D-Aminopeptidase (EC 220.127.116.11) from Ochrobactrum an-thropi was discovered, and its primary structure has been found to have similarity to the / -lactamases and penicillin-binding proteins (21,28). The enzyme acts mostly on pep-tides with D-Ala at the N-terminus to yield D-amino acids; it does not act on D-amino acid derivatives with bulkier substituents. We proposed that D-aminopeptidase is a new penicillin-recognizing enzyme (11) based on its primary structure, inhibition by / -lactam compounds, and the ability to catalyze peptide bond formation in organic solvents, although the enzyme does not show / -lactamase activity (21,47).
Here, we describe the screening of soil microorganisms for D-stereospecific endopeptidases using a synthetic pep-tide (D-Phe)4, characterization of the new enzyme alkaline D-peptidase (ADP), as well as cloning and sequencing of the adp gene from B. cereus strain DF4-B.
Isolation and Properties of the Alkaline d-Peptidase
An enrichment culture in LB medium containing a synthetic substrate (D-Phe)4 led to the isolation of a bacterial strain Bacillus cereus DF4-B. The extracellular enzyme hydrolyzing (D-Phe)4 was purified and characterized (48). The Mr of the subunit calculated was about 36,000 by gel electrophoresis (SDS-PAGE), 37,000 by HPLC, and 37,952 by mass spectrophotometry. The absorption of the purified enzyme in 0.01 M potassium phosphate buffer, pH 7.0, was maximal at 281 nm. The enzyme is an endopeptidase that acts D-stereospecifically on peptides composed of aromatic D-amino acids, recognizing the D-configuration of the amino acid whose carboxy-terminal peptide bond is hydro-lyzed. The enzyme had an optimum pH at around 10.3. Thus, the enzyme was named alkaline D-peptidase (D-stereospecific peptide hydrolase [EC 3.4.11.-]). The substrate specificity of the enzyme was examined as shown in Table 3. Each substrate (2 mM) was incubated under standard enzyme assay conditions (48). The enzyme was active
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