Optimization of Starch and Tryptone Concentrations in Medium Composition for Lipase Production by Submerged Fermentation of Rhizopus arrhizus Using Response Surface Methodology

Abstract

Aims: The aim of this study is optimization of the concentrations of carbon and nitrogen sources for lipase production by Rhizopus arrhizus using response surface methodology. Study Design: For this work optimal 22 composite design was used for studying the optimal concentrations of corn starch and tryptone for lipase production by submerged fermentation. A series of planned experiments in three replications was carried out and a mathematical model was developed which was used to describe the process. Optimal levels of studied independent variables were calculated by using the model and the conversion rate. Place and Duration of Study: This study is a part of PhD dissertation developed in University of Food Technologies, Bulgaria, Department of Biochemistry and Molecular Biology. Methodology: Maximum lipase activity was achieved by an optimization of some components of the fermentation medium. Corn starch (in concentrations 5.0, 10.0, 15.0 g.dm-3) and tryptone (2.0, 5.0, 8.0 g.dm-3) as independent variables were chosen. Lipase activity was determined by a spectrophotometric assay using synthetic substrate p-nitrophenyl palmitate. Results: A planned mathematical experiment was carried out and a regression model was developed. The value of R2 was 95.65% which showed that the model had high correlation with the experimental results. The effects of every independent variable had an optimal value while the interaction effects led toenhancement of lipolytical activity. In this case the enzyme activity increased rapidly to 1100 U.dm-3. For lipase activity above that value, large enhancements of the corn starch and tryptone concentrations were needed. In order to use the medium substrates properly there the conversion rate was calculated and it was also considered for the optimization. Conclusion: By carrying out an optimal composite design a mathematical model was derived, with the aid of which, optimum values of tryptone (6.6 g.dm-3) and corn starch (10.5 g.dm-3) were determined, when the conversion rate and the first derivative of enzyme activity were considered. Those results were confirmed by triplicate experiments at the optimal concentrations. Lipase activity of Ȳ=1340.74 U.dm-3 was achieved, which was very close to the predicted one Ŷ=1235.26 U.dm-3.