Partial purification and characterization of an inducible indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans
- Department of Agriculture, Beltsville, MD (United States)
- Department of Agriculture, Beltsville, MD (United States); and others
Indole-3-acetyl-amino acid conjugate hydrolases are believed to be important in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. To isolate bacterial strains exhibiting significant indole-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge inoculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglomerans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-Asp but not by IAA, (NH{sub 4}){sub 2}SO{sub 4}, urea, or indoleacetamide. Among a total of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp of 13.5 mM. The optimal pH for this enzyme was between 8.0 and 8.5. In extraction buffer containing 0.8 mM Mg{sup 2+} the hydrolase activity was inhibited to 80% by 1 mM dithiothreitol and to 60% by 1 mm CuSO{sub 4}; the activity was increased by 40% with 1mM MnSO{sub 4}. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50% by either 1 mM dithiothreitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Francisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 45 refs., 5 figs., 3 tabs.
- OSTI ID:
- 495299
- Journal Information:
- Plant Physiology (Bethesda), Vol. 112, Issue 3; Other Information: PBD: Nov 1996
- Country of Publication:
- United States
- Language:
- English
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