title: Genetic and biochemical characterization of a new extracellular lipase from Streptomyces cinnamomeus.
abstract: Streptomyces cinnamomeus Tü89 secretes a 30-kDa esterase and a 50-kDa lipase. The lipase-encoding gene, lipA, was cloned from genomic DNA into Streptomyces lividans TK23 with plasmid vector pIJ702. Two lipase-positive clones were identified; each recombinant plasmid had a 5.2-kb MboI insert that contained the complete lipA gene. The two plasmids differed in the orientation of the insert and the degree of lipolytic activity produced. The lipA gene was sequenced; lipA encodes a proprotein of 275 amino acids (29,213 Da) with a pI of 5.35. The LipA signal peptide is 30 amino acids long, and the mature lipase sequence is 245 amino acids long (26.2 kDa) and contains six cysteine residues. The conserved catalytic serine residue of LipA is in position 125. Sequence similarity of the mature lipases (29% identity, 60% similarity) was observed mainly in the N-terminal 104 amino acids with the group II Pseudomonas lipases; no similarity to the two Streptomyces lipase sequences was found. lipA was also expressed in Escherichia coli under the control of lacZ promoter. In the presence of the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG), growth of the E. coli clone was severely affected, and the cells lysed in liquid medium. Lipase activity in the E. coli clone was found mainly in the pellet fraction. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, three additional protein bands of 50, 29, and 27 kDa were visible. The 27-kDa protein showed lipolytic activity and represents the mature lipase; the 29- and 50-kDa forms showed no activity and very probably represent the unprocessed form and a dimeric misfolded form, respectively. For higher expression of lipA in S. lividans, the gene was cloned next to the strong aphII promoter. In contrast to the lipA-expressing E. coli clone, S. cinnamomeus and the corresponding S. lividans clone secreted only an active protein of 50 kDa. The lipase showed highest activity with C6 and C18 triglycerides; no activity was observed with phospholipids, Tween 20, or p-nitrophenylesters. Upstream of lipA and in the same orientation, an open reading frame, orfA, is found whose deduced protein sequence (519 amino acids) shows similarity to various membrane-localized transporters. Downstream of lipA and in the opposite orientation, an open reading frame, orfB (encoding a 199-amino-acid protein) is found, which shows no conspicuous sequence similarity to known proteins, other than an NAD and flavin adenine dinucleotide binding-site sequence.
authors.affiliation: Universität Tübingen, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Sommer
authors.forename: C
authors.initials: C
authors.lastname: Bormann
authors.forename: F
authors.initials: F
authors.lastname: Götz
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007526:Isoelectric Point; D008049:Lipase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D004269:DNA, Bacterial; D021382:Protein Sorting Signals; D008049:Lipase