Product optimization, purification and characterization of a novel polygalacturonase produced by Macrophomina phaseolina

Introduction: Production of a novel polygalacturonase (PG) active at pH = 3.0 and suitable to be used in fruit juice industries from Macrophomina phaseolina was evaluated. Suitable carbon, nitrogen and phosphorous forms were determined and the condition was optimized for higher PG production. Materials and methods: Macrophomina phaseolina was cultured in so called production medium. The secretome was separated from fungal cells and polygalacturonase was isolated via column chromatography. The biochemical activity of both secretome and isolated polygalacturonase were assayed calorimetrically. The production of polygalacturonase was optimized via changes in culture medium in terms of contents, pH and temperature and Taguchi analysis of data. Enzyme kinetics was partially performed followed by the determination of pH and temperature stability . Results: A range of sugars except glucose and chitin demonstrated to improve the PG production. Ammonium sulfate and peptone demonstrated to be suitable nitrogen sources. Amongst phosphorous sources, dipotassium hydrogen phosphate had the greatest effect. Taguchi’s orthogonal array demonstrated that pH, temperature, ammonium sulfate and trace elements had significant effect on PG production. Furthermore, mean comparisons showed that the optimum condition achieved at pH 6.0, 35 °C, 2 g.l -1 ammonium sulfate and 2 mg.l -1 trace elements . Discussion and conclusion: The purified PG with a relative molecular mass of 70 kDa was demonstrated its highest activity at pH = 3, and 30 ° C. Amongst tested cations and chemicals, Fe 2+ improved the enzyme activity by 2 fold, while the secretome responded differently .