Archives

  • 2018-07
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05
  • 2024-06
  • 2024-07
  • As noted above yeasts particularly S cerevisiae Frey and R

    2023-09-27

    As noted above, yeasts, particularly S. cerevisiae (Frey and Röhm 1978; Trumbly and Bradley 1983), produce APs, but these are intracellular enzymes located in the vacuolar compartment, with the exception of aminopeptidase II. About 40% of aminopeptidase II activity is detected as external enzyme, like periplasmic hydrolase, but not secreted in culture medium (Frey and Röhm 1979). Here we report the isolation of a yeast, Pseudozyma hubeiensis 31-B, which produces AP as the extracellular peptidase. Purification of this AP is described and its ability to reduce the bitterness of peptides is characterized, indicating that this AP holds promise for improving the flavor of dairy products such as yogurt or cheese.
    Materials and methods
    Results
    Discussion We isolated a yeast, P. hubeiensis 31-B, that produces extracellular AP and carboxypeptidase, but we did not detect any proteinase activities in the culture filtrate using azocasein, milk casein, hemoglobin, or SPI as the substrate. Inouye and Nagai (2004) Fmoc-Gln(Trt)-OH reported a novel and convenient proteinase assay method using SPI. Using this method, they have detected activity of many kinds of proteinase, such as subtilisin Carlsberg, subtilisin BPN′, thermolysin, α-chymotrypsin, bromelain, and pronase, but we were not able to detect proteinase activity in the culture filtrate of P. hubeiensis 31-B using this method. Pseudozyma hubeiensis is a known producer of glycolipid biosurfactants (Sari et al. 2013), and xylanase (Adsul et al. 2009), but this is the first report of AP from P. hubeiensis. The AP was produced from the exponential phase and was almost synchronized with the growth of yeast Fmoc-Gln(Trt)-OH in a jar fermenter. The produced AP seemed to utilize and hydrolyze proteins or peptides in medium for cell growth. There are many reports about APs from yeasts such as S. cerevisiae (Masuda et al. 1975; Trumbly and Bradley 1983) and Candida albicans (Moudni et al. 1995), but almost all APs are produced as intracellular enzymes. As an example, S. cerevisiae cannot produce extracellular glucoamylase, but S. diastaticus can, as it possesses the STA gene, a glucoamylase structure gene with secretion signal sequence (Tamaki 1978). Yamashita et al. (1985) reported that STA1 gene expression is controlled by the MATa2 gene at the posttranscriptional level. Pseudozyma hubeiensis may also have such a gene that produces extracellular AP. We assayed the properties of AP31-B and compared it with that of two other yeast APs with similar properties reported previously (Table 4). The three APs are quite different from each other in terms of molecular mass (Table 4); according to other reports, the molecular masses of vacuolar aminopeptidase I and periplasmic aminopeptidase II from S. cerevisiae are 640 kDa (Metz and Röhm 1976) and 85 kDa (Frey and Röhm 1978), respectively. Table 4 shows that the maximum activities of each of the three APs are pH 7–8, and the activity strongly inhibited with EDTA and Zn2+. The serine protease inhibitor Petabloc, the cysteine protease inhibitor E-64, and the aspartic protease inhibitor Pepstacine A did not affect the activity of AP31-B, but activity was inhibited by the cysteine protease inhibitor PCMB (Table 2). Aminopeptidase P from Escherichia coli is also inhibited by EDTA and PCMB (Yoshimoto et al. 1988). Leu-p-NA and Arg-p-NA were better substrates for AP31-B and aminopeptidase Y, but Pro-p-NA was essentially not hydrolyzed by those APs. Experiments characterizing the hydrolysis of oligopeptides showed that AP31-B cannot hydrolyze X-Pro like aminopeptidase Y can, when proline is the 2nd amino acid in the sequence (Fig. 4). The utility of AP from P. hubeiensis to food processing applications was investigated by characterizing the decrease in the bitterness of a casein peptide hydrolyzate prepared using bacterial proteinase, then treated with AP31-B. Milk casein can be strongly bitter when digested with proteolytic enzymes due to hydrophobic amino acids (Matoba and Hata 1972). Hydrophobic amino acids are generally found in the protein interior and are exposed when the protein is proteolyzed (Li et al. 2012). N- or C-terminal hydrophobic amino acids in peptides in particular result in a bitter taste. A peptide sample with a bitterness equivalent to 2% caffeine has been produced from milk casein by Prochin SD-AY 10, a Bacillus proteinase, and there are many reports about bitter peptide formation with Bacillus proteinase (Ichikawa et al. 1959; Minamiura et al. 1972), papain and trypsin (Minagawa et al. 1989), and other proteinases. Incubation of the bitter taste hydrolyzate with AP31-B for 3 h reduced the bitterness according to released amino acids (Fig. 5). Izawa et al. (1997) reported that an aminopeptidase could reduce the bitterness of a proteinase hydrolyzate of milk casein. Nishiwaki et al. (2002) also reported the debittering of soy protein and milk casein hydrolyzate incubated pepsin using Grifola frondosa aminopeptidase. In their report, as free amino acids increased, bitterness decreased and hydrophobic amino acids such as valine and leucine were preferentially released from the bitter peptides.