Because about half of Japanese population have been suffering from hypertension or are about to suffer from it, hypertension is a typical lifestyle related disease in Japan. Therefore, it is important to eat foods with antihypertensive effect in order to prevent the onset or aggravation of hypertension. It is known that soy foods such as soy sauce, miso, and natto show antihypertensive effect by inhibiting angiotensin converting enzyme (ACE), the key enzyme in renin-angiotensin system. ACE inhibitory substances contained in soy sauce and miso are nicotianamine and the peptide Ser-Trp respectively, but there is almost no evidence about ACE inhibitors in natto. We purified and identified 5 novel ACE inhibitory peptides containing Ile-Ile and Ile-Asp from protease-treated hikiwari-natto, whose ACE inhibitory activity was about 1.4 times higher than that of protease-untreated hikiwari-natto. In addition, we identified 8 novel ACE inhibitory peptides containing Phe-Phe-Tyr-Tyr and Trp-His-Pro derived from protease-treated soymilk, whose ACE inhibitory activity was about 36 times higher than that of protease-untreated soymilk.

View full abstract

From the culture supernatant of a thermophilic actinomycete KP1246, which grows at 45°C to 66°C, two electrophoretically homogenous p-nitrophenyl-β-D-glucopyranoside (pNPG)-degrading β-glucosidases (BGL1, BGL2) were obtained by the combination of an ion -exchange and gel-filtration chromatography. The molecular weight, Stokes radius, and sedimentation coefficient of BGL1 were found to be 51,000, 2.92, and 4.4S, respectively, which were similar to those of BGL2 (50,000, 3.06, and 4.2S, respectively). However, differences were observed in the isoelectric point (pI) (5.4 for BGL1 and 4.2 for BGL2) and in the optimum pH for activity (6.6 for BGL1 and 5.5 for BGL2). The optimum temperature for activity was found to be 70°C for BGL1 and 55°C for BGL2. In the study on the retention of heat resistance after 30 min of processing, BGL1 was demonstrated to retain 100% of the original activity over a wide range of pH, from 5.0 to 11.0. Meanwhile, the stability range of pH was found to be narrower, i.e., 7 to 9.5, for BGL2. For both BGL1 and BGL2, pNPG, as well as cellobios e and salicin, were found to be good substrates. Neithe r BGL1 nor BGL2 reacted with Avicel, carboxymethylcellulose (CM-cellulose), or maltose. Enzymatic reaction of BGL1 was not at all inhibited by glucose; however, in the enzymatic r eaction of BGL2, glucose acted as a competitive inhibitor (Ki = 408 mM). Cellobiose acted as a noncompetitive inhibitor to BGL1 (Ki = 50 mM) and showed a similar inhibition format to BGL2 (Ki = 15.6 mM). Addition of 2 mM EDTA, Pb2 + , Fe 2 + , Mn2 + , or Mg2 + did not at all inhibit the activity of BGL1; however, these resulted in 26.1%, 2 5.2%, 22.3%, 17.5%, and 10.5% inhibition of BGL2 activity, respectively. pCMB (p-chloromercuribennzoate) was found to inhibit BGL1 activity by 100%; however, the level of inhibition was only 32.5% for BGL2. The results of this kinetic study, which is the first of its kind, on the thermophilic actinomycete T. curvata suggest that at least 2 different types of heat-resistant BGLs function in the final stage of the cellulase degradation system of the KP1246 strain and that the strain can be classified as T. curvata.

View full abstract