Food Science and Biotechnology

Korean Society of Food Science and Technology (한국식품과학회)
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Purification and Characterization of a Novel Salt-tolerant Protease Produced by Saccharomyces sp. B101 Isolated from Baker's Dough Yeast

  • Hwang, Joo-Yeon (Department of Applied Biology and Chemistry, Konkuk University) ;
  • Kim, Sang-Moo (Faculty of Marine Bioscience and Technology, Kangneung National University) ;
  • Heo, Seok (Department of Applied Biology and Chemistry, Konkuk University) ;
  • Kim, Cheon-Jei (Department of Food Science and Biotechnolgy of Animal Resources, Konkuk University) ;
  • Lee, Chi-Ho (Department of Food Science and Biotechnolgy of Animal Resources, Konkuk University) ;
  • Lee, Si-Kyung (Department of Applied Biology and Chemistry, Konkuk University)
  • Published : 2008.08.31
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Abstract

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

Keywords

References

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