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.
Similar content being viewed by others
References
Aoyama M, Yasuda M, Nakachi K, Kobamoto N, Oku H, Kato F (2000) Soybean-milk-coagulating activity of Bacillus pumilus derives from a serine proteinase. Appl Microbiol Biotechnol 53:390–395
Buchanan R, Gibbons N (1974) Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore
Chaiyanan S, Chaiyanan S, Maugel T, Huq A, Robb FT, Colwell RR (1999) Polyphasic taxonomy of a novel Halobacillus, Halobacillus thailandensis sp. nov. isolated from fish sauce. Syst Appl Microbiol 22:360–365
Cho S, Oh S, Pridmore R, Juillerat M, Lee C (2003) Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter. J Agric Food Chem 51:7664–7670
Choorit W, Prasertsan P (1992) Characterization of proteases produced by newly isolated and identified proteolytic microorganisms from fermented fish (Budu). World J Microbiol Biotechnol 8:284–286
Curtis RI (2009) Umami and the foods of classical antiquity. Am J Clin Nutr 90:712s–718s
Dastgheib SMM, Amoozegar MA, Elahi E, Asad S, Banat IM (2008) Bioemulsifier production by a halothermophilic Bacillus strain with potential applications in microbially enhanced oil recovery. Biotechnol Lett 30:263–270
Edman P, Begg G (1967) A protein sequenator. Eur J Biochem 1:80–91
Hiraga K, Nishikata Y, Namwong S, Tanasupawat S, Takada K, Oda K (2005) Purification and characterization of serine proteinase from a halophilic bacterium, Filobacillus sp. RF2-5. Biosci Biotechnol Biochem 69:38–44
Jacobs M, Eliasson M, Uhlén M, Flock JI (1985) Cloning, sequencing and expression of subtilisin Carlsberg from Bacillus licheniformis. Nucleic Acids Res 13:8913–8926
Klomklao S, Benjakul S, Visessanguan W, Kishimura H, Simpson BK (2006) Proteolytic degradation of sardine (Sardinella gibbosa) proteins by trypsin from skipjack tuna (Katsuwonus pelamis) spleen. Food Chem 98:14–22
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Markland FS, Smith EL (1971) Subtilisins: primary structure, chemical and physical properties. The enzymes 3:561–608
McIver R, Brooks R, Reineccius G (1982) Flavor of fermented fish sauce. J Agric Food Chem 30:1017–1020
Morihara K, Tsuzuki H (1969) Comparison of the specificities of various serine proteinases from microorganisms. Arch Biochem Biophys 129:620–634
Nakamura T, Yamagata Y, Ichishima E (1992) Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci Biotechnol Biochem 56:1869–1871
Namwong S, Hiraga K, Takada K, Tsunemi M, Tanasupawat S, Oda K (2006) A halophilic serine proteinase from Halobacillus sp. SR5-3 isolated from fish sauce: Purification and characterization. Biosci Biotechnol Biochem 70:1395–1401
Nguyen AD, Nungaray J, Martel A, Le Goffic F, Mollé D, Léonil J (1998) Purification and characterization of the main pepsinogen from the shark, Centroscymnus coelolepis. J Biochem 124:287–293
Nishiyama Y, Maeda H, Nagashima T, Watanabe T, Mura K (2003) Partial amino acid sequence of protease I produced by Bacillus sp. 11-4 isolated from Vietnamese fish sauces. Food Preservation Sci 29:25–31
Okamoto M, Yonejima Y, Tsujimoto Y, Suzuki Y, Watanabe K (2001) A thermostable collagenolytic protease with a very large molecular mass produced by thermophilic Bacillus sp. strain MO-1. Appl Microbiol Biotechnol 57:103–108
Park Y, Kobayashi H, Kusakabe I, Murakami K (1987) Purification and characterization of soymilk-clotting enzymes from Bacillus sp. K-295G-7. Agric Biol Chem 51:2343–2349
Park JN, Ishida K, Watanabe T, Endoh K, Watanabe K, Murakami M, Abe H (2002) Taste effects of oligopeptides in a Vietnamese fish sauce. Fish Sci 68:921–928
Ricks E, Estrada-Valdés M, McLean T, Iacobucci GA (1992) Highly enantioselective hydrolysis of (R, S)-phenylalanine isopropyl ester by subtilisin carlsberg. Continuous synthesis of (S)-phenylalanine in a hollow fiber/liquid membrane reactor. Biotechnol Prog 8:197–203
Setyorini E, Takenaka S, Murakami S, Aoki K (2006) Purification and characterization of two novel halotolerant extracellular proteases from Bacillus subtilis strain FP-133. Biosci Biotechnol Biochem 70:433–440
Taira W, Funatsu Y, Satomi M, Takano T, Abe H (2007) Changes in extractive components and microbial proliferation during fermentation of fish sauce from underutilized fish species and quality of final products. Fish Sci 73:913–923
Takahashi M, Sekine T, Kuba N, Nakamori S, Yasuda M, Takagi H (2004) The production of recombinant APRP, an alkaline protease derived from Bacillus pumilus TYO-67, by in vitro refolding of pro-enzyme fixed on a solid surface. J Biochem 136:549–556
Yasuda M, Sakaguchi M (1998) Degradation of soybean protein by an acid proteinase from Monascus anka. Food Sci Technol Int (Tokyo) 4:6–8
Yasuda M, Aoyama M, Sakaguchi M, Nakachi K, Kobamoto N (1999) Purification and characterization of a soybean-milk-coagulating enzyme from Bacillus pumilus TYO-67. Appl Microbiol Biotechnol 51:474–479
Yongsawatdigul J, Rodtong S, Raksakulthai N (2007) Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J Food Sci 72:M382–M390
Yossan S, Reungsang A, Yasuda M (2006) Purification and characterization of alkaline protease from Bacillus megaterium isolated from Thai fish sauce fermentation process. Sci Asia 32:377–383
Acknowledgments
This work was supported by JSPS Core University Program. The authors wish to thank Dr. Toki Taira for his helpful advice and technical assistance.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Toyokawa, Y., Takahara, H., Reungsang, A. et al. Purification and characterization of a halotolerant serine proteinase from thermotolerant Bacillus licheniformis RKK-04 isolated from Thai fish sauce. Appl Microbiol Biotechnol 86, 1867–1875 (2010). https://doi.org/10.1007/s00253-009-2434-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-009-2434-5