A novel streptomycete lipase: cloning, sequencing and high-level expression of the Streptomyces rimosus GDS(L)-lipase gene

Abstract

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.

Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix‐assisted laser desorption/ionization time‐of‐flight and matrix‐assisted laser desorption/ionization quadrupole ion trap reflectron time‐of‐flight mass spectrometry: localization of the active site serine

“…The amino acid sequence showed 268 amino acid residues, including 34 of the signal peptide (TrEMBL accession number Q93MW7). 21 The enzyme contains six cysteines. All of them were shown to form intramolecular disulfide bonds (C-27-C-52, C-93-C-101 and C-151-C-198) and therefore contribute to the observed high stability.…”

“…20 In order to explore biotechnological applications and to characterize the structure on the protein level, large quantities of enzyme are required. Therefore, cloning, sequencing and high-level expression of S. rimosus lipase gene was carried out, 21 and the derived nucleotide sequence was analyzed by bioinformatic approaches. 21,22 Recently, capillary electrophoresis-on-a-chip, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and matrix-assisted laser desorption/ionization quadrupole ion trap reflection time-of-flight (MALDI-QIT-RTOF) mass spectrometry were used to characterize the primary structure of the S. rimosus lipase at the protein level and to prove the identity of the lipase from the wild-type and the overexpressing strain.…”

“…Therefore, cloning, sequencing and high-level expression of S. rimosus lipase gene was carried out, 21 and the derived nucleotide sequence was analyzed by bioinformatic approaches. 21,22 Recently, capillary electrophoresis-on-a-chip, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and matrix-assisted laser desorption/ionization quadrupole ion trap reflection time-of-flight (MALDI-QIT-RTOF) mass spectrometry were used to characterize the primary structure of the S. rimosus lipase at the protein level and to prove the identity of the lipase from the wild-type and the overexpressing strain. 23 These previous studies point out that this enzyme is of considerable interest, because it exhibits several properties that indicate its significant biotechnological potential and because it seems to differ in structure from most other known lipases.…”

Inhibition of extracellular lipase fromStreptomyces rimosuswith 3,4-dichloroisocoumarin

“…Overexpressed lipase was purified from culture filtrate of S. rimosus strain R6-ZGL3(pDJ7) that contains a high-copy-number plasmid harboring S. rimosus lipase gene 35 , as already described 36 . The purity of prepared enzyme solution was confirmed by capillary gel electrophoresis using lab-on-a-chip technology (Agilent Technologies).…”

A Conserved Inverted Repeat, the LipR Box, Mediates Transcriptional Activation of the Streptomyces exfoliatus Lipase Gene by LipR, a Member of the STAND Class of P-Loop Nucleoside Triphosphatases

“…More recently, it has been shown that two hydrolases from Acidovorax delafieldii and Thermobifida fusca capable of degrading synthetic polyesters exhibit high levels of similarity to these Streptomyces lipases (13,29). Other lipase-encoding genes have been cloned from Streptomyces species, including the gene for a Streptomyces cinnamomeus lipase related to Pseudomonas lipases (26) and a Streptomyces rimosus gene encoding an enzyme representing a third lipase family of Streptomyces (31).…”