Date of Award:

5-1-1972

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Department name when degree awarded

Bacteriology

Committee Chair(s)

Larre N. Egbert

Committee

Larre N. Egbert

Committee

Rex Spendlove

Committee

Carl A. Westby

Committee

Bruce F. Burnham

Committee

John R. Simmons

Abstract

L-asparaginase was isolated and purified 60 fold from Bacillus kaustophilus by heat treatment, ammonium sulfate fractionation, adsorption chromatography on glass beads and preparative electrophoresis. The purified sample from ammonium sulfate precipitation and adsorption chromatography on glass beads could be stored at 4 C for several weeks and could withstand freezing and thawing without loss of activity. Kinetic studies showed that this L-asparaginase has a temperature optimum of 74 C, an activation energy of 11,400 calories, a pH optimum of 9.2 in sodium carbonate-bicarbonate buffer and Michaelis constant for asparagine of 3.7 x 10-3 Mat 74 C and pH 9.2. The enzyme showed no activity in the highly purified state unless monovalent cations such as Na+, K+ or Li+ were included in the reaction mixture. A hyperbolic pattern of activation was shown to be exerted by the monovalent cations. The enzyme showed a considerable resistance to the action of urea. It also displayed a sigmoidal kinetic pattern for the Michaelis-Menten plot, A molecular weight of 120,000-125,000 was obtained for the active enzyme from gel filtration data. Electrophoretically prepared L-asparaginase showed several protein bands on electrophoresis in the presence of sodium dodecyl sulfate with a molecular weight ranging from 32,500-46,000. Using the polyacrylamide gel electrophoresis and isoelectric focusing techniques, it was possible to demonstrate the presence of several bands with enzymatic activity.

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