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Enzymatic characterization of a novel Xaa-Pro aminopeptidase XpmA from Aspergillus oryzae expressed in Escherichia coli
- Matsushita-Morita, Mayumi, Tada, Sawaki, Suzuki, Satoshi, Hattori, Ryota, Kusumoto, Ken-Ichi
- Journal of bioscience and bioengineering 2017 v.124 no.5 pp. 534-541
- Aspergillus oryzae, EDTA (chelating agent), Escherichia coli, aminopeptidases, cobalt, databases, dipeptides, divalent metals, enzyme activity, fungi, gel chromatography, genes, hydrolysis, magnesium, manganese, metal ions, metalloproteinases, molecular weight, pH, polyacrylamide gel electrophoresis, proline, sodium chloride
- 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.