Propionibacterium acnes, a resident of lipid-rich human skin, produces a 33 kDa extracellular lipase encoded by gehA

Abstract

Five independent clones of thePropionibacterium acnesP-37 lipase gene (gehA) were obtained inEscherichia coli,and the gene was localized to a 2.75 kbXholfragment by subcloning. The five clones were shown to contain the same gene by Southern blotting with a DIG-labelled probe togehA.The nucleotide sequence ofgehAwas determined, and shown to contain a single ORF of 1017 kb, encoding a protein of 339 amino acids. The predicted molecular mass was 36 kDa. A 33 kDa (PAGE) radiolabeled polypeptide was detected fromE. coliminicell preparations harbouringgehA,which could correspond to GehA after cleavage of the putative 26 amino acid residue signal peptide.gehAwas overexpressed inE. coliunder the control of the bacteriophage T7 promoter, and the corresponding polypeptide was found to be present in insoluble aggregates. Active lipase was produced when the overexpressing strain was incubated at a reduced temperature in the presence of sucrose. Purification of lipase fromP. acnesculture supernatant fluids confirmed the production of a 33 kDa (PAGE) lipase.