Enzymatic characterization of a novel Xaa-Pro aminopeptidase XpmA from Aspergillus oryzae expressed in Escherichia coli

• Published in: Journal of bioscience and bioengineering Vol. 124; no. 5; pp. 534 - 541
• Main Authors: Matsushita-Morita, Mayumi, Tada, Sawaki, Suzuki, Satoshi, Hattori, Ryota, Kusumoto, Ken-Ichi
• Format: Journal Article
• Language: English
• Published: Japan Elsevier B.V 01.11.2017
• Subjects: Amino Acid Sequence; aminopeptidases; Aminopeptidases - biosynthesis; Aminopeptidases - genetics; Aminopeptidases - isolation & purification; Aminopeptidases - metabolism; Aspergillus oryzae; Aspergillus oryzae - enzymology; Aspergillus oryzae - genetics; cobalt; dipeptides; Dipeptides - metabolism; Dipeptides - pharmacology; divalent metals; EDTA (chelating agent); Electrophoresis, Polyacrylamide Gel; enzyme activity; Escherichia coli; Escherichia coli - genetics; fungi; gel chromatography; genes; Halophilicity; Histidine; Hydrogen-Ion Concentration; hydrolysis; Hydrolysis - drug effects; M24B family; magnesium; manganese; metal ions; Metallopeptidase; metalloproteinases; Molecular Weight; Oligopeptides; polyacrylamide gel electrophoresis; Proline; Proline - analogs & derivatives; Proline - metabolism; Proline - pharmacology; sodium chloride; Substrate Specificity - drug effects; Temperature; Xaa-Pro aminopeptidase; Xaa-Pro aminopeptidase; Proline; Metallopeptidase; Halophilicity; M24B family; Aspergillus oryzae
• ISSN: 1389-1723; 1347-4421; 1347-4421
• DOI: 10.1016/j.jbiosc.2017.06.007

Xaa-Pro aminopeptidases are peptidases responsible for the cleavage of any amino acid N-terminally adjacent to a proline residue. We identified a gene encoding a putative Xaa-Pro aminopeptidase in the genome of the filamentous fungus Aspergillus oryzae (genome database number: AO090701000720) and named this gene xpmA. We produced its enzyme in a C-terminally His6-tag-fused form in an Escherichia coli expression system and purified it. The purified recombinant XpmA (rXpmA) showed hydrolysis activity toward Xaa-Pro-oligopeptides, especially the two dipeptides Ala-Pro and Phe-Pro. The molecular weight of rXpmA was estimated to be 69 kDa by SDS-PAGE and 126 kDa by gel filtration, suggesting that it is a homodimer. The enzyme was activated by various divalent metal ions such as Mn2+, Co2+, and Mg2+; in particular, the enzyme activity was increased 27.6-times relative to the no-addition control by 1 mM Mn2+. Additionally, 10 mM EDTA suppressed its activity to 0.26-times of the control level. Therefore, rXpmA was a metalloprotease. Optimal hydrolytic activity of rXpmA was observed at 50°C and pH 8.5–9.0. The enzyme was stable up to 50°C and from pH 4.0 to 11.0. rXpmA showed substrate inhibition by Leu-Pro, Ser-Pro and Arg-Pro at concentrations over 4 mM, 10 mM, and 3 mM, respectively. NaCl increased the enzyme activity in the concentration range 0.5–3.0 M, suggesting that the enzyme is halophilic. •XpmA is characterized as a novel Xaa-Pro aminopeptidase of Aspergillus oryzae.•XpmA is activated by various divalent metal ions, especially Mn2+.•XpmA can act in a broad alkaline pH range from 7.0 to 12.0.•XpmA shows thermostability up to 50°C.•XpmA is a halophilic aminopeptidase.