Figure 1.
Substrate spectrum of LipS and LipT shown as relative activity on 4-nitrophenyl (pNP) esters with fatty acid chains of 4 to 18 C-atoms.
Reactions were incubated at 70°C (LipS) or 75°C (LipT) with final substrate concentrations of 1 mM in potassium phosphate buffer (PB, 0.1 M, pH 8.0). Extinction was measured at 405 nm against an enzyme-free blank. Data are mean values of at least three independent measurements and bars indicate the standard deviation.
Table 1.
Biochemical parameters of recombinant LipT and LipS determined using 4-nitrophenol-decanoate (C10) for LipT and –octanoate (C8) for LipS.
Figure 2.
Temperature optimum (A) and thermal stability (B) of LipS and LipT.
Data are mean values of at least three independent measurements and bars indicate the standard deviation. Temperature range and optimum of LipS and LipT were measured with pNP-dodecanoate at temperatures ranging from 20°C to 90°C for 10 min. Assays were performed by incubation of the enzymes at 70°C for up to 72 hours and by measuring residual activities with pNP-dodecanoate at 70°C (LipS) and 75°C (LipT).
Table 2.
Specific activity* (U/mg) of LipT and LipS on pNP esters.
Figure 3.
HPLC-MS measurement of LipS catalyzing (R)-selectively the hydrolysis of ibuprofen phenyl ester.
The products of the reaction were converted to the corresponding methyl esters for measurement.
Figure 4.
Esterification reactions between 1-propanol and lauric acid (20 mmol each) as well as 1-tetradecanol and myristic acid (15 mmol each).
Synthesis reactions were catalyzed by LipS and CalB (purchased from Sigma-Aldrich, Buchs, Switzerland) under solvent-free conditions at 70°C. Specific activities of LipS and CalB refer to the dry-weights of the lyophilisates. Data are mean values of at least three independent measurements and bars indicate the standard deviation.
Figure 5.
A) Ribbon representation of the LipS monomer colored according to secondary structure elements. The inserted lid-domain is indicated in red. The catalytic triad residues Ser126, His257 and Asp227 are shown as stick representation. B) Surface representation of the LipS monomer with the lid-domain (β6, β7, αD1′) shown as a cartoon representation in red. The active site S126 (in yellow) is completely occluded from the bulk solvent and only accessible through a narrow tunnel. The active site pocket identified by CASTp server is colored in green. Amino acids building a pocket as part of the inserted domain are shown in orange. C) The catalytic triad residues of LipS are properly placed to establish hydrogen bonds.
Figure 6.
Topology of the inserted domains of α/β-hydrolases.
Superimposition of the inserted domain of LipS (in red) with A) Est1E (2WTM, orange) and LJ0536 (3PF8, turquoise), B) human MGL (3PE6, purple) and C) EstD (3DKR, blue) and Est30 (1TQH, green). The core structure of LipS is indicated in grey and catalytic S126 in yellow. The core structures of LipS homologues are not shown for simplicity.
Figure 7.
Phylogenetic tree illustrating the sorting of 40 metagenome derived lipase/esterase sequences into the eight known lipase/esterase families [61].
The eight families are color coded and labeled with the respective family name (LipS, LipT) or number (I-VIII). The five subfamilies containing the 11 unassignable metagenome lipase/esterase sequences are shown in white and are labeled with the respective family name (UF1-UF5). For the reference sequences, the full organism name as well as the accession number is given at the respective clade. Metagenome sequences are labeled with their protein name and accession number, respectively.
Table 3.
Activities of LipS and LipT in comparison with other characterized and published bacterial thermostable lipases.