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.
Subject(s)
Acinetobacter calcoaceticus/enzymology , Genes, Bacterial , Lipase/chemistry , Acinetobacter calcoaceticus/genetics , Acinetobacter calcoaceticus/growth & development , Amino Acid Sequence , Base Sequence , Binding Sites , Cloning, Molecular , Culture Media , Hydrogen-Ion Concentration , Immunoblotting , Kinetics , Lipase/genetics , Lipase/isolation & purification , Lipase/metabolism , Molecular Sequence Data , Open Reading Frames/genetics , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Alignment , Sequence Analysis , Substrate SpecificityABSTRACT
Hemocytes from two species of crabs, Eriocheir sinensis and Carcinus maenas, have been submitted to different cytochemical reactions in order to bring out the nature of granules' content, either by specific coloration or be enzymatic digestion. Light as well as electron microscopy has been used. The granules appeared to be made essentially of basic proteins, rich in arginin and disulfide bridges but poor in tyrosin. These proteins are linked with nonglycogenic carbohydrates. Glycogen deposits of varied size are restricted to the cytoplasm in all the different cell-types but have never been detected inside the granules themselves. Lipids are poorly represented. The bulk of the granule material is made of neutral mucopolysaccharides although minute amounts of acid mucosubstances could be found in some instances.
