Applied Microbiology and Biotechnology

, Volume 86, Issue 6, pp 1867–1875

Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce

  • Yoichi Toyokawa
  • Hiroaki Takahara
  • Alissara Reungsang
  • Masakazu Fukuta
  • Yuki Hachimine
  • Shinjiro Tachibana
  • Masaaki Yasuda
Biotechnologically Relevant Enzymes and Proteins

DOI: 10.1007/s00253-009-2434-5

Cite this article as:
Toyokawa, Y., Takahara, H., Reungsang, A. et al. Appl Microbiol Biotechnol (2010) 86: 1867. doi:10.1007/s00253-009-2434-5

Abstract

A gram-positive thermotolerant bacterium, designated strain RKK-04, was isolated from a fermented Thai fish sauce broth as it demonstrated high proteolytic activity. A phylogenetic analysis based on comparisons of 16S rRNA gene sequences showed that strain RKK-04 is Bacillus licheniformis. The proteolytic enzyme, which was purified 80-fold with 18% yield, has a molecular mass of 31 kDa and an isoelectric point higher than 9.3. The optimum pH and temperature of the enzyme activity were found to be 10.0 and 50°C, respectively. The addition of diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride completely inhibited enzymatic activity. These results showed that the enzyme is a subtilisin-like alkaline serine proteinase. On the other hand, the enzyme exhibited unique cleavage sites in oxidized insulin B-chain that differed from those of other subtilisin-like proteases. High enzymatic activity was also retained under high salt conditions (30% NaCl). The myosin heavy chain of fish protein was completely digested by reaction with this enzyme. Thus the halotolerant proteinase from B. licheniformis RKK-04 is a key enzyme for fish sauce fermentation.

Keywords

Bacillus licheniformis Fish sauce Halotolerant Serine proteinase Thermotolerant 

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Yoichi Toyokawa
    • 1
  • Hiroaki Takahara
    • 1
  • Alissara Reungsang
    • 2
  • Masakazu Fukuta
    • 1
  • Yuki Hachimine
    • 1
  • Shinjiro Tachibana
    • 1
  • Masaaki Yasuda
    • 1
  1. 1.Department of Bioscience and Biotechnology, Faculty of AgricultureUniversity of the RyukyusOkinawaJapan
  2. 2.Fermentation Research Centre for Value Added Agriculture Products, Department of Biotechnology, Faculty of TechnologyKhon Kaen UniversityKhon KaenThailand

Personalised recommendations