Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli

Abstract

Environmental DNA libraries prepared from three different soil samples were screened for genes conferring lipolytic activity on Escherichia coli clones. Screening on triolein agar revealed 1 positive clone out of 730,000 clones, and screening on tributyrin agar revealed 3 positive clones out of 286,000 E. coli clones. Substrate specificity analysis revealed that one recombinant strain harbored a lipase and the other three contained esterases. The genes responsible for the lipolytic activity were identified and characterized.

FIG. 1

Restriction maps of the inserts of pAH110 to pAH113 and of the corresponding subcloned inserts of pAH114 to pAH117. The arrows represent length, location, and orientation of the genes lip, orf111, orf112, and orf113.

FIG. 2

Lipase activity staining after SDS-polyacrylamide gel electrophoresis of E. coli/pAH110 crude extract. Lanes: 1, marker proteins; 2, Coomassie blue-stained crude extract of E. coli/pAH110; 3, activity-stained crude extract of E. coli/pAH110; 4, activity-stained crude extract of E. coli/pSK(+). In lanes 3 and 4, tricaprylin was used as a substrate.

FIG. 3

Amino acid alignment of the protein regions of the deduced lip, orf111, orf112, and orf113 gene products containing the conserved motif around the proposed active-site serine of lipolytic enzymes. Matching amino acids of the consensus sequence for this region ([LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]) (5) are shaded.