Abstract
BackgroundThe concentration of urinary oxalate is more influential to the formation of calcium oxalate urolithiasis than is urinary calcium concentration. YvrK gene encodes a 43 KD-sized oxalate decarboxylase. We previously developed the recombinant Escherichia coli (E. coli) expressing Yvrk gene from Bacillus subtilis and named it as pBy. The aim of this study was to purify the recombinant oxalate decarboxylase overexpressed in E. coli and evaluate the oxalate-degrading activity of the purified enzyme.ResultsThe oxalate-degrading activity of pBy was highest when cultured at pH 5. The activity of purified oxalate decarboxylase was determined after incubation with sodium oxalate and the optimal pH and temperature of oxalate decarboxylase were determined. Purified oxalate decarboxylase degraded more than 50% of oxalate when incubated with MnCl2 and sodium oxalate in atmospheric O2. The optimal pH of recombinant oxalate decarboxylase was 5 and the optimal temperature was 28°C. Eight-week-old Sprague–Dawley male rats were used as a transient hyperoxaluric rat model. Suprapubic catheter was inserted into the bladder of each rat and urine was collected hourly before and 3 hours after oral oxalate intake in the absence and presence of homogenates of pBy and non-recombinant E. coli as the control. After the oral intake of sodium oxalate, the concentration of oxalate in urine increased exponentially for 3 hours. The oxalate concentration in urine was decreased significantly by pBy homogenates compared to control.ConclusionsWe constructed the recombinant E. coli expressing YvrK gene and purified the recombinant oxalate decarboxylase successfully. Purified recombinant oxalate decarboxylase, as well as recombinant E. coli named pBy, showed the oxalate-degrading activity in in vitro and in vivo model.
Highlights
Studies of the pathophysiology of urinary stones have focused on oxalate, since the concentration of urinary oxalate is more influential to the formation of calcium oxalate urolithiasis than is urinary calcium concentration [1]
Production of Yvrk-recombinant E. coli As we previously reported [11], 1.2 kb sized Yvrk gene was cloned from genomic DNA of B. subtilis by polymerase chain reaction (PCR)
Automated DNA sequencing identified the same DNA sequence of Yvrk gene-recombinant pBAD/gIII-A vector to that of the Yvrk gene registered in National Center for Biotechnology Information
Summary
The concentration of urinary oxalate is more influential to the formation of calcium oxalate urolithiasis than is urinary calcium concentration. We previously developed the recombinant Escherichia coli (E. coli) expressing Yvrk gene from Bacillus subtilis and named it as pBy. The aim of this study was to purify the recombinant oxalate decarboxylase overexpressed in E. coli and evaluate the oxalate-degrading activity of the purified enzyme. Studies of the pathophysiology of urinary stones have focused on oxalate, since the concentration of urinary oxalate is more influential to the formation of calcium oxalate urolithiasis than is urinary calcium concentration [1]. The majority of urinary oxalate is derived from the endogenous metabolism of glycine, glyoxylate, and ascorbic acid, and 40-50% is Oxalobacter formigenes is an oxalate degrading bacterium colonizing the gastrointestinal tract of vertebrates including humans. O. formigenes is difficult to culture and isolate, and has complex pathways of oxalate degradation
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