Screening And Enzyme Properties Of A Highly Effective Oxalate-Degrading Strain

Oxalate-degrading strains and oxalate-degrading enzymes have a number of potential applications, including the control of sclerotinia stem rot in economically important plants caused by fungus Sclerotinia sclerotiorum, and in making plants resistant to the white mold disease by oxalate-degrading enzyme transgene, including medical diagnosis and treatments for hyperoxaluria and other oxalate-related diseases, bioremediation of the environment. The paper is to isolate the bacteria that can degrade oxalate, study degrading enzyme characteristics and the degrading mechanism.A method for the determination of oxalic acid with methylene blue - dichromate system had been developed. It was based on the catalytic effect of oxalic acid on the oxidation of methylene blue by dichromate in acidic media. The absorbance of methylene blue at the maximum wavelength of 660 nm was recorded at fixed reaction times. The absorbance differences (â–³A ) of the methylene blue - dichromate system with and without oxalic acid were linearly correlated with the oxalic acid concentration. The linear range of the calibration graph for oxalic acid was 0 - 80μg/mL with a correlation coefficient of 0.9986. The optimum concentrations for methylene blue, H₂SO₄ and K₂Cr₂O₇ were 3.752×10^-5 mol/L, 0.12 mol/L and 2 mmol/L, respectively. The developed method was simple, sensitive and inexpensive.One new strain designated as OXJ-11 was isolated from soil samples, which could grow in the medium with oxalate as the sole carbon and energy source. The bacteria colonies on plate appeared milky, circular and the isolate OXJ-11 was Gram-negative straight rod. It occured singly and was motile by means of a double polar flagellum. Catalase was positive and nitrate was not reduced. It growed aerobically at 25℃-30℃and pH 6.0 - 10.0, respectively. The polyphasic taxonomic data along with 16S rRNA sequence comparison demonstrated that the isolate OXJ-11 should belong to the genus Pandoraea and represented a new member in this family. The optimal fermentation medium composition was 0.5% sodium oxalate, 0.3% yeast extract powder, 0.05% MgSO₄·7H₂O. And the optimal temperature and pH for the bacterium's enzyme production were 30℃and pH 6.0-7.0. Pandoraea sp.OXJ-11 had been shown to produce the degrading enzyme which could be induced by the oxalate in medium. Quantitating the reduction of oxalate by the methylene blue - dichromate system, a method to assay the oxalate-degrading activity was constructed. The key enzyme was cell wall enzyme, the conditions of ultrasonic cell-break were: output power 200w - 300w, ultrasonic time 0.5s,intermission time 5s, total working time 7min. Tests showed that the optimal initial substrate concentration for oxalate-degrading enzyme was 0.7mg/mL, while the optimal temperature and pH was 30℃and 5.5 - 8.0; the enzyme activity was stable below 30℃and 7.0-8.0. The enzyme activity was inhibited by Fe²⁺ , Cr³⁺ and SDS had strong inhibition on it, while 10mmol/L Mg²⁺ could increase the enzyme activity. 0.15mol/L and 0.3mol/L NaCl reduced the enzyme activity by 50% and 100%, respectively.An enzymatic hydrolysate of the oxalate-degrading was carbon dioxide, and carbonate was formed by reaction between CO₂ and the cation in the system. Hydrogen peroxide was not the enzymatic hydrolysate. Formate was another enzymatic hydrolysate and it was degraded into carbon dioxide by formate dehydrogenase. Oxalate-degrading enzyme in OXJ-11 required molecular oxygen for catalytic turnover. Oxalate-degrading enzyme could use oxalic acid and succinic acid as exchangeable substrate. Both ATP and ADP could inhibit the enzyme activity.