Applied Microbiology and Biotechnology

, Volume 26, Issue 4, pp 318–322 | Cite as

Purification of extracellular alkaline phosphatase released by Escherichia coli excretory mutants

  • Danielle Atlan
  • Raymond Portalier


Alkaline phosphatase (APase) is the major protein released into the extracellular medium by strain 706, a periplasmic-excretory (lky) mutant of Escherichia coli K12. We developed a rapid three step procedure for APase purification from culture supernatants of lky mutants. Two ultrafiltration stages and an heat treatment were sufficient to obtain a 99% pure enzyme preparation. Batch culture conditions of strain 706 in a 15 l fermentor leading to an extracellular APase yield of 1250 U/ml were determined.


Enzyme Escherichia Coli Purification Heat Treatment Alkaline Phosphatase 
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E. coli alkaline phosphatase


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  1. Argast M, Boos W (1980) Co-regulation in Escherichia coli of a novel transport system for sn-glycerol-3-phosphate and outer membrane protein Ic (e, E) with alkaline phosphatase and phosphate binding protein. J Bacteriol 143:142–150Google Scholar
  2. Atlan D, Portalier R (1984) Optimized extracellular production of alkaline phosphatase by lky mutants of Escherichia coli K12. Appl Microbiol Biotechnol 19:5–12Google Scholar
  3. Atlan D, Lazzaroni JC, Portalier R (1986) Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12. J Gen Microbiology 132:171–181Google Scholar
  4. Beacham I (1979) Periplasmic enzymes in gram-negative bacteria. Int J Biochem 10:877–883Google Scholar
  5. Gleiser IE, Bauer S (1981) Growth of E. coli W to high cell concentration by oxygen level linked control of carbon source concentration. Biotechnol Bioeng 23:1015–1021Google Scholar
  6. Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–865PubMedGoogle Scholar
  7. Lazzaroni JC, Portalier R (1981) Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K12. J Bacteriol 145:1351–1358Google Scholar
  8. Lazzaroni JC, Portalier R (1985) Regulation of the lkyB gene expression in Escherichia coli K12 strains carrying an lkyBlacZ gene fusion. Mol Gen Genet 201:323–328Google Scholar
  9. Levitz R, Klar A, Yagil E (1984) A new locus in the phosphate specific transport (PST) region of Escherichia coli. Mol Gen Genet 197:98–103Google Scholar
  10. Malamy M, Horecker B (1961) The localization of alkaline phosphatase in E. coli K12. Biochem Biophys Res Commun 5:104–108Google Scholar
  11. Malamy M, Horecker BL (1964) Release of alkaline phosphatase from cells of Escherichia coli upon lysozyme spheroplast formation. Biochemistry 3:1889–1893Google Scholar
  12. Maniatis T, Fritsh E, Sambrook J (eds) (1982) In: Cold Spring Harbor Laboratory. Molecular cloning. Cold Spring Harbor, New York, p 13Google Scholar
  13. Maxam AM, Gilbert W (1977) A new method for sequencing DNA. Proc Natl Acad Sci USA 74:560–564Google Scholar
  14. Miller J (ed) (1972) Experiments in molecular genetics, Cold Spring Harbor Laboratory. Cold Spring Harbor, New York 352–355Google Scholar
  15. Nakata A, Amemura M, Shinagawa H (1984) Regulation of the phosphate regulon in Escherichia coli K12: Regulation of the negative regulatory gene phoU and identification of the gene product. J Bacteriol 159:979–985Google Scholar
  16. Neu HC, Heppel LA (1965) The release of enzymes from Escherichia coli by osmotic shock and during the formation of spheroplasts. J Biol Chem 240:3685–3692Google Scholar
  17. Rao N, Wang E, Yashphe J, Torriani A (1986) Nucleotide pool in pho regulon mutants and alkaline phosphatase synthesis in Escherichia coli. J Bacteriol 166:205–211Google Scholar
  18. Schacterle G, Pollack R (1973) A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal Biochem 51:654–655PubMedGoogle Scholar
  19. Tommassen J, Lugtenberg B (1982) pho regulon of Escherichia coli K12: a minireview. Ann Microbiol (Institut Pasteur) 133A:243–249Google Scholar
  20. Torriani A (1960) Influence of inorganic phosphate in the formation of phosphatases in Escherichia coli. Biochim Biophys Acta 38:460–479Google Scholar
  21. Torriani A (1967) Alkaline phosphatase from Escherichia coli. In: Cantoni GL, Davies DR (eds) Procedures in nucleic acid research. Harper and Row Publishers Inc, New York, pp 224–235Google Scholar

Copyright information

© Springer-Verlag 1987

Authors and Affiliations

  • Danielle Atlan
    • 1
  • Raymond Portalier
    • 1
  1. 1.Atelier de Biotechnologie Enzymatique et Microbienne (UM 24)Université Claude BernardVilleurbanne CedexFrance

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