Skip to main content
SpringerLink
Log in

Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Alkaline phosphatase catalyzes the hydrolysis of phosphomonoesters and is widely used in molecular biology techniques and clinical diagnostics. We expressed a recombinant alkaline phosphatase of the marine bacterium, Cobetia marina, in Escherichia coli BL21 (DE3). The recombinant protein was purified with a specific activity of 12,700 U/mg protein, which is the highest activity reported of any bacterial alkaline phosphatase studied to date. The molecular mass of the recombinant protein was 55–60 kDa, as determined by SDS–PAGE, and was observed to be a dimer by gel filtration analysis. The enzyme was optimally active at 45°C and the recombinant alkaline phosphatase efficiently hydrolyzed a phosphoric acid ester in luminescent and fluorescent substrates. Therefore, this enzyme can be considered to be extremely useful as a label conjugated to an antibody.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Chang CN, Kuang WJ, Chen EY (1986) Nucleotide sequence of the alkaline phosphatase gene of Escherichia coli. Gene 44:121–125

    Article  PubMed  CAS  Google Scholar 

  • Hauksson JB, Andrésson OS, Ásgeirsson B (2000) Heat-labile bacterial alkaline phosphatase from a marine Vibrio sp. Enzyme Microb Technol 27:66–73

    Article  PubMed  CAS  Google Scholar 

  • Helianti I, Okubo T, Morita Y, Tamiya E (2007) Characterization of thermostable native alkaline phosphatase from an aerobic hyperthermophilic archaeon, Aeropyrum pernix K1. Appl Microbiol Biotechnol 74:107–112

    Article  PubMed  CAS  Google Scholar 

  • Hirokawa K, Ichiyanagi A, Kajiyama N (2008) Enhancement of thermostability of fungal deglycating enzymes by directed evolution. Appl Microbiol Biotechnol 78:775–781

    Article  PubMed  CAS  Google Scholar 

  • Janeway CM, Xu X, Murphy JE, Chaidaroglou A, Kantrowitz ER (1993) Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis. Biochemistry 32:1601–1609

    Article  PubMed  CAS  Google Scholar 

  • Kim EE, Wyckoff HW (1991) Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis. J Mol Biol 218:449–464

    Article  PubMed  CAS  Google Scholar 

  • Manes T, Hoylaerts MF, Müller R, Lottspeich F, Hölke W, Millán JL (1998) Genetic complexity, structure, and characterization of highly active bovine intestinal alkaline phosphatases. J Biol Chem 273:23353–23360

    Article  PubMed  CAS  Google Scholar 

  • Murakawa T, Yamagata H, Tsuruta H, Aizono Y (2002) Cloning of cold-active alkaline phosphatase gene of a psychrophile, Shewanella sp., and expression of the recombinant enzyme. Biosci Biotechnol Biochem 66:754–761

    Article  PubMed  CAS  Google Scholar 

  • Murphy JE, Kantrowitz ER (1994) Why are mammalian alkaline phosphatase much more active than bacterial alkaline phosphatase. Mol Microbiol 12:351–357

    Article  PubMed  CAS  Google Scholar 

  • Murphy JE, Xu X, Kantrowitz ER (1993) Conversion of a magnesium binding site into a zinc binding site by a single amino acid substitution in Escherichia coli alkaline phosphatase. J Biol Chem 268:21497–21500

    PubMed  CAS  Google Scholar 

  • Plisova EY, Balabanova LA, Ivanova EP, Kozhemyako VB, Mikhailov VV, Agafonova EV, Rasskazov VA (2005) A highly active alkaline phosphatase from the marine bacterium cobetia. Mar Biotechnol 7:173–178

    Article  CAS  Google Scholar 

  • Rainer M, Johann PT, Frank G, Werner H, Stephan G, Hellmut E, Thomas K, Bettina B (2003) Expression of alkaline phosphatase in yeast. US Patent Application: 20030096341 A1 (22, May, 2003)

  • Rina M, Pozidis C, Mavromatis K, Tzanodaskalaki M, Kokkinidis M, Bouriotis V (2000) Alkaline phosphatase from the antarctic strain TAB 5. Properties and psychrophilic adaptations. Eur J Biochem 267:1230–1238

    Article  PubMed  CAS  Google Scholar 

  • Suzuki Y, Mizutani Y, Tsuji T, Ohtani N, Takano K, Haruki M, Morikawa M, Kanaya S (2005) Gene cloning, overproduction, and characterization of thermolabile alkaline phosphatase from a psychrotrophic bacterium. Biosci Biotechnol Biochem 69:364–373

    Article  PubMed  CAS  Google Scholar 

  • Zappa S, Rolland JL, Flament D, Gueguen Y, Boudrant J, Dietrich J (2001) Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi. Appl Environ Microbiol 67:4504–4511

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Noda Development Group, Planning & Administration Department, Kikkoman Biochemifa Company, 376-2, Kamihanawa, Nodashi, Chiba, 278-0033, Japan

    Eriko Nasu

  2. Research and Development Division, Kikkoman Corporation, 399, Noda, Nodashi, Chiba, 278-0037, Japan

    Atsushi Ichiyanagi & Keiko Gomi

Authors
  1. Eriko Nasu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Atsushi Ichiyanagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Keiko Gomi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Eriko Nasu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nasu, E., Ichiyanagi, A. & Gomi, K. Cloning and expression of a highly active recombinant alkaline phosphatase from psychrotrophic Cobetia marina . Biotechnol Lett 34, 321–328 (2012). https://doi.org/10.1007/s10529-011-0772-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-011-0772-8

Keywords

Search

Navigation