Cloned (Comment) | Organism |
---|---|
overexpression of wild-type enzymes, and mutant LipA and LipH mutant 1H8 in Escherichia coli strain BL21(DE3) in inclusion bodies, subcloning in Escherichia coli strain DH5alpha, expression of mutant enzymes in Pseudomonase aeruginosa strains PABS1 and PABST7.1 | Pseudomonas aeruginosa |
Protein Variants | Comment | Organism |
---|---|---|
additional information | construction of a truncated lipH gene in which a 5'-deletion of 60 nucleotides encoding a 20 amino acid membrane anchor domain is replaced by a His10-tag and cloned into pET19b, resulting in plasmid pEHTHis19 | Pseudomonas aeruginosa |
S53P/L162G | optimizing enantioselectivity of LipA by comprehensive directed evolution approach | Pseudomonas aeruginosa |
T180I/T234S | identification of a mutant LipH 1H8 with increased (S)-enantioselectivity for model substrate 2-methyldecanoic acid 4-nitrophenyl ester compared to the wild-type enzyme, overview. The mutant shows low secretion efficiency, identification of two amino acid substitutions located on the protein surface, which significantly impair lipase secretion, the amino acid substitutions T180I and T234S of lipase variant 1H8 are located in close vicinity to these cysteines, suggesting that these substitutions may impair its ability to form disulfide bonds | Pseudomonas aeruginosa |
Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|
extracellular | the enzyme is secreted, the formation of one disulfide bond between residues C183 and C235 is essential for secretion of lipase, and residues D20 and T180 also affect secretion of lipase, overview | Pseudomonas aeruginosa | - |
- |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Pseudomonas aeruginosa | - |
genes lipA and lipH | - |
Purification (Comment) | Organism |
---|---|
recombinant wild-type LipA and LipH mutant 1H8 from Escherichia coli strain BL21(DE3) solubilized inclusion bodies by | Pseudomonas aeruginosa |
Renatured (Comment) | Organism |
---|---|
recombinant wild-type LipA and LipH mutant 1H8 from Escherichia coli strain BL21(DE3) inclusion bodies, denaturation by treatment for 1 h at 37°C with 100 mM Tris-HCl, pH 8.0, 8 M urea, followed by in vitro refolding to enzymatic activity performed for 3 h by adding equimolar ratios of LipH and sudden 10fold dilution in a buffer containing 50 mM Tris-HCl, pH 8.0, 3.5 mM CaCl2, 0.7 mM laurylmaltoside and 45% v/v glycerol | Pseudomonas aeruginosa |
Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|
2-methyldecanoic acid 4-nitrophenyl ester + H2O | model substrate, enantioselectivity in the asymmetric hydrolysis with preference for the S-enantiomer | Pseudomonas aeruginosa | (S)-2-methyldecanoate + 4-nitrophenol + (R)-2-methyldecanoic acid 4-nitrophenyl ester | - |
? |
Synonyms | Comment | Organism |
---|---|---|
LipA | - |
Pseudomonas aeruginosa |
lipase | - |
Pseudomonas aeruginosa |
LipH | - |
Pseudomonas aeruginosa |