Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii.
The aminopeptidase A (PepA; EC 3.4.11.7) is an intracellular exopeptidase present in lactic acid bacteria. The PepA cleaves glutamyl/aspartyl residues from the N-terminal end of peptides and can, therefore, be applied for the production of protein hydrolysates with an increased amount of these amino acids, which results in a savory taste (umami). The first PepA from a lactobacilli strain was recombinantly expressed in Escherichia coli in a recently published study and harbored a C-terminal His6-tag for easier purification. Due to the fact that a His-tag might influence the properties of an enzyme, a simple purification method for the non-His-tagged PepA was required. Surprisingly, the PepA precipitated at a very low ammonium sulfate concentration of 5%. Unusual for a precipitating step, the purity of PepA was over 95% and the obtained activity yield was 110%. The high purity allows biochemical characterization and kinetic investigation. As a result, the optimum pH (6.0-6.5) and temperature (60-65 °C) were comparable to the His6-tag harboring PepA; the KMvalue was at 0.79 mM slightly lower compared to 1.21 mM, respectively. Since PepA is a homo dodecamer, it has a high molecular mass of approximately 480 kDa. Therefore, a subsequent preparative size-exclusion chromatography (SEC) step seemed promising. The PepA after SEC was purified to homogeneity. In summary, the simple two-step purification method presented can be applied to purify high amounts of PepA that will allow the performance of experiments in the future to crystalize PepA for the first time.
Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany. Electronic address: t.stressler@uni-hohenheim.de.
Timo
T
Stressler
Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
Coralie
C
Tanzer
Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
Jacob
J
Ewert
Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
Wolfgang
W
Claaßen
Institute of Food Science and Biotechnology, Department of Biotechnology and Enzyme Science, University of Hohenheim, Stuttgart, Germany.
Lutz
L
Fischer
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004795:Enzyme Stability; D043384:Glutamyl Aminopeptidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D048668:Lactobacillus delbrueckii / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D043384:Glutamyl Aminopeptidase
Ammonium sulfate precipitation; Characterization; Lactobacillus delbrueckii; PepA; Size-exclusion chromatography