L-MALIC ACID FORMATION BY IMMOBILIZED SACCHAROMYCES-CEREVISIAE AMPLIFIED FOR FUMARASE

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to L-malic acid at a rate of 65 mM g-1 h-1 in free cell experiments, and approximately 87% of the fumaric acid was converted to L-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871-mu-m mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-mu-m diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.