PARTIAL-PURIFICATION AND CHARACTERIZATION OF AN INDUCIBLE INDOLE-3-ACETYL-L-ASPARTIC ACID HYDROLASE FROM ENTEROBACTER-AGGLOMERANS

Indole-3-acetyl-amino acid conjugate hydrolases are believed to be imp ortant in the regulation of indole-3-acetic acid (IAA) metabolism in p lants and therefore have potential uses for the alteration of plant IA A metabolism. To isolate bacterial strains exhibiting significant indo le-3-acetyl-aspartate (IAA-Asp) hydrolase activity, a sewage sludge in oculation was cultured under conditions in which IAA-Asp served as the sole source of carbon and nitrogen. One isolate, Enterobacter agglome rans, showed hydrolase activity inducible by IAA-L-Asp or N-acetyl-L-A sp but not by IAA, (NH4)(2)SO4, urea, or indoleacetamide. Among a tota l of 17 IAA conjugates tested as potential substrates, the enzyme had an exclusively high substrate specificity for IAA-L-Asp. Substrate con centration curves and Lineweaver-Burk plots of the kinetic data showed a Michaelis constant value for IAA-L-Asp of 13.5 mM. The optimal pH f or this enzyme was between 8.0 and 8.5. In extraction buffer containin g 0.8 mM Mg2+ the hydrolase activity was inhibited to 80% by 1 mM dith iothreitol and to 60% by 1 mm CuSO4; the activity was increased by 40% with 1 mM MnSO4. However, in extraction buffer with no trace elements , the hydrolase activity was inhibited to 50% by either 1 mM dithiothr eitol or 1% Triton X-100 (Sigma). These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the h ydrolase by hydroxyapatite and TSK-phenyl (HP-Genenchem, South San Fra ncisco, CA) preparative high-performance liquid chromatography yielded a major 45-kD polypeptide as shown by sodium dodecyl sulfate-polyacry lamide gel electrophoresis.