Skip to main content
ARS Home » Research » Publications at this Location » Publication #70998
Title: PARTIAL PURIFICATION AND CHARACTERIZATION OF AN INDUCIBLE INDOLE-3-ACETYL-L-ASPARTIC ACID HYDROLASE FROM ENTEROBACTER AGGLOMERANS

Author
item CHOU, JYH-CHING - UNIVERSITY OF MARYLAND
item KULECK, GARY - LOYOLA MARYMOUNT UNIV.
item Cohen, Jerry
item MULBRY, WALTER - 1270-35-00

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/23/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Fruit growth and ripening are controlled by the interaction of several signaling compounds (plant hormones), the most important of which are auxin and ethylene. Reduction in auxin levels toward the end of fruit growth results in a rapid rate of the biochemical processes known as ripening. In order to develop the technology to alter auxin levels in fruit, a bacterial lenzyme that releases free auxin from bound forms was isolated and characterized. These findings will be important for the development of improved genetic traits for the production of tomato fruit with better ripening and postharvest qualities. These results will be useful to plant biochemists, plant breeders and molecular biologists interested in improving the quality and ripening characteristics of tomato and to consumers interested in the potential for obtaining variety improvements.

Technical Abstract: Indole-3-acetyl-amino acid conjugate hydrolases are believed to play important roles in the regulation of indole-3-acetic acid (IAA) metabolism in plants and therefore have potential uses for the alteration of plant IAA metabolism. In order 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-aspartic acid, but not by IAA, (NH4)2SO4, 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. Substrate concentration curves and Lineweaver-Burk plots of the kinetic data showed a Km value for IAA-L-Asp at 13.5 mM. The optimal pH for this enzyme was between pH 8.0 to 8.5. In extraction buffer containing 0.8 mM Mg2+, the hydrolase activity was inhibited to 80 percent by 1 mM DTT and 60 percent by 1 mM CuSO4, and the activity was increased by 40 percent with 1 mM MnSO4. However, in extraction buffer with no trace elements, the hydrolase activity was inhibited to 50 percent by either 1 mM DTT and 1 percent Triton X-100. These results suggest that disulfide bonding might be essential for enzyme activity. Purification of the hydrolase by hydroxyapatite and TSK-phenyl preparative HPLC yielded a major 45 kD polypeptide as shown by SDS-PAGE.