Simple purification method for a recombinantly expressed native His-tag-free aminopeptidase A from Lactobacillus delbrueckii

Publication Type

Journal contribution (peer reviewed)

Authors

Stressler, T.; Tanzer, C.; Ewert, J.; Claaßen, W.; Fischer, L.

Year of publication

2017

Published in

Protein Expression and Purification

Pubisher

Elsevier Inc.

ISBN / ISSN / eISSN

1046-5928

DOI

10.1016/j.pep.2016.10.010

Page (from - to)

7-15

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 K_M value 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.