Purification and Partial Characterization of an X-prolyl-dipeptidyl Aminopeptidase from Lactobacillus gasseri ME-284

• Published in: FOOD SCIENCE AND TECHNOLOGY RESEARCH Vol. 21; no. 3; pp. 445 - 451
• Main Authors: Miyabe, Ryosuke, Takahashi, Yuto, Matsufuji, Hiroshi, Ogihara, Jun, Itou, Kentarou, Kawai, Yasushi, Masuda, Tetsuya, Suzuki, Koichi, Oda, Munehiro
• Format: Journal Article
• Language: English; Japanese
• Published: Tsukuba Japanese Society for Food Science and Technology 2015; The Japanese Society for Food Science and Technology; Japan Science and Technology Agency
• Subjects: Amino acids; beta-casomorphin; Chromatography; Degradation; Enzymes; Fragments; Inhibition; L. gasseri; Lactobacillus gasseri; Lactobacillus sakei; Mercury (metal); Peptides; purification; X-prolyl-dipeptidyl aminopeptidase
• ISSN: 1344-6606; 1881-3984
• DOI: 10.3136/fstr.21.445

An X-prolyl-dipeptidyl aminopeptidase has been purified from Lactobacillus gasseri by lysozyme treatment and seven chromatographic steps. The yield was 1.4% and the specific activity increased 265-fold over the crude enzyme extract. SDS-PAGE provided a single band with a MW of approximately 82 kDa. Gel filtration chromatography showed that the native enzyme was approximately 173 kDa, suggesting that the protein is a dimer. The amino acid sequences of the peptide fragments obtained after tryptic treatment of the purified enzyme were determined and found to be consistent with that of an X-prolyl-dipeptidyl aminopeptidase from L. gasseri ATCC 33323 (LGAS_0712, coverage: 31.74%). Optimal activity was observed at pH 7.0 and 55°C. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonylfluoride, and by divalent cations (Cu2+, Hg2+, and Zn2+). From these inhibition characteristics, the enzyme is likely a serine proteinase. Beta-casomorphin 7 (BCM-7, Tyr-Pro-Phe-Pro-Gly-Pro-Ile) was hydrolyzed into X-proline fragments from the N-terminus of the peptide. The characteristics of the enzyme are very similar to those of the enzyme from Lactobacillus sakei. However, as free amino acids were detected among the degradation products of BCM-7, some aspects of substrate specificity require further clarification.