Identification and molecular modeling of a novel lipase from an Antarctic soil metagenomic library.
In this work, we present the construction of a metagenomic library in Escherichia coli using pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Screening on agar supplemented with olive oil and rhodamine B revealed one clone with lipolytic activity (Lip1) out of 1000 E. coli clones. This clone harbored a plasmid, pLip1, which has an insert of 4722 bp that was completely sequenced from both directions. Further analysis of the insert showed three open reading frames (ORFs). ORF2 encoded a protein (Lip1 ) of 469 amino acids with 93% identity to the uncultured Pseudomonas sp. lipase LipJ03. Amino acid sequence comparison and phylogenetic analysis indicated that Lip1 lipase was closely related to family I subfamily 3. Furthermore, we present a three-dimensional model of lipase Lip1 which was generated based on the two known structures of mesophilic lipases from Pseudomonas sp. MIS 38 (PML lipase, PDB; 2Z8X) and Serratia marcescens (SML lipase, PDB: 2QUB). Finally, we report the results of comparisons between lipase Lip1 and mesophilic lipases and point out similarities and differences in the catalytic site and in other parts of the analyzed structures.
Cieśliński
Hubert
H
Department of Microbiology, Chemical Faculty, Gdańsk University of Technology, Gdańsk, Poland. hcieslin@pg.gda.pl
Białkowskaa
Aneta
A
Tkaczuk
Karolina
K
Długołecka
Anna
A
Kur
Józef
J
Turkiewicz
Marianna
M
D000595:Amino Acid Sequence; D000864:Antarctic Regions; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D023281:Genomics; D008049:Lipase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012988:Soil Microbiology
D001426:Bacterial Proteins; D008049:Lipase