Studies On The Biosynthesis Of Polyglucosides Of Aureobasidium Pullulans

Date of Award

1982

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Abstract

The elaboration of polyglucosides by A. pullulans was studied at three levels. These include the elaboration of glycogen, an intracellular carbohydrate reserve polymer; pullulan, an extracellular polysaccharide; and (beta)-(1 (--->) 3)-glucan, a water insoluble structural polysaccharide of the cell wall.;Protoplasts were prepared form log-phase blastospores of A. pullulans using Driselase, a lytic preparation from the Basidiomycete, Irpex lacteus. The protoplasts were shown to be capable of regenerating walls in liquid media. The reversion process was studied with the fluorescent stain, Calcofluor M2R.;Glycogen was shown to be accumulated and reutilized in A. pullulans. Glycogen synthetase was recovered from extracts of both protoplasts and whole cells of A. pullulans. This activity used UDPG as a glycosyl donor and had a strict requirement for exogenous glycogen as primer. Glucose-6-phosphate activated the enzyme. The enzyme underwent a time and temperature dependent increase in activity. This effect could be mimicked by trypsin and inhibited by the protease inhibitors TPCK, TLCK, and ZPCK.;Extracellular polysaccharide (pullulan) elaboration by A. pullulans blastospores was studied by a highly efficient micro-assay. The induction and decay of the elaborative process was studied with respect to the effects of glycolysis intermediates. The inhibition of polysaccharide elaboration by acetate was shown to be a non-specific effect due to interference in glucose metabolism. A similar explanation accounted for the inhibition of elaboration by TPCK and ZPCK.;Protoplasts of A. pullulans elaborated extracellular polysaccharide including pullulan (50%), indicating that the cell wall is not a requirement for elaboration. The pullulan produced was, however, of much lower molecular weight than that produced by whole cells suggesting some change in the elaborative process.;A cell-free system for the synthesis of (beta)-glucan was developed using the particulate fraction of whole cell extracts as the source of enzyme. UDPG was the glycosyl donor and Mg('++) and sucrose (1M) were required to stabilize the enzyme. BSA and cellobiose enhanced the activity. The possibility that this enzyme exists as a zymogen was suggested by an increase in activity after trypsinization. The glucan produced was insoluble in water, soluble sodium hydroxide (.5M), and susceptible to hydrolysis by (beta)-(1 (--->) 3)-glucanses.

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