Overexpression and Characterization of an Extracellular Leucine Aminopeptidase from Aspergillus oryzae

Abstract

Leucine aminopeptidase (LAP), an enzyme used in the food industry, is an exopeptidase that removes an amino acid residue, primarily leucine (Leu), from the N-terminus of peptides and protein substrates. In this study, we focused on the leucine aminopeptidase A (lapA) gene from Aspergillus oryzae RIB40. To purify and characterize the LapA, lapA was overexpressed in A. oryzae RIB40 using the amyB promoter. LAP activity in the culture supernatant of one transformant harboring the lapA expression plasmid was 33 times that of the host strain. LapA was purified from the culture supernatant of this lapA-overexpressing strain by column chromatography. The purified recombinant LapA had a molecular mass of 33 kDa, and its N-terminal amino acid was the tyrosine at position 80 of the deduced amino acid sequence. Optimal enzyme activity was observed at 60°C and pH 8.5, and the enzyme was stable at temperatures up to 60°C and in the pH range 7.5-11. In transcriptional analysis, lapA was induced under alkaline conditions and expressed at a relatively low level under normal conditions. LapA showed maximum hydrolyzing activity for the substrate leucine para-nitroanilide (Leu-pNA), followed by substrates Phe-pNA (39% activity compared with Leu-pNA), Met-pNA, Lys-pNA, and Arg-pNA. In addition, LapA preferentially hydrolyzed peptides longer than tripeptides.

Protease fromAspergillus oryzae: Biochemical Characterization and Application as a Potential Biocatalyst for Production of Protein Hydrolysates with Antioxidant Activities

“…The enzyme was not inhibited by the addition of EDTA, indicating that metal ions are not necessary for the activity of the protease from A. oryzae LBA 01. Matsushita-Morita et al [6] reported that a protease from A. oryzae ATCC 42149 was activated in the presence of the metal ions Zn 2+ and Co 2+ at final concentrations of 1 mM. In the same study, the addition of 1 mM Ca 2+ , Mg 2+ , or Mn 2+ inhibited the enzyme.…”

“…Aspergillus oryzae (A. oryzae) is a filamentous fungus listed as a "Generally Recognized as Safe (GRAS)" organism by the US Food and Drug Administration. It has a long history of use in the food industry in the production of traditional fermented foods, due to its high proteolytic activity and its use of sugar [5,6]. According to Machida et al [7] the molecular history of the organism shows that A. oryzae has the largest expansion of hydrolytic genes (135 proteinase genes).…”

Flavourzyme, an Enzyme Preparation with Industrial Relevance: Automated Nine-Step Purification and Partial Characterization of Eight Enzymes

“…An extracellular leucine aminopeptidase from A. oryzae RIB40 was described previously by Matsushita et al 40 Here, the aminopeptidase exhibited the highest activity at pH 8.5 and 60°C and showed a molecular weight of 33 kDa by SDS-PAGE analysis. 40 A leucine aminopeptidase 1 from A. oryzae ATCC 20386 was purified and characterized by Nakadai et al 41 Again, the enzyme exhibited the highest enzyme activity at pH 8.5 and 60°C. However, in contrast to our observations, the native size determined was 26.5 kDa by SEC analysis.…”

Genomic Analysis of Koji Mold Aspergillus oryzae and Investigation of Novel Peptidases by Post-genomic Approach

“…Consequently, the genome analysis of A. oryzae and its post-genome research revealed novel enzymes that may be involved in the degradation of proteins of the start material of fermented food. In the same approach, a novel leucine aminopeptidase from A. oryzae, which has broad substrate specificity mainly for the release of leucine from the amino-termini of peptides, were identified in A. oryzae 12 . Some of the novel proteases are also identified 13,14 in the same strategy.…”