Synthesis of d-phenylalanine oligopeptides catalyzed by alkaline d-peptidase from Bacillus cereus DF4-B1
Introduction
Enzyme-catalyzed peptide bond formation represents a promising alternative to chemical synthesis procedures 1, 2, 3. Several attempts have been reported on the in vitro synthesis of the d-amino acid containing peptides and amides 4, 5, 6, 7, 8, 9, 10. Naturally, however, these enzymatic reactions have an inevitable drawback as they are not stereospecific for substrates with D-configurations. A d-stereospecific peptidase, d-aminopeptidase was isolated from Ochrobactrum anthropi and characterized 11, 12. The enzyme can be applied for kinetic resolution of racemic amino acid amides to yield d-amino acids 11, 13, synthesis of N-alkylamides from dl-amino acid esters and amines [14], and synthesis of d-alanine oligopeptides from d-alanine methylester [15]. We also characterized a novel extracellular d-stereospecific endopeptidase `Alkaline d-peptidase (ADP)' from a bacterium Bacillus cereus strain DF4-B, isolated with a synthetic substrate (d-Phe)4 [16]. The enzyme (Mr: 37,952, monomer) acts as a dipeptidyl endopeptidase strictly d-stereospecific toward oligopeptides such as (d-Phe)3 and (d-Phe)4. The ADP gene (adp) was isolated and its deduced amino acid sequence revealed that the ADP could be classified as a member of penicillin-recognizing enzymes.
Since the ADP was found to be a serine peptidase [16], we attempted to use it for kinetically controlled peptide synthesis. In this study, we describe for the first time the synthesis of d-phenylalanine oligomers by use of the ADP as catalyst in an alkaline aqueous medium.
Section snippets
Materials
d-Phenylalanine methylester as a substrate and authentic (d-Phe)2, (d-Phe)3, and (d-Phe)4 were chemically synthesized from d-phenylalanine by the procedure as described previously [16]. -NMR spectrum was recorded in CD3OD using a JEOL JNM-EX400 spectrometer (Tokyo, Japan), with tetramethylsilane as the internal standard. Mass spectrum was recorded on a JEOL JMS-AX500 mass spectrometer (Tokyo, Japan) under fast atom bombardment (FAB) conditions. DEAE-Toyopearl 650 M and Butyl-Toyopearl 650 M
Enzymatic oligomerization in aqueous solution
We have previously described the characterization of the alkaline d-peptidase (ADP) purified from B. cereus DF4-B [16]. The enzyme not only hydrolyzed (d-Phe)3 and (d-Phe)4 to form (d-Phe)2 and d-phenylalanine, but also acted on d-phenylalanine methylester and d-phenylalanine amide to form (d-Phe)2. This finding gave us the opportunity to further investigate the synthesis of d-phenylalanine oligopeptides from d-phenylalanine methylester by use of the ADP.
ADP was purified from E. coli
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Alkaline d-Peptidase
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D-amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity
2005, Journal of Bioscience and BioengineeringGenes for an alkaline D-stereospecific endopeptidase and its homolog are located in tandem on Bacillus cereus genome
2003, FEMS Microbiology LettersEnhancement of the thermostability and catalytic activity of D-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3 by directed evolution
2003, Journal of Molecular Catalysis B: Enzymatic
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Dedicated to Professor Hideaki Yamada in honor of his 70th birthday.