Optimization of Growth Conditions for Oxalate Decarboxylase Production from Pseudomonas sp. OXDC12 and in vitro Inhibition of Calcium Oxalate Crystallization by Oxalate Decarboxylase

Abstract

Background: Kidney stones have become a common disease worldwide, and their incidence and recurrence rates have drastically increased over the past few decades. Oxalate decarboxylase (OxDC) enzyme which catalyzes the disproportionation reaction of oxalate monoanions into formate and CO2 could exhibit significant potential in the treatment of hyperoxaluria. Objective: The present work describes isolation and screening of new OxDC producing bacterial strain from oxalate-rich soils and one-factor-at-a-time (OFAT) and response surface methodology (RSM) statistical approaches were used to optimize the production media to obtain an improved intracellular OxDC production. Methods and Results: An OxDC producing bacterial strain, isolated from spinach soil sample(s), was identified to be Pseudomonas sp. OXDC12 by 16S rRNA sequencing. The OFAT approach was used to determine the effect of supplementation of carbon, nitrogen, and other physical conditions like pH, temperature, etc. on intracellular OxDC production by Pseudomonas sp. OXDC12. The three factors screened by Plackett Burman design (PBD) were further used by the central composite design (CCD) approach of RSM to determine their interactive effects on OxDC production. The anti-urolithiatic activity of the enzyme OxDC was determined by carrying out in vitro calcium oxalate crystallization in the presence and absence of OxDC. The factorial values selected by 23 CCD for OxDC were temperature 30ºC, manganese ion concentration 5 mmol l-1, and inoculum size 3.25% (v/v). The highest predicted value of OxDC was 5.7 U ml-1 while the actual value obtained was 6.7 U ml-1 which was 79.1% and 2.92 fold greater than the initial activity of OxDC produced by Pseudomonas sp. OXDC12. As depicted by the light micrographs, OxDC displayed a significant reduction in the crystallization and formation of calcium oxalate stones as compared to the control under in vitro conditions. Conclusion: OFAT and RSM statistical optimization approaches led to improved OxDC production with a final activity of 6.7 U/mL and a 2.92 fold increase in the enzyme activity. The study suggests that OFAT and RSM optimization approaches significantly enhanced OxDC production from Pseudomonas sp. OXDC12. The enzyme may serve as a potential therapeutic agent for hyperoxaluria or kidney stones as it significantly inhibited the formation of calcium oxalate crystals under in vitro conditions.