Publication Date

5-1989

Advisor(s) - Committee Chair

David Hartman, Frank Toman, Martin Houston

Degree Program

Department of Biology

Degree Type

Master of Science

Abstract

Various properties of a carboxylesterase of the greater wax moth larva, Galleria melionella (L.), and a commercial carboxylic ester hydrolase (EC 3.1.1.1) from porcine liver were examined. The larval enzyme was partially purified. Esterase activities were determined with acetylsalicylic acid, 0-acetyl-m-hydroxybenzoic acid, and 0-acetyl-phydroxybenzoic acid in tris buffer (0.05 M tris, 0.1 M EDTA, pH 7.85). The larval enzyme was partially purified by a combination of acetone powder preparation, ammonium sulfate precipitation, anion exchange chromatography, 0-200 gel filtration, and polyacrylamide gel electrophoresis (PAGE).

The acetone powder preparation was initially obtained and tested for esterolytic activity. The ammonium sulfate fractionation greatly increased the stability of the enzyme preparation. After 18 days of refrigerated storage, 63% of the original specific activity remained. The specific activity was increased from 0.108 umol/min/mg to 1.415 umol/min/mg by the 50% ammonium sulfate fractionation. This step also decreased the total protein content by 50%. The enzyme was further purified by a combination of chromatographic techniques. Fach procedure showed an increase in specific activity with a considerable reduction in the total protein content. Various chromatographic fractions were separated by polyacrylamide gel electrophoresis. There was a large reduction in the number of protein bands observed, with only one strong band of protein present after the final purification procedure was performed.

The kinetic behavior of the larval enzyme and the commercial esterase with different substrates was studied. Using the commercial enzyme, the Vmax and Km values were found to be 0.046 umol/min/mg and 7.62 Um, 0.227 umol/min/mg and 1.61 uM, 1.11 umol/minimg and 1.67 uM, for acetylsalicylic acid, 0-acetyl-m-hydroxybenzoic acid, and O-acetyl- p-hydroxybenzoic acid used as substrate molecules respectively. Using the larval enzyme, the Vmax and Km values were found to be 0.116 umol/min/mg and 9.09 uM when the substrate being used was O-acetyl-p-hydroxybenzcic acid.

Spontaneous hydrolysis of the various hydroxybenzoic acid ester substrate molecules was observed with varying values in pH. The acetylsalicylic acid showed considerable hydrolytic rates over a broad range in pH (6.5-8.6). The Cacetyl- p-hydrcxybenzoic and 0-acetyl-m-hydroxybenzoic acid demonstrated considerable rates of hydrolysis at pH values above 8.0, and negligible rates at pH values below 8.0.

Spontaneous hydrolysis of the various substrate molecules was observed in 50% saturated ammonium sulfate solutions. The rate of hydrolysis was significant and interfered with low specific activity enzyme preparations.

Disciplines

Biology | Life Sciences

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