Purification and Characterization of the Enzymes

The DCases from Agrobacterium sp. KNK712 and Pseudomonas sp. KNK003A were purified after large-scale incubation by ammonium sulfate precipitation and chroma-tography such as ion exchange and gel filtration.

DCase activity was assayed by measurement of D-p-hydroxyphenyl glycine (d-HPG) production from A-carba-moyl-D-HPG (c-D-HPG) (12). The reaction was started by the addition of 100 uL of an enzyme solution diluted with 100 mM potassium phosphate buffer, pH 7.0 (hereinafter abbreviated as KP buffer), containing 5 mM dithiothreitol (DTT) to the assay mixture containing 47.6umol c-D-HPG and 100 umol KP buffer in a total volume of 1 mL. After 20 min incubation at 40 °C, the reaction was stopped by the addition of 0.25 uL of 20% (w/v) trichloroacetic acid. The d-HPG produced was analyzed by means of the following high-performance liquid chromatography (HPLC) method. A-Carbamoyl-amino acids and amino acids were detected and quantified by HPLC at 210 nm on a reversephase HPLC column (e.g., Finepack SIL C18-5 column; Nipponbunko), using 36.7 mM KH2PO4 containing 15% methanol adjusted to pH 2.5 with phosphoric acid. One unit of the enzyme was defined as the amount of enzyme that catalyzed the formation of d-HPG at the rate of 1 imol/min under the assay conditions already mentioned.

The reactivity of the DCase of Agrobacterium sp. KNK712 is about 20 times higher than that of Pseudomonas sp. KNK003A with c-D-HPG as the substrate, but as concerns heat stability, in contrast, the Pseudomonas sp. KNK003A enzyme is more stable, with an about 10 °C increase in view of its denaturation temperature.

The molecular weights of these DCases were found to be about 37,000 and 38,000 Da, respectively, on SDS-PAGE. The properties of these two enzymes are shown in Table 2.

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