was investigated. The experimental results showed that the EDTA adsorption on calcium oxalate monohydrate crystals increased with its initial concentration in the aqueous solutions. High temperature was beneficial to the adsorption process. However, the adsorption capacity of EDTA on calcium oxalate monohydrate crystals decreased as the pH increased from 5 to 8. The kinetic data fitting analysis suggested the pseudo-secondorder kinetic model described the adsorption process better. The adsorption equilibrium data analysis indicated that the Langmuir isotherm model fit better than the Freundlich isotherm model, based on the R2, RMSE and χ2 values. Thermodynamic parameters such as ΔG0, ΔH0 and ΔS0 for EDTA adsorption had also been estimated. The results demonstrated that the adsorption nature was feasible, spontaneous and endothermic.3. Chemical modification of Oxdc with ethylenediaminetetraacetic dianhydride（EDTAD） was investigated in this study. The results of response surface methodology（RSM） showed that the extent of modification and the recovery rate of the enzymatic activity can are reached 70.23% and 82.06%, respectively, when the reaction time is 7 h, the molar ratio of EDTAD/Oxdc is 62:1, pH 6.2, and the temperature is 25℃. The chemical modification strategies and the enzymology changes were described, and the kinetic characteristics of enzymatic reaction and adsorption properties for adsorbing on CaOx monohydrate crystals were also discussed. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDSPAGE） and ultra performance liquid chromatography tandem mass spectrometry（UPLC-MS） analysis results demonstrated that Oxdc and EDTAD have been covalently bound, and suggested that the chemical modification occurred at the free amino of the side chain and the α-amine of the N-terminus of Oxdc. Ultravioletvisible spectrum（UV）, fluorescene and circular dichroic（CD） measurement showed that the structure and conformation of Oxdc were tinily altered after modification by EDTAD. The analysis of kinetic parameters indicated that the EDTAD-modified Oxdc showed a higher affinity towards the substrate. The optimum pH of EDTADmodified Oxdc was shifted to the alkaline side about 1.5 unit and it has a higher thermostability. Through modification the adsorption capacity of Oxdc onto calcium oxalate monohydrate crystals was increased by 42.42%.