CHARACTERIZATION OF THE EXTRACELLULAR LIPASE, LIPA, OF ACINETOBACTER-CALCOACETICUS BD413 AND SEQUENCE-ANALYSIS OF THE CLONED STRUCTURAL GENE

The extracellular lipase from Acinetobacter calcoaceticus BD413 was pu rified to homogeneity, via hydrophobic-interaction fast performance li quid chromatography (FPLC), from cultures grown in mineral medium with hexadecane as the sole carbon source, The enzyme has an apparent mole cular mass of 32 kDa on SDS-polyacrylamide gels and hydrolyses long ac yl chain p-nitrophenol (pNP) esters, like pNP palmitate (pNPP), with o ptimal activity between pH 7.8 and 8.8. Additionally, the enzyme shows activity towards triglycerides such as olive oil and tributyrin and t owards egg-yolk emulsions. The N-terminal amino acid sequence of the m ature 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 pro tein shows high similarity to known lipases, especially Pseudomonas li pases, that are exported in a two-step secretion mechanism and require a lipase-specific chaperone. The identification of an export signal s equence at the N-terminus of the mature lipase suggests that the lipas e of Acinetobacter is also exported via a two-step translocation mecha nism. 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 disulphide 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 second ary-structure elements in AcLipA, The active site serine of AclipA was changed to an alanine, via site-directed mutagenesis, resulting in pr oduction of an inactive extracellular lipase.