Skip to main content

Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli

Applied Microbiology and Biotechnology volume 97, pages 5793–5800 (2013)Cite this article

Abstract

Lipoxygenase (LOX; EC 1.13.11.12,) is an enzyme that is widely used in food industry to improve aroma, rheological, or baking properties of foods. In this study, we described the expression and characterization of Pseudomonas aeruginosa LOX in Escherichia coli. The recombinant LOX was successfully expressed and secreted by E. coli using its endogenous signal peptide. When induced with 1 mM isopropyl β-d-1-thiogalactopyranoside (final concentration) at 20 °C for 47 h, the titer of the recombinant enzyme reached 3.89 U/mL. In order to characterize the catalytic properties, the recombinant LOX was purified to homogeneity on Q High Performance and Mono Q5/50GL sequentially. The molecular weight of the LOX was estimated as 70 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The K m and V max of the recombinant enzyme were 48.9 μM and 0.226 μmol/min, respectively. The purified enzyme exhibited a maximum activity at 25 °C and pH 7.5. High-performance liquid chromatography analysis of the linoleic acid hydroperoxides produced by recombinant LOX revealed that the LOX from P. aeruginosa falls into linoleic acid 13(S)-LOX. To the best of our knowledge, this is the first report on the overexpression of extracellular LOX in microorganisms, and the achieved LOX yield is the highest ever reported.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

References

  • Bae JH, Hou CT, Kim HR (2010) Thermostable lipoxygenase is a key enzyme in the conversion of linoleic acid to trihydroxy-octadecenoic acid by Pseudomonas aeruginosa PR3. Biotechnol Bioproc E 15:1022–1030

    Article  CAS  Google Scholar 

  • Bansal S, Sudha ML (2011) Nutritional, microstructural, rheological and quality characteristics of biscuits using processed wheat germ. Int J Food Sci Nutr 62:474–479

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Buchhaupt M, Guder JC, Etschmann MM, Schrader J (2012) Synthesis of green note aroma compounds by biotransformation of fatty acids using yeast cells coexpressing lipoxygenase and hydroperoxide lyase. Appl Microbiol Biot 93:159–168

    Article  Google Scholar 

  • Busquets M, Deroncele V, Vidal-Mas J, Rodriguez E, Guerrero A, Manresa A (2004) Isolation and characterization of a lipoxygenase from Pseudomonas 42A2 responsible for the biotransformation of oleic acid into (S)-(E)-10-hydroxy-8-octadecenoic acid. Anton Leeuw Int J G 85:129–139

    Article  CAS  Google Scholar 

  • Busto M, Owusu Apenten R, Robinson D, Wu Z, Casey R, Hughes R (1999) Kinetics of thermal inactivation of pea seed lipoxygenases and the effect of additives on their thermostability. Food Chem 65:323–329

    Article  CAS  Google Scholar 

  • Casey R, Hughes RK (2004) Recombinant lipoxygenases and oxylipin metabolism in relation to food quality. Food Biotechnol 18:135–170

    Article  CAS  Google Scholar 

  • Coffa G, Brash AR (2004) A single active site residue directs oxygenation stereospecificity in lipoxygenases: stereocontrol is linked to the position of oxygenation. P Natl Acad Sci USA 101:15579–15584

    Article  CAS  Google Scholar 

  • Cristea M, Engstrom K, Su C, Hornsten L, Oliw EH (2005) Expression of manganese lipoxygenase in Pichia pastoris and site-directed mutagenesis of putative metal ligands. Arch Biochem Biophys 434:201–211

    Article  CAS  Google Scholar 

  • Fukushige H, Hildebrand DF (2005) A simple and efficient system for green note compound biogenesis by use of certain lipoxygenase and hydroperoxide lyase sources. J Agr Food Chem 53:6877–6882

    Article  CAS  Google Scholar 

  • Funk MO, Carroll RT, Thompson JF, Dunham WR (1986) The lipoxygenases in developing soybean seeds, their characterization and synthesis in vitro. Plant Physiol 82:1139–1144

    Article  CAS  Google Scholar 

  • Guerrero A, Casals I, Busquets M, Leon Y, Manresa A (1997) Oxydation of oleic acid to (E)-10-hydroperoxy-8-octadecenoic and (E)-10-hydroxy-8-octadecenoic acids by Pseudomonas sp. 42A2. BBA-Lipids and Lipid Metabolism 1347:75–81

    Article  CAS  Google Scholar 

  • Hornung E, Kunze S, Liavonchanka A, Zimmermann G, Kuhn D, Fritsche K, Renz A, Kuhn H, Feussner I (2008) Identification of an amino acid determinant of pH regiospecificity in a seed lipoxygenase from Momordica charantia. Phytochemistry 69:2774–2780

    Article  CAS  Google Scholar 

  • Hughes RK, Wu Z, Robinson DS, Hardy D, West SI, Fairhurst SA, Casey R (1998) Characterization of authentic recombinant pea-seed lipoxygenases with distinct properties and reaction mechanisms. Biochem J 333:33–43

    CAS  Google Scholar 

  • Ivanov I, Heydeck D, Hofheinz K, Roffeis J, O'Donnell VB, Kuhn H, Walther M (2010) Molecular enzymology of lipoxygenases. Arch Biochem Biophys 503:161–174

    Article  CAS  Google Scholar 

  • Izumi T, Hoshiko S, Radmark O, Samuelsson B (1990) Cloning of the cDNA for human 12-lipoxygenase. P Natl Acad Sci USA 87:7477–7481

    Article  CAS  Google Scholar 

  • Johannesson M, Backman L, Claesson HE, Forsell PKA (2010) Cloning, purification and characterization of non-human primate 12/15-lipoxygenases. Prostag Leukotr Ess 82:121–129

    Article  CAS  Google Scholar 

  • Jonasson P, Liljeqvist S, Nygren PA, Stahl S (2002) Genetic design for facilitated production and recovery of recombinant proteins in Escherichia coli. Biotechnol appl bioc 35:91–105

    Article  CAS  Google Scholar 

  • Knust B, Wettstein D (1992) Expression and secretion of pea-seed lipoxygenase isoenzymes in Saccharomyces cerevisiae. Appl Microbiol Biot 37:342–351

    Article  CAS  Google Scholar 

  • Kuhn H, Saam J, Eibach S, Holzhutter HG, Ivanov I, Walther M (2005) Structural biology of mammalian lipoxygenases: enzymatic consequences of targeted alterations of the protein structure. Biochem Bioph Res Co 338:93–101

    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 

  • Liavonchanka A, Feussner I (2006) Lipoxygenases: occurrence, functions and catalysis. J Plant Physiol 163:348–357

    Article  CAS  Google Scholar 

  • Maccarrone M, Salucci ML, van Zadelhoff G, Malatesta F, Veldink G, Vliegenthart JF, Finazzi-Agro A (2001) Tryptic digestion of soybean lipoxygenase-1 generates a 60 kDa fragment with improved activity and membrane binding ability. Biochemistry 40:6819–6827

    Article  CAS  Google Scholar 

  • Mergulhao FJ, Summers DK, Monteiro GA (2005) Recombinant protein secretion in Escherichia coli. Biotechnol Adv 23:177–202

    Article  CAS  Google Scholar 

  • Nakamura M, Matsumoto T, Noguchi M, Yamashita I, Noma M (1990) Expression of a cDNA encoding human 5-lipoxygenase under control of the STA1 promoter in Saccharomyces cerevisiae. Gene 89:231–237

    Article  CAS  Google Scholar 

  • Oldham ML, Brash AR, Newcomer ME (2005) Insights from the X-ray crystal structure of coral 8R-lipoxygenase: calcium activation via a C2-like domain and a structural basis of product chirality. J Biol Chem 280:39545–39552

    Article  CAS  Google Scholar 

  • Oliw EH (2002) Plant and fungal lipoxygenases. Prostag Oth Lipid M 68:313–323

    Article  Google Scholar 

  • Porta H, Rocha-Sosa M (2001) Lipoxygenase in bacteria: a horizontal transfer event? Microbiology 147:3199–3200

    CAS  Google Scholar 

  • Reddy RG, Yoshimoto T, Yamamoto S, Marnett LJ (1994) Expression, purification, and characterization of porcine leukocyte 12-lipoxygenase produced in the methylotrophic yeast, Pichia pastoris. Biochem Bioph Res Co 205:381–388

    Article  CAS  Google Scholar 

  • Romanov S, Wiesner R, Myagkova G, Kuhn H, Ivanov I (2006) Affinity labeling of the rabbit 12/15-lipoxygenase using azido derivatives of arachidonic acid. Biochemistry 45:3554–3562

    Article  CAS  Google Scholar 

  • Rouzer CA, Samuelsson B (1985) On the nature of the 5-lipoxygenase reaction in human leukocytes: enzyme purification and requirement for multiple stimulatory factors. P Natl Acad Sci USA 82:6040–6044

    Article  CAS  Google Scholar 

  • Sloane DL, Leung R, Craik CS, Sigal E (1991) A primary determinant for lipoxygenase positional specificity. Nature 354:149–152

    Article  CAS  Google Scholar 

  • Sorensen HP, Mortensen KK (2005) Advanced genetic strategies for recombinant protein expression in Escherichia coli. J Biotechnol 115:113–128

    Article  CAS  Google Scholar 

  • Steczko J, Donoho GA, Dixon JE, Sugimoto T, Axelrod B (1991) Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in Escherichia coli. Protein Expr Purif 2:221–227

    Article  CAS  Google Scholar 

  • Vance R, Hong S, Gronert K, Serhan C, Mekalanos J (2004) The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase. P Natl Acad Sci USA 101:2135–2139

    Article  CAS  Google Scholar 

  • Vidal-Mas J, Busquets M, Manresa A (2005) Cloning and expression of a lipoxygenase from Pseudomonas aeruginosa 42A2. Anton Leeuw Int J G 87:245–251

    Article  CAS  Google Scholar 

  • Walther M, Anton M, Wiedmann M, Fletterick R, Kuhn H (2002) The N-terminal domain of the reticulocyte-type 15-lipoxygenase is not essential for enzymatic activity but contains determinants for membrane binding. J Biol Chem 277:27360–27366

    Article  CAS  Google Scholar 

  • Wu Z, Robinson DS, Hughes RK, Casey R, Hardy D, West SI (1999) Co-oxidation of beta-carotene catalyzed by soybean and recombinant pea lipoxygenases. J Agr Food Chem 47:4899–4906

    Article  CAS  Google Scholar 

  • Zhang C, Tao T, Ying Q, Zhang D, Lu F, Bie X, Lu Z (2012) Extracellular production of lipoxygenase from Anabaena sp. PCC 7120 in Bacillus subtilis and its effect on wheat protein. Appl Microbiol Biot 94:949–958

    Google Scholar 

Download references

Acknowledgments

We would like to thank Professor Byong H. Lee for critical reading and suggestions on the manuscript. This project was financially supported by the Key Program of National Natural Science Foundation of China (no. 20836003), the National Natural Science Foundation of China (nos. 30900013 and 31171639), the National Key Technology R&D Program in the 12th Five-Year Plan of China (no. 2011BAK10B03), the National High Technology Research and Development Program of China (863 Program, no. 2011AA100905), and the Self-determined Research Program of Jiangnan University (nos. JUSRP 21009 and JUSRP11215).

Author information

Affiliations

  1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Xinyao Lu, Juan Zhang, Song Liu, Dongxu Zhang, Zhi Xu, Jianghua Li & Guocheng Du

  2. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Jian Chen

  3. School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Xinyao Lu, Juan Zhang, Song Liu, Dongxu Zhang, Zhi Xu, Jing Wu, Jianghua Li, Guocheng Du & Jian Chen

  4. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China

    Jing Wu

Authors
  1. Xinyao Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Juan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Song Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dongxu Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Zhi Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jianghua Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guocheng Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jian Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Juan Zhang or Guocheng Du.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Lu, X., Zhang, J., Liu, S. et al. Overproduction, purification, and characterization of extracellular lipoxygenase of Pseudomonas aeruginosa in Escherichia coli . Appl Microbiol Biotechnol 97, 5793–5800 (2013). https://doi.org/10.1007/s00253-012-4457-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4457-6

Keywords

  • Lipoxygenase
  • Secretory expression
  • Escherichia coli
  • Catalytic properties