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Biotransformation of fumaric acid to L-malic acid by the whole cells of baker's yeast

L-malic acid, an intermediate of cell metabolism, is used in pharmaceutical industry, as well as in food and cosmetic industries likes an acidulent (a substitute for citric acid). It is very effective in treating liver diseases, especially hyperammonemia, and it is used as one of the components in amino acid infusions1. In this work, biotransformation of fumaric acid to L-malic acid by the whole cells of baker's yeast was studied. Enzyme fumarase is a constituent part of cell metabolism (present in the cycle of citric acid)2, and baker's yeast3 is an accessible and inexpensive source of that enzyme. Two types of experiments of biotransformation of fumaric acid to L-malic acid were performed: experiments in a shaker and experiments in a laboratory bioreactor. Experiments on a shaker were performed using different concentrations of fumaric acid solution and different concentrations of baker's yeast suspension. This procedure was utilized in order to determine the effect of concentration of fumaric acid and concentration of baker's yeast on volumetric productivity and conversion, and to estimate kinetic parameters of the process model. An experiment with non-permeabilized yeast was performed to determinate the effect of permeabilization on process performances. Biotransformation of fumaric acid to L-malic acid in a laboratory bioreactor was conducted as batch, fed-batch, and a continuous experiment with biomass recycle. The mathematical model of the process includes a kinetic model4 and a reactor model, or in other words mass balances for every component, which depend on the type of the reactor. Kinetics of biotransformation of fumaric acid is described by the Michaelis-Menten equation with a competitive inhibition by the product, L-malic acid. Additionally, in the model for continuous bioreactor, a time-dependant deactivation of the enzyme was included. Model parameters were estimated by simplex or least squares method using SCIENTIST software package. Model applicability was demonstrated by comparing experimental results with the results obtained from model simulations. Figure 1 shows results of the experiments conducted in a shaker using different starting concentrations of the permeabilized cells of baker’ s yeast. The concentration of baker's yeast suspension influences the time needed to establish equilibrium and accomplish maximum conversion. From the reported results (Figure 1), it can be also seen that the maximum conversion is practically independent of the concentration of the permeabilized cells of baker’ s yeast and is approximately 80 % in all experiments. This model can be thus effectively used to describe the results of biotransformation of fumaric acid to L-malic acid for a wide range of concentrations of the permeabilized cells of baker’ s yeast, as well as for different starting concentrations of fumaric acid. Due to the established inhibition by the product, in addition to the experiments on a shaker and in a batch bioreactor, a continuous experiment with biomass recycle in a bioreactor was conducted (Figure 2). In this continuous process, volumetric productivity decreases due to the time-dependent deactivation of enzyme fumarase in the cells of baker’ s yeast, and the related reduced speed of the reaction was observed. With equal concentrations of baker’ s yeast, 10 g dm-3, in all experiments, volumetric productivity is the highest in a continuous process. This makes a continuous bioreactor the optimal configuration for the observed process of biotransformation of fumaric acid. Simulation results of biotransformation of fumaric acid to L-malic acid using the proposed mathematical model fit the experimental data well. 1. Wang, X. ; Gong, C.S. ; Tsao, G.T. L-malic acid production from fumaric acid by a laboratory Saccharomyces cerevisiae strain SHY2, Bio. Letters, 18 (1996) 1441, 1442. 2. Belafi-Bako, K. ; Nemestothy, N. ; Gubicza, L. A study of applications of membrane techniques in bioconversion of fumaric acid to L-malic acid, Desalination, 162 (2004) 301-303. 3. Vrsalović Presečki, A. Studij procesa pridobivanja enzima u rastućim stanicama pekarskog kvasca, Master thesis, Zagreb, 2003. 4. Vasić-Rački, Đ. ; Kragl, U. ; Liese, A. Benefits of Enzyme Kinetics Modelling, Chem. Biochem. Eng., 17 (2003) 7-18.

biotransformation; L-malic acid; baker's yeast; mathematical model

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