Purification and identification of proteolytic enzymes from Aspergillus oryzae capable of producing the antihypertensive peptide Ile-Pro-Pro.
Two proteolytic enzymes capable of releasing the angiotensin I-converting enzyme (ACE) inhibitor Ile-Pro-Pro from casein were identified by purification of an Aspergillus oryzae extract by three-step column chromatography. First, proteins capable of producing Ile-Pro-Pro from beta-casein were eluted using a DEAE-sepharose FF column with a linear sodium chloride gradient. An endopeptidase capable of releasing Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro from Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln and an aminopeptidase producing Ile-Pro-Pro from Gln-Asn-Ile-Pro-Pro were separated from the resultant fraction using a hydroxyapatite column. Each active enzyme was then loaded onto a Develosil 300Diol gel filtration column for high performance liquid chromatography (HPLC) and purified to homogeneity. The endopeptidase had a molecular mass of approximately 46,000 Da and exhibited an N-terminal amino acid sequence identical to that of neutral protease I (NP I) of A. oryzae. Meanwhile, the aminopeptidase had a molecular mass of 36,000 Da and an N-terminal amino acid sequence similar to that of Leucine aminopeptidase (LAP), as reported in Aspergillus sojae and A. oryzae. The eluted endopeptidase and aminopeptidase were thus identified as NP I and LAP, respectively. Analysis of peptide production using synthetic proteins containing an Ile-Pro-Pro sequence showed that NP I processed the C-terminal end and LAP processed the N terminus to produce Ile-Pro-Pro. While Ile-Pro-Pro was successfully produced from casein by the addition of these two purified enzymes, it was not generated with the addition of only a single enzyme. Based on our experimental findings, we suggest that NP I and LAP are key proteolytic enzymes in the release of Ile-Pro-Pro from casein in A. oryzae.
Gotou
Takanobu
T
Functional Food and Drink Development Laboratory, Calpis Co., Ltd., 11-10, 5-Chome, Fuchinobe, Sagamihara-shi, Kanagawa 229-0006, Japan.
Shinoda
Tadashi
T
Mizuno
Seiichi
S
Yamamoto
Naoyuki
N
D000595:Amino Acid Sequence; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism*; D000959:Antihypertensive Agents / Q000378:metabolism*; D001236:Aspergillus oryzae / Q000201:enzymology*; D002364:Caseins / Q000378:metabolism; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*
D000806:Angiotensin-Converting Enzyme Inhibitors; D000959:Antihypertensive Agents; D002364:Caseins; D005656:Fungal Proteins; D009842:Oligopeptides; C489032:isoleucyl-prolyl-proline; D010450:Endopeptidases