During the course of studies on the enzyme-catalyzed organic synthesis of D-amino acid derivatives, a D-stereospe-cific aminopeptidase was needed. It was speculated that microorganisms might be a likely source for such an enzyme, given their important role in the environment as synthesizers and degraders of a wide variety of substances.
An enrichment culture in a medium containing a synthetic substrate (D-Ala-NH2) as the sole nitrogen source led to isolation of a bacterial strain, Ochrobactrum anthropi SCRC C1-38. The enzyme hydrolyzing D-Ala-NH2, named D-aminopeptidase (EC 126.96.36.199), was purified to homogeneity from the cell-free extract of the strain and characterized in detail (21). The molecular weight of the native enzyme was estimated to be approximately 122,000, with two identical subunits of molecular weight of about 59,000.
A typical time course for the stereoselective hydrolysis of D-alanine amide is shown in Figure 1, with racemic alanine amide as a substrate. D-Alanine amide was completely hydrolyzed by the action of the enzyme, whereas the L-enantiomer remained unhydrolyzed in the reaction mixture. The rate of the hydrolysis of L-alanine amide was less than 0.01% that of D-alanine amide. The enzyme showed strict chemo- and stereospecificities toward D-amino acid amides, peptides, and esters, as shown in Table 1. Each substrate (100 mM) was incubated under the standard enzyme assay condition (21). The substrates include D-alanine amide (relative velocity: 100%, Km value: 0.65 mM); glycine amide (44%, 22.3 mM); D-a-aminobutyram-ide (30%, 18.3 mM);D-serine amide (29%, 27.0 mM); D-alanine 3-aminopentane amide (32%, 2.27 mM); D-alanine-p-nitroanilide (96%, 0.51 mM); D-alanine methyl ester (75%), dimer (21%, 10.2 mM), trimer (92%, 0.57 mM), and tetramer (89%, 0.32 mM) of D-alanine; D-alanylglycine (95%, 0.98 mM), D-alanylglycylglycine (45%, 0.37 mM); and D-alanyl-L-alanyl-L-alanine (100%, 0.65 mM). These results show that the enzyme has higher affinity toward peptide substrates than amino acid amides. The enzyme showed neither endopeptidase nor carboxypeptidase activity. The mode of action of the enzyme toward a peptide substrate was studied with D-alanylglycylglycine. The reaction was followed over time, and it was observed that alanine and glycylglycine were released until the substrate tripeptide was nearly completely consumed, whereupon glycine release began, and finally alanine and glycine were produced. This result shows that the enzyme catalyzes the hydrolysis of a single amino acid from the N-terminus of a peptide. Because the mode of action is typical of an ami-nopeptidase, we named the enzyme D-aminopeptidase.
The D-aminopeptidase from O. anthropi SCRC C1-38 was utilized for a stereoselective synthesis of D-alanine A-alkylamide from an amine and D-amino acid amide or D-amino acid methyl ester (22). In water, D-alanine A-alkylamide once formed by the enzyme was successively hydrolyzed to yield D-alanine. The enzyme was immobilized by urethane prepolymer PU-6. When the aminolysis reaction was performed in water-saturated organic solvents such as butylacetate, benzene, and 1,1,1-trichloro-ethane with the immobilized enzyme as a catalyst, it progressed well quantitatively in a highly D-stereoselective manner, to give optically pure D-alanine A-alkylamides. The acyl donor specificity of this reaction was rather limited, with D-alanine, glycine, and D-a-amino butyric acid as substrates, while the acyl acceptor specificity was relatively wide, with bulky, aromatic-containing, and straight-chain amines (Table 2). The catalytic center activity (kcat) of this reaction, 7,700 (min-1), was much higher than in the case of the nonstereoselective synthesis of D-amino acid-containing peptides (4,5).
Alkaline conditions are usually essential for peptide bond formation in kinetically controlled systems (23,24). Several attempts to oligomerize D-alanine methyl ester with D-aminopeptidase in an alkaline aqueous medium were unsuccessful, probably because the substrate is unstable under alkaline conditions. The immobilized D-aminopeptidase catalyzed the synthesis of D-alanine oligomers from D-alanine methylester in organic solvents (25). Dimer and trimer of D-alanine were obtained in 58% and 6% yield, respectively, when urethane prepolymer PU-6 immobilized D-aminopeptidase (1.5 U/mL) was incubated
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