Overexpression and characterization of an extracellular leucine aminopeptidase from Aspergillus oryzae.
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.
Matsushita-Morita
Mayumi
M
National Food Research Institute, Tsukuba, Ibaraki, 305-8642, Japan.
Tada
Sawaki
S
Suzuki
Satoshi
S
Hattori
Ryota
R
Marui
Junichiro
J
Furukawa
Ikuyo
I
Yamagata
Youhei
Y
Amano
Hitoshi
H
Ishida
Hiroki
H
Takeuchi
Michio
M
Kashiwagi
Yutaka
Y
Kusumoto
Ken-Ichi
K
D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004795:Enzyme Stability; D015870:Gene Expression; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity; D013696:Temperature
D003470:Culture Media; D007931:Leucyl Aminopeptidase
Nature. 2005 Dec 22;438(7071):1157-61
16372010
Anal Biochem. 1976 May 7;72:248-54
942051
J Appl Microbiol. 2008 Nov;105(5):1711-9
18828788
J Med Food. 2005 Winter;8(4):423-30
16379551
FEMS Microbiol Rev. 1996 Jul;18(4):319-44
8703509
J Mol Biol. 1983 Jun 5;166(4):557-80
6345791
Appl Environ Microbiol. 1984 Jan;47(1):177-83
16346456
Microbiology (Reading). 2005 Jan;151(Pt 1):145-155
15632434
Fungal Genet Biol. 2000 Mar;29(2):61-71
10919375
EMBO J. 1995 Feb 15;14(4):779-90
7882981
Biosci Biotechnol Biochem. 2010;74(1):188-90
20057127
Appl Microbiol Biotechnol. 2009 Nov;85(2):335-46
19557408
J Dairy Sci. 1995 Jun;78(6):1253-7
7673515
Annu Rev Biochem. 1981;50:555-83
7023366
J Appl Microbiol. 2010 Jul;109(1):156-65
20028436
Nature. 1970 Aug 15;227(5259):680-5
5432063
DNA Res. 2007 Apr;14(2):47-57
17540709
J Biosci Bioeng. 2009 Jun;107(6):615-9
19447337
Mol Gen Genet. 1989 Oct;219(1-2):33-8
2693947
Biochim Biophys Acta. 2002 Jun 7;1576(1-2):119-26
12031491