The Research Of Lactic Acid Bacteria Recombined With The Coding Gene Of Oxalate-degrading Enzymes In Reducing Hyperoxaluria

Objective: Calcium oxalate stone is one of the most common urological diseases, hyperoxaluria is the most important pathological factor, till now there is no efficient method for the treatment and prevention of hyperoxaluria. Dietary oxalate is the main source of urinary oxalate, and when there is less oxalate in food, the hyperoxaluria should be reduced. Oxalate decarboxylase(ODC) and oxalate oxidase(Ox O) are efficient and specific oxalate-degrading enzymes, and they both show potential in the treatment of calcium oxalate stones. In this research, we construct lactic acid bacteria(Lab) recombined with the coding gene of ODC and Ox O and express them to degrade oxalate continuously. And then the dietary oxalate would be degraded by recombinant Lab through oral administration, consequently, urinary oxalate should be reduced and stones should be prevented.Methods: The coding gene of ODC and Ox O was combined with expressing plasmid p MG36 e, which was transformed into Lab strain MG1363. To improve the express level, we used a p H-regulating promotor p170. The recombinant Lab was generated in culture medium containing 100mmol/L oxalate in 37℃ for 48 hours. We examined the growth and oxalate degradation by testing media’s absorbance using spectrophotometer and testing media’s oxalate concentration using high performance liquid chromatography(HPLC). The rats were fed with 5% oxalate diet for one month and at the same time, the recombinant Lab was given. The 24-hour urine was collected at the 0, 7th, 14 th, 21 st, 28 th days, the oxalate in urine was measured. At the30th day, the kidneys were removed and the sections from each kidney were stained with silver nitrate. Then the Ca Ox crystals were examined under light microscope.Results: The Lab recombined with ODC(ODC-Lab) and that recombined with Ox O(Ox O-Lab) could both grow in the media with 100mmol/L oxalate, but only ODC-Lab could degrade the oxalate, and after adding the promotor p170(p170-ODC-Lab), the degrading efficiency improved. In the animal study, ODC-Lab and p170-ODC-Lab reduced the urinary oxalate and prevented the formation of Ca Ox cystals. However, Ox O-Lab and p170-Ox O-Lab could not degrade the oxalate in media, and they had no function to reduce urinary oxalate, neither to prevent the formation of Ca Ox cystals.Conclusion: Recombinant Lab with ODC could efficiently degrade oxalate in vitro, and p170 could improve its biological function. ODC-Lab and p170-ODC-Lab could decrease the absorption of dietary oxalate by oral administration, and they had the potential for the treatment and prevention of calcium oxalate stones.