L-aminopeptidase

L-Amino adds could be produced from D,L-aminonitriles with 50% conversion using Pseudomonas putida and Brembacterium sp respectively, the remainder being the corresponding D-amino add amide. However, this does not prove the presence of a stereoselective nitrilase. It is more likely that the nitrile hydratase converts the D,L-nitrile into the D,L-amino add amide, where upon a L-spedfic amidase converts the amide further into 50% L-amino add and 50% D-amino add amide. In this respect the method has no real advantage over the process of using a stereospecific L-aminopeptidase (vide supra). 

Two other very interesting applications of proteases are firstly the use of a protease to convert porcine insulin into human insulin, and secondly the use of L-aminopeptidase to produce the pharmaceutically useful substance p-phenylglycine by selectively hydrolyzing L-phenylglycinamide in a racemic mixture. 

L. Dijkhuizen, and E. M. Meijer, Purification and characterization of an L-aminopeptidase from Pseudomonas pulida ATCC 12633, Appl. Environm. Microbiol. 1993, 59, 4330-4334. 

Production of optically pure amino acids using their amides as racemic precursors and L-aminopeptidases as catalysts is a well-established commercial process. To overcome the limit of 50% conversion, D-aminopeptidases and amino acid amide racemases were also developed [182]. 

Figure 1 Scheme of the optimized purification protocol of an L-aminopeptidase from P. putida ATCC 12633. 

Characterization of the purified protein by nondenaturing gel filtration reveals a native molecular mass of approximately 400 kDa. SDS-PAGE analysis of the pure enzyme shows a single band with a molecular mass of about 52 kDa. This indicates that the l-aminopeptidase most probably is a homo-octameric enzyme. The isoelectric point of the protein is estimated as pH 10.5 using an isoelectric focusing (lEF) 3-9 Ph tgel with an expanded pH range. 

The L-aminopeptidase is sensitive to various classes of proteinase inhibitors. Strong inhibition of the enzyme is observed by treatment with the thiol reagents / -chloromercu-ribenzoate (pCMB) and iodoacetamide. The inhibition by pCMB can be reversed by subsequent treatment with dithiothreitol. In addition, the enzyme is inhibited by the metalchelating compounds EDTA and o-phenanthroline and the serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropylfluorophosphate. These phenomena point to an essential serine or cysteine residue in the active site furthermore, divalent cations seem to be involved in the catalytic mechanism and/or are important for the stability of the enzyme. 

The L-aminopeptidase from P. putida displays clear amide-hydrolyzing activity between pH 7 and 11, with the highest activity at pH 9.0-9.5. Furthermore, the enzyme has maximum activity at 40°C (Fig. 2). The thermal stability of this enzyme in purified form is rather limited after a 60-min incubation period at 30 C, only 65% of the initial activity remains. Addition of substrate, however, dramatically enhances the thermal stability (60 min at 30°C, 100% activity left). 

Table 4 Similarity of P. putida L-Aminopeptidase with Other Members of the Family of Leucine Aminopeptidases...

Because all of these important residues for the blLAP catalytic mechanism are found to be fully conserved in the E. coli [34] and Rickettsia prowazekii [37] pepA-encoded leucine aminopeptidases, a sequence alignment has been performed to see whether these residues are also present in the P. putida L-aminopeptidase. This indeed appears to be the case (see Fig. 4). Interestingly, it was observed that these residues were fully conserved in all currently known leucine aminopeptidases that are significantly homologous to the P. putida L-aminopeptidase. Because of these apparent similarities in (1) overall primary structure (Table 4) and (2) key active site residues among the members of the leucine... 

To construct a more efficient whole-cell L-aminopeptidase biocatalyst, it was decided to clone the P. putida L-aminopeptidase gene in an E. coli K-12 host microorganism. This bacterium was chosen because of its favorable fermentation proj rties and the availability of a large number of specialized expression vectors. 

Figure 5 Restriction map of the expression vector pTrcLAR In this vector the expression of the R putida L-aminopeptidase gene (pepA) is controlled by the P e-ZacF combination.

The performance of the E. co/i/pTrpLAP whole cells, which were obtained by the fermentation described above, has been tested in a standard resolution reaction of an arbitrary a-hydrogen amino acid amide, at the optimum reaction conditions for the P. putida l-aminopeptidase (40°C, pH 9.5). 

The strategy used in this case for the cloning and heterologous expression of P. putida pepA did not result in any mutation on protein level. Therefore, the wild-type P. putida enzyme will be formed in the recombinant E. coli strain. This means tiiat all intrinsic properties of this L-aminopeptidase (e.g., pH optimum, substrate range, enantio-specificity) are unaltered in comparison with the properties of the enzyme found in P. putida. 

---