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 lipasepositive 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-␤-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 29and 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 C 6 and C 18 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, the 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 bindingsite sequence.

Crystal structure of a lipase from Streptomyces sp. strain W007 – implications for thermostability and regiospecificity

“…Sequence alignment analysis of lipases from the I.7 subfamily showed that MAS1 shares 80%, 47%, 45% and 41% sequence identity, respectively, with lipases from Streptomyces avermitilis (LpsA2, Uniprot entry: Q828V2) [23], Propionibacterium acnes (PAL, Uniprot entry: Q6A601) [24], Janibacter sp. strain HTCC2649 (MAJ1, Uniprot entry: A3TMR7) [25] and Streptomyces cinnamoneus (LipA, UniParc entry: UPI0009042970) [26]. The MAS1 structure is the first reported of the I.7 subfamily.…”

Bacterial lipolytic enzymes: classification and properties

“…The lipases from Propionibacterium acnes (339 residues) [35] and from Streptomyces cinnamoneus (275 residues) [36] show significant similarity to each other (39 % identity, 50 % similarity). The central region of these proteins (residues 50-150) is approx.…”

A High Salt-Tolerant Thermoactive Esterase from Golden Grey Mullet: Purification, Characterization and Kinetic Properties

“…Protein transfer was performed for 1 h at 1 mA/cm 2 at room temperature. Zymogram was prepared from an emulsion of 1% TC4 in 150 mM NaCl, 2 mM Tris-HCl (pH 8) (Sommer et al 1997). Activity staining was accomplished by overlaying native gel with the zymogram in a closed glass dish and incubated at 40C from 4 to 12 h. Esterase activity against TC4 was recorded by the appearance of a clear zone.…”