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

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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.

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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

    Article  CAS  Google Scholar 

  • Buchanan R, Gibbons N (1974) Bergey's manual of determinative bacteriology. Williams and Wilkins, Baltimore

    Google Scholar 

  • 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

    CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Curtis RI (2009) Umami and the foods of classical antiquity. Am J Clin Nutr 90:712s–718s

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Edman P, Begg G (1967) A protein sequenator. Eur J Biochem 1:80–91

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  Google Scholar 

  • Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    CAS  Google Scholar 

  • Markland FS, Smith EL (1971) Subtilisins: primary structure, chemical and physical properties. The enzymes 3:561–608

    Google Scholar 

  • McIver R, Brooks R, Reineccius G (1982) Flavor of fermented fish sauce. J Agric Food Chem 30:1017–1020

    Article  CAS  Google Scholar 

  • Morihara K, Tsuzuki H (1969) Comparison of the specificities of various serine proteinases from microorganisms. Arch Biochem Biophys 129:620–634

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • 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

    Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • Yasuda M, Sakaguchi M (1998) Degradation of soybean protein by an acid proteinase from Monascus anka. Food Sci Technol Int (Tokyo) 4:6–8

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

  • 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

    Article  CAS  Google Scholar 

Download references

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.

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Correspondence to Masaaki Yasuda.

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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

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  • DOI: https://doi.org/10.1007/s00253-009-2434-5

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