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

Abstract

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 signal 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 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 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.

Production, purification, characterization, and applications of lipases

“…Production of an extracellular lipase by Aci. calcoaceticus BD 413 was enhanced when the medium was supplemented with Mg 2 + , Ca 2 + , Cu 2 + , and Co 2 + (Kok et al, 1995). The enzyme hydrolyzed long acyl chain p-nitrophenol ( pNP) esters, such as pNPP, and its optimal activity occurred between pH 7.8 and 8.8 (Kok et al, 1995).…”

“…The structural lipase gene from a gene library of Aci. calcoaceticus BD413 DNA was cloned in Escherichia coli phage M13 by Kok et al (1995). The sequence analysis of 2.1-kb chromosomal DNA fragment revealed one complete open reading frame, lip A, encoding a mature protein with a predicted molecular mass of 32.1 kDa.…”

“…calcoaceticus BD 413 was purified to homogeneity using hydrophobic interaction fast performance liquid chromatography (FPLC) (Kok et al, 1995). The enzyme had an apparent molecular mass of 32 kDa on SDS-PAGE and an optimal activity pH of between 7.8 and 8.8 (Kok et al, 1995). Also, a lipase from Pe.…”

Characterization of lipase-deficient mutants of Acinetobacter calcoaceticus BD413: identification of a periplasmic lipase chaperone essential for the production of extracellular lipase

“…BD413 estA, encoding one of the cell-bound esterases (40), and lipA, encoding the extracellular lipase (43), have been cloned and characterized. The regulation of their expression is currently being investigated.…”

“…The extracellular lipase of A. calcoaceticus BD413 presumably is exported from the cell via a two-step translocation process (43): across the cytoplasmic membrane, this translocation will be mediated by the Sec system (59,66), whereas translocation through the outer membrane proceeds via a translocation complex similar to the Xcp system in Pseudomonas spp. (69) and the Pul system in Klebsiella spp.…”