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Secretory expression of a heterologous nattokinase in Lactococcus lactis

  • Biotechnologically Relevant Enzymes and Proteins
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Abstract

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter PnisZ and signal peptide SPUsp were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.

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References

  • Arnau J, Hjerl-Hansen E, Israelsen H (1997) Heterologous gene expression of bovine plasmin in Lactococcus lactis. Appl Microbiol Biotechnol 48:331–338

    Article  CAS  Google Scholar 

  • Astrup T, Sterndorff I (1952) The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 40:346–351

    Article  CAS  Google Scholar 

  • Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrlich SD, Sorokin A (2001) The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. Lactis IL 1403. Genome Res 11:731–753

    Article  CAS  Google Scholar 

  • Bruinenberg PG, Vos P, de Vos WM (1992) Proteinase overproduction in Lactococcus lactis: regulation and effect on growth and acidification in milk. Appl Environ Microbiol 58(1):78–84

    Article  CAS  Google Scholar 

  • Chen XZ, Hu HJ, Yang W, Huan LD (2001) Cloning and expression of nisin Z gene in Lactococcus lactis. Acta Genet Sin 28:285–290

    CAS  PubMed  Google Scholar 

  • Chiang CJ, Chen HC, Chao YP, Tzen JT (2005) Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli. J Agric Food Chem 53(12):4799–4804

    Article  CAS  Google Scholar 

  • de Ruyter PG, Kuipers OP, de Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62:3662–3667

    Article  Google Scholar 

  • Enouf V, Langella P, Commissaire J, Cohen J, Corthier G (2001) Bovine rotavirus nonstructural protein 4 produced by Lactococcus lactis is antigenic and immunogenic. Appl Environ Microbiol 67:1423–1428

    Article  CAS  Google Scholar 

  • Gasson MJ (1983) Plasmid complements of Streptococcus lactis NCDO 712 and other lactis streptococci after protoplast-induced curing. J Bacteriol 154:1–9

    Article  CAS  Google Scholar 

  • Holo H, Nes IF (1989) High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microbiol 55:3119–3123

    Article  CAS  Google Scholar 

  • Kim W, Choi K, Kim Y, Park H, Choi J, Lee Y, Oh H, Kwon I, Lee S (1996) Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from chungkook-jang. Appl Environ Microbiol 62:2482–2488

    Article  CAS  Google Scholar 

  • Kim TY, Choi YL, Cho YS, Kim CH, Lee YC (2002) Independently expressed N-terminal pro-domain of aqualysin 1 precursor complements the folding of its mature domain to active form in Escherichia coli. J Basic Microbiol 42(3):181–189

    Article  CAS  Google Scholar 

  • Kuipers OP, de Ruyter PGGA, Kleerebezem M, de Vos WM (1998) Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64:15–21

    Article  CAS  Google Scholar 

  • Le Loir Y, Azevedo V, Oliverira SC, Freitas DA, Miyoshi A, Bermudez-Humaran LG, Nouaille S, Ribeiro LA, Leclercq S, Gabriel JE, Guimaraes VD, Oliveira MN, Charlier C, Gautier M, Langella P (2005) Protein secretion in Lactococcus lactis: an efficient way to increase the over all heterologous protein production. Microb Cell Fact 4:2

    Article  Google Scholar 

  • Liu BY, Song HY (2002) Molecular cloning and expression of nattokinase gene in Bacillus subtilis. Acta Biochim Biophys Sin (Shanghai) 34(3):338–340

    CAS  Google Scholar 

  • Markland FS, Smith EL (1967) Subtilisin BPN. VII. Isolation of cyanogen bromide peptides and the complete amino acid sequence. J Biol Chem 242:5198–5211

    CAS  PubMed  Google Scholar 

  • Milner M, Makise K (2002) Natto and its active ingredient nattokinase-A potent and safe thrombolytic agent. Altern Complement Ther 8(3):157–194

    Article  Google Scholar 

  • Nouaille S, Ribeiro LA, Miyoshi A, Pontes D, Le Loir Y, Oliverira SC, Langella P, Azevedo V (2003) Heterologous protein production and delivery systems for Lactococcus lactis. Genet Mol Res 2:102–111

    PubMed  Google Scholar 

  • O’sullivan DJ, Klaenhammer TR (1993) Rapid mini-prep isolation of high-quality plasmid DNA from Lactococcus and Lactobacillus spp. Appl Environ Microbiol 59:2730–2733

    Article  Google Scholar 

  • Peng Y, Huang Q, Zhang RH, Zhang YZ (2003) Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B Biochem Mol Biol 134:45–52

    Article  Google Scholar 

  • Robinson K, Chamberlain LM, Schofield KM, Wells JM, Page RWFL (1997) Oral vaccination of mice against tetanus with recombinant Lactococcus lactis. Nat Biotechnol 15:653–657

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Shinde U, Inouye M (1993) Intramolecular chaperones and protein folding. Trends Biochem Sci 18:442–446

    Article  CAS  Google Scholar 

  • Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E (2000) Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 289:1352–1355

    Article  CAS  Google Scholar 

  • Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H (1987) A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese natto; a typical and popular soybean food in the Japanese diet. Experientia 43:1110–1111

    Article  CAS  Google Scholar 

  • Sumi H, Hamada H, Nakanishi K, Hiratani H (1990) Enhancement of the fibrinolytic activity in plasma by oral administration of nattokinase. Acta Haematol 84:139–143

    Article  CAS  Google Scholar 

  • Suzuki Y, Kondo K, Matsumoto Y, Zhao BQ, Otsuguro K, Maeda T, Tsukamoto Y, Urano T, Umemura K (2003) Dietary supplementation of fermented soybean, natto, suppresses intimal thickening and modulates the lysis of mural thrombi after endothelial injury in rat femoral artery. Life Sci 73:1289–1298

    Article  CAS  Google Scholar 

  • Tang B, Nirasawa S, Kitaoka M, Marie-Claire C, Hayashi K (2003) General function of N-terminal propeptide on assisting protein folding and inhibiting catalytic activity based on observations with a chimeric thermolysin-like protease. Biochem Biophys Res Commun 301:1093–1098

    Article  CAS  Google Scholar 

  • van de Guchte M, van de Vossen JMBM, Kok J, Venema G (1989) Construction of a Lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55:224–228

    Article  Google Scholar 

  • van de Guchte M, Kodde J, van der Vossen JM, Kok J, Venema G (1990) Heterologous gene expression in Lactococcus lactis subsp. Lactis: synthesis, secretion, and processing of the Bacillus subtilis neutral protease. Appl Environ Microbiol 56:2606–2611

    Article  Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Article  CAS  Google Scholar 

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Acknowledgment

This project was funded by the Top Field Program of Institute of Microbiology, Chinese Academy of Sciences: KSCXZ-SW-113.

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Authors and Affiliations

  1. Center for Metabolic Engineering of Microorganisms, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, People’s Republic of China

    Xiaobo Liang, Lixin Zhang & Jin Zhong

  2. State Key Laboratory of Microbial Resource, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100080, People’s Republic of China

    Liandong Huan

  3. Graduate School of the Chinese Academy of Sciences, Beijing, 100039, People’s Republic of China

    Xiaobo Liang

  4. South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, People’s Republic of China

    Lixin Zhang

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  1. Xiaobo Liang
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  2. Lixin Zhang
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  3. Jin Zhong
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  4. Liandong Huan
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Correspondence to Jin Zhong or Liandong Huan.

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Liang, X., Zhang, L., Zhong, J. et al. Secretory expression of a heterologous nattokinase in Lactococcus lactis . Appl Microbiol Biotechnol 75, 95–101 (2007). https://doi.org/10.1007/s00253-006-0809-4

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  • DOI: https://doi.org/10.1007/s00253-006-0809-4

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