Whole-cell aminopeptidase biocatalyst

H. Application of the Improved Whole-Cell Aminopeptidase Biocatalyst... 

As the graphs in Fig. 8 show, the hydrolysis reaction with the recombinant E. coli cells proceeds much faster than with the P. putida wild-type cells. Therefore, the use of this improved whole-cell aminopeptidase biocatalyst results in significantly shorter reaction times. The reason for this better performance of the newly constructed whole-cell biocatalyst is its much higher aminopeptidase content, reflected by a 25-fold increase in specific activity compared to P. putida. This is caused by improved expression of the P. putida pep A gene, due to the use of a jstrong promoter in combination with a multicopy situation. 

The aminopeptidase from Pseudomonas putida ATCC 12633 has also recently been cloned and overexpressed in E. coli resulting in a highly efficient whole-cell biocatalyst for industrial applications 1291. The specific activity of this new biocatalyst is substantially increased (25 times) compared with the specific activity of the P. putida wild type cells without changing the other positive characteristics of the aminopeptidase. Even though the aminopeptidase from Pseudomonas putida exhibits the relaxed substrate specificity described above, an a-hydrogen atom in the substrate is an essential structural feature for the enzymatic activity. Therefore this enzyme can not be used for the resolution of higher substituted amino acids. 

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. 

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