Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene

  1. Ruben G. Kok1,
  2. Jasper J. van Thor1,
  3. Inge M. Nugteren-Roodzant1,
  4. Marc B. W. Brouwer1,
  5. Maarten R. Egmond2,
  6. Clara B. Nudel3,
  7. Ben Vosman4,
  8. Klaas J. Hellingwerf1,*

Article first published online: 27 OCT 2006

DOI: 10.1111/j.1365-2958.1995.tb02351.x

Issue

Molecular Microbiology

Molecular Microbiology

Volume 15, Issue 5, pages 803–818, March 1995

Additional Information

How to Cite

Kok, R. G., van Thor, J. J., Nugteren-Roodzant, I. M., Brouwer, M. B. W., Egmond, M. R., Nudel, C. B., Vosman, B. and Hellingwerf, K. J. (1995), Characterization of the extracellular lipase, LipA, of Acinetobacter calcoaceticus BD413 and sequence analysis of the cloned structural gene. Molecular Microbiology, 15: 803–818. doi: 10.1111/j.1365-2958.1995.tb02351.x

Author Information

  1. 1

    Department of Microbiology, E.C. Slater Institute, BioCentrum Amsterdam, Nieuwe Achtergracht 127, 1018 WS Amsterdam, the Netherlands.

  2. 2

    Unilever Research Laboratories (URL) Vlaardingen, PO Box 114, 3130 AC Vlaardingen, the Netherlands.

  3. 3

    Universidad de Buenos Aires, Facultad de Farmacia y Bioquimica, Orlentacion Biotechnologia, Junin 956, 6° Piso, 1113 Capital-Buenos Aires, Argentina 96-6411.

  4. 4

    Centre for Plant Breeding and Reproduction Research (CPRO-DLO), PO Box 16, 6700 AA Wageningen, the Netherlands.

* For correspondence, E-mall a417hell@horus.sara.nl; Tel. (20) 5257055; Fax (20) 5257056.

Publication History

  1. Issue published online: 27 OCT 2006
  2. Article first published online: 27 OCT 2006
  3. Received 23 August, 1994; revised 7 November, 1994; accepted 10 November, 1994.

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Summary

The extracellular lipase from Acinetobacter calcoaceticus BD413 was purified to homogeneity, via hydrophobic-interaction fast performance liquid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source. The enzyme has an apparent molecular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long acyl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with optimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and towards egg-yolk emulsions. The N-terminal amino acid sequence of the mature protein was determined, and via reverse genetics the structural lipase gene was cloned from a gene library of A. calcoaceticus DNA in Escherichia coli phage M13. Sequence analysis of a 2.1 kb chromosomal DNA fragment revealed one complete open reading frame, lipA, encoding a mature protein with a predicted molecular mass of 32.1 kDa. This protein shows high similarity to known lipases, especially Pseudomonas lipases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signai sequence at the N-terminus of the mature lipase suggests that the lipase of Acinetobacter is also exported via a two-step translocation mechanism. However, no chaperone-encoding gene was found downstream of lipA, unlike the situation in Pseudomonas. Analysis of an A. calcoaceticus mutant showing reduced lipase production revealed that a periplasmic disutphide oxidoreductase is involved in processing of the lipase. Via sequence alignments, based upon the crystal structure of the closely related Pseudomonas glumae lipase, a model has been made of the secondary-structure elements in AcLipA. The active site serine of AcLipA was changed to an alanine, via site-directed mutagenesis, resulting in production of an inactive extracellular lipase.

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