Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola III. Kinetics of Levan and Alginate Formation in Batch Culture and Demonstration of Levansucrase Activity in Crude EPS

Abstract

AbstractIn batch culture Pseudomonas phaseolicola converted ca. 8–11% of the substrate (sucrose) into extracellular polysaccharides (EPS). The percentage of conversion was independent of the concentration of sucrose in the medium. However, the increase of total EPS observed with enhanced substrate concentration was largely due to a drastic stimulation of the levan produced. Production of alginate, in contrast, was stimulated to a much lower extent. EPS production was highest during the middle phaseof exponential growth and gradually terminated as the cultures grew older. With sucrose as substrate, the formation of alginate followed the levan synthesis. With glucose or gluconate, however, there was no production of levan, and the alginate synthesis, started immediately after the lag‐phase of the culture.Levansucrase activity was detected in the protein fraction of crude EPS produced by a culture grown in sucrose containing medium. Enzyme activity was demonstrated both by the release of glucose and the formation of levan from sucrose. Upon ion exchange chromatography on DEAE‐fractogel levansucrase behaved as a rather acidic protein. Native polyacrylamide gel electrophoresis of the enzyme fraction revealed a major band with an apparent molecular weight of ca. 68,000 d. Several grams of levan were synthesized with a partly purified enzyme preparation in the absence of bacteria.