Modelling of growth kinetics of isolated Pseudomonas sp. and optimisation of parameters for enhancement of xanthine oxidoreductase production by statistical design of experiments

Abstract

This report presents the substrate inhibitory effect of xanthine (XN) on microbial growth and optimisation of effective parameters to achieve high enzyme activity of xanthine oxidoreductase (XOR) through statistical design. Three efficient isolated strains (Pseudomonas aeruginosa CEBP1 and CEBP2, Pseudomonas sp. CEB1G) were screened for growth kinetic studies. Substrate inhibitory models (eg. Aiba, Edward) could explain the growth kinetics of CEBP1, CEBP2 and CEB1G very well with various initial [XN] (S₀), e.g., 0.1-35 g L^-1. Highest XOR activity was obtained at stationary phase when biomass yield was high. Highest catalytic efficiency (k_cat/K_M) of XOR was obtained by CEBP1 at optimum specific growth rate of 0.082 h^-1 and biomass yield of 0.196 g g^-1 at S₀ = 5 g L^-1. The effects of S₀, pH and temperature were studied by Box-Behnken experimental design to evaluate the interactive effects of the significant variables influencing XOR production by CEBP1. ANOVA with high correlation coefficient (R² > 0.99) and lower 'Prob > F'value (< 0.05) validated the second order polynomial model for the enzyme production. The highest XOR activity of 31.2 KU min^-1 mg^-1 was achieved by CEBP1 under optimised conditions (35 °C; S₀=5 g L^-1; pH = 7.0) as compared to any report in literature. A sevenfold substrate affinity of the enzyme was observed after purification.

Keywords: Box-Behnken design; Xanthine oxidoreductase; growth kinetics models; ion exchange chromatography; substrate inhibition.