Enzymatic depolymerization of emulsan.

Abstract

Emulsan, the polyanionic emulsifying agent synthesized by Acinetobacter calcoaceticus RAG-1, was depolymerized by an enzyme obtained from a soil bacterium YUV-1. The extracellular emulsan depolymerase was produced when strains RAG-1 and YUV-1 were grown together on agar medium. The enzyme was extracted from the agar and concentrated by ultrafiltration and ammonium sulfate precipitation. The molecular weight of the enzyme was estimated to be 89,000. Emulsan depolymerase activity was due to an eliminase reaction which split glycosidic linkages within the heteropolysaccharide backbone of emulsan to generate reducing groups and alpha, beta-unsaturated uronides with an absorbance maximum of 233 nm. Deesterified emulsan was degraded by emulsan depolymerase at only 27% of the rate of the native polymer. The treatment of emulsan solutions with emulsan depolymerase for brief periods caused a rapid and parallel drop in viscosity and emulsifying activity. More than 75% of the viscosity and emulsifying activity was lost at a time when less than 0.5% of the glycosidic linkages were broken. These data indicate that (i) emulsan depolymerase is an endoglycosidase and (ii) the higher the molecular weight of emulsan, the greater its emulsifying activity. Exhaustive digestion of emulsan with emulsan depolymerase produced oligosaccharides with a number average molecular weight of about 3,000. The fractionation of the digest on Bio-Gel P-6 yielded four broad peaks. The pooled fractions from each of the peaks contained the same relative amounts of reducing sugar and had an absorbance at 233 nm. The molar ratio of esterified sugar to reducing groups was close to 2 in each fraction.