Gene_locus Report for: serma-lipasA

Lipase LipA from Serratia marcescens is a 613-amino acid enzyme belonging to family I.3 of lipolytic enzymes that has an important biotechnological application in the production of a chiral precursor for the coronary vasodilator diltiazem. Like other family I.3 lipases, LipA is secreted by Gram-negative bacteria via a type I secretion system and possesses 13 copies of a calcium binding tandem repeat motif, GGXGXDXUX (U, hydrophobic amino acids), in the C-terminal part of the polypeptide chain. The 1.8-A crystal structure of LipA reveals a close relation to eukaryotic lipases, whereas family I.1 and I.2 enzymes appear to be more distantly related. Interestingly, the structure shows for the N-terminal lipase domain a variation on the canonical alpha/beta hydrolase fold in an open conformation, where the putative lid helix is anchored by a Ca(2+) ion essential for activity. Another novel feature observed in this lipase structure is the presence of a helical hairpin additional to the putative lid helix that exposes a hydrophobic surface to the aqueous medium and might function as an additional lid. The tandem repeats form two separated parallel beta-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second beta-roll result in an asymmetric Ca(2+) binding on only one side of the roll. The analysis of the properties of the beta-roll domains suggests an intramolecular chaperone function.

The gene encoding extracellular lipase of Serratia marcescens has been identified from a phage lambda genomic library. Formation of orange-red fluorescent plaques on rhodamine B-triolein plates was used to identify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcloned into a plasmid, and lipase was expressed in Escherichia coli. Extracellular lipase was detected in the presence of the secretion plasmid pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotide sequence of the entire cloned fragment revealed an open reading frame coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (around 70%) to lipases of Pseudomonas fluorescens strains. The lipase-specific consensus sequence G-X1-S-X2-G resided in the amino-terminal part of the protein, and carboxyl-terminal consensus sequences were an L-X-G-G-B-G-B-B-X repeat motif and a so-called aspartate box, respectively, which are both found in proteins secreted by the class I secretion pathway. Lipase was purified from the supernatant of a culture carrying a lipase expression vector, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marcescens belongs to the group of extracellular enzyme proteins secreted by the class I secretion pathway.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.

An enantioselective lipase gene (esf) for the kinetic resolution of optically active (S)-flurbiprofen was cloned from the new strain Serratia marcescens ES-2. The esf gene was composed of a 1,845-bp open reading frame encoding 614 amino acid residues with a calculated molecular mass of 64,978 Da. The lipase expressed in E. coli was purified by a three-step procedure, and it showed preferential substrate specificity toward the medium-chain-length fatty acids. The esf gene encoding the enantioselective lipase was reintroduced into the parent strain S. marcescens ES-2 for secretory overexpression. The transformant S. marcescens BESF secreted up to 217 kU/ ml of the enantioselective lipase, about 54-fold more than the parent strain, after supplementing 3.0% Triton X-207. The kinetic resolution of (S)-flurbiprofen was carried out even at an extremely high (R,S)-flurbiprofen ethyl ester [(R,S)-FEE] concentration of 500 mM, 130 kU of the S. marcescens ES-2 lipase per mmol of (R,S)-FEE, and 1,000 mM of succinyl beta-cyclodextrin as the dispenser at 37 degrees C for 12 h, achieving the high enantiomeric excess and conversion yield of 98% and 48%, respectively.

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000-6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an eep (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% eep and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.

Lipase LipA from Serratia marcescens is a 613-amino acid enzyme belonging to family I.3 of lipolytic enzymes that has an important biotechnological application in the production of a chiral precursor for the coronary vasodilator diltiazem. Like other family I.3 lipases, LipA is secreted by Gram-negative bacteria via a type I secretion system and possesses 13 copies of a calcium binding tandem repeat motif, GGXGXDXUX (U, hydrophobic amino acids), in the C-terminal part of the polypeptide chain. The 1.8-A crystal structure of LipA reveals a close relation to eukaryotic lipases, whereas family I.1 and I.2 enzymes appear to be more distantly related. Interestingly, the structure shows for the N-terminal lipase domain a variation on the canonical alpha/beta hydrolase fold in an open conformation, where the putative lid helix is anchored by a Ca(2+) ion essential for activity. Another novel feature observed in this lipase structure is the presence of a helical hairpin additional to the putative lid helix that exposes a hydrophobic surface to the aqueous medium and might function as an additional lid. The tandem repeats form two separated parallel beta-roll domains that pack tightly against each other. Variations of the consensus sequence of the tandem repeats within the second beta-roll result in an asymmetric Ca(2+) binding on only one side of the roll. The analysis of the properties of the beta-roll domains suggests an intramolecular chaperone function.

The gene encoding extracellular lipase of Serratia marcescens has been identified from a phage lambda genomic library. Formation of orange-red fluorescent plaques on rhodamine B-triolein plates was used to identify phages carrying the lipase gene. A 2.8-kb SalI fragment was subcloned into a plasmid, and lipase was expressed in Escherichia coli. Extracellular lipase was detected in the presence of the secretion plasmid pGSD6 carrying the genes prtD, -E, and -F, which guide the secretion of protease from Erwinia chrysanthemi. Determination of the nucleotide sequence of the entire cloned fragment revealed an open reading frame coding for a 613-amino-acid protein with a predicted M(r) of 64,800. Analysis of the amino acid sequence revealed significant homology (around 70%) to lipases of Pseudomonas fluorescens strains. The lipase-specific consensus sequence G-X1-S-X2-G resided in the amino-terminal part of the protein, and carboxyl-terminal consensus sequences were an L-X-G-G-B-G-B-B-X repeat motif and a so-called aspartate box, respectively, which are both found in proteins secreted by the class I secretion pathway. Lipase was purified from the supernatant of a culture carrying a lipase expression vector, and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an M(r) of 64,000 for the purified protein. Our results suggest that the lipase of S. marcescens belongs to the group of extracellular enzyme proteins secreted by the class I secretion pathway.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.