Gotou Takanobu, Shinoda Tadashi, Mizuno Seiichi, Yamamoto Naoyuki
Functional Food and Drink Development Laboratory, Calpis Co., Ltd., 11-10, 5-Chome, Fuchinobe, Sagamihara-shi, Kanagawa 229-0006, Japan.
J Biosci Bioeng. 2009 Jun;107(6):615-9. doi: 10.1016/j.jbiosc.2009.01.022.
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.
通过三步柱色谱法对米曲霉提取物进行纯化，鉴定出两种能够从酪蛋白中释放血管紧张素I转换酶（ACE）抑制剂Ile-Pro-Pro的蛋白水解酶。首先，使用具有线性氯化钠梯度的DEAE-琼脂糖FF柱洗脱能够从β-酪蛋白产生Ile-Pro-Pro的蛋白质。使用羟基磷灰石柱从所得级分中分离出一种能够从Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln释放Pro-Ile-Pro-Gln-Ser-Leu-Pro-Gln-Asn-Ile-Pro-Pro的内肽酶和一种从Gln-Asn-Ile-Pro-Pro产生Ile-Pro-Pro的氨肽酶。然后将每种活性酶加载到Develosil 300Diol凝胶过滤柱上进行高效液相色谱（HPLC），并纯化至同质。该内肽酶的分子量约为46,000 Da，其N端氨基酸序列与米曲霉的中性蛋白酶I（NP I）相同。同时，该氨肽酶的分子量为36,000 Da，其N端氨基酸序列与酱油曲霉和米曲霉中报道的亮氨酸氨肽酶（LAP）相似。因此，洗脱的内肽酶和氨肽酶分别被鉴定为NP I和LAP。使用含有Ile-Pro-Pro序列的合成蛋白进行肽产生分析表明，NP I处理C末端，LAP处理N末端以产生Ile-Pro-Pro。虽然通过添加这两种纯化的酶成功地从酪蛋白中产生了Ile-Pro-Pro，但仅添加单一酶时并未产生。基于我们的实验结果，我们认为NP I和LAP是米曲霉中从酪蛋白释放Ile-Pro-Pro的关键蛋白水解酶。
