The Metagenome-Derived Enzymes LipS and LipT Increase the Diversity of Known Lipases

Triacylglycerol lipases (EC 3.1.1.3) catalyze both hydrolysis and synthesis reactions with a broad spectrum of substrates rendering them especially suitable for many biotechnological applications. Most lipases used today originate from mesophilic organisms and are susceptible to thermal denaturation whereas only few possess high thermotolerance. Here, we report on the identification and characterization of two novel thermostable bacterial lipases identified by functional metagenomic screenings. Metagenomic libraries were constructed from enrichment cultures maintained at 65 to 75°C and screened resulting in the identification of initially 10 clones with lipolytic activities. Subsequently, two ORFs were identified encoding lipases, LipS and LipT. Comparative sequence analyses suggested that both enzymes are members of novel lipase families. LipS is a 30.2 kDa protein and revealed a half-life of 48 h at 70°C. The lipT gene encoded for a multimeric enzyme with a half-life of 3 h at 70°C. LipS had an optimum temperature at 70°C and LipT at 75°C. Both enzymes catalyzed hydrolysis of long-chain (C12 and C14) fatty acid esters and additionally hydrolyzed a number of industry-relevant substrates. LipS was highly specific for (R)-ibuprofen-phenyl ester with an enantiomeric excess (ee) of 99%. Furthermore, LipS was able to synthesize 1-propyl laurate and 1-tetradecyl myristate at 70°C with rates similar to those of the lipase CalB from Candida antarctica. LipS represents the first example of a thermostable metagenome-derived lipase with significant synthesis activities. Its X-ray structure was solved with a resolution of 1.99 Å revealing an unusually compact lid structure

Cooking birch pollen-related food: divergent consequences for IgE- and T cell-mediated reactivity in vitro and in vivo

BACKGROUND: The major birch pollen allergen Bet v 1 cross-reacts with homologous food allergens, resulting in IgE-mediated oral allergy syndromes (OASs). To avoid this food, allergy allergologists and guidebooks advise patients to consume birch pollen-related foods after heating. OBJECTIVE: We sought to evaluate whether cooked Bet v 1-related food allergens induce IgE- and T cell-mediated reactions in vitro and in vivo. METHODS: Recombinant Bet v 1, Mal d 1 (apple), Api g 1 (celery), and Dau c 1 (carrot) were incubated at increasing temperatures. Protein structures were determined by means of circular dichroism. Mediator release was tested in basophil activation assays. PBMCs and Bet v 1-specific T-cell lines with known epitope specificity were stimulated with native and cooked food allergens. Patients with birch pollen allergy who experienced OAS and the exacerbation of atopic dermatitis (AD) on ingestion of fresh apple, celery, or carrot were retested in double-blind, placebo-controlled food challenges with the respective foods in cooked form. RESULTS: In vitro, cooked food allergens lost the capacity to bind IgE and to induce mediator release but had the same potency to activate Bet v 1-specific T cells as native proteins. In vivo, ingestion of cooked birch pollen-related foods did not induce OAS but caused atopic eczema to worsen. CONCLUSION: T-cell cross-reactivity between Bet v 1 and related food allergens occurs independently of IgE cross-reactivity in vitro and in vivo. In patients with AD, the resulting immune reaction can even manifest as late eczematous skin reactions. Therefore the view that cooked pollen-related foods can be consumed without allergologic consequences should be reconsidered. CLINICAL IMPLICATIONS: Symptom-free consumed pollen-related food allergens might cause T cell-mediated late-phase skin reactions in patients with pollen allergy and A

Biochemical parameters of recombinant LipT and LipS determined using 4-nitrophenol-decanoate (C10) for LipT and –octanoate (C8) for LipS.

<p>The measurements were performed at 75 and 70°C, respectively, in 0.1 M PB pH 8.0.</p><p>Data are mean values of three independent measurements.</p

Esterification reactions between 1-propanol and lauric acid (20 mmol each) as well as 1-tetradecanol and myristic acid (15 mmol each).

<p>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.</p

Phylogenetic tree illustrating the sorting of 40 metagenome derived lipase/esterase sequences into the eight known lipase/esterase families [<b>61</b>].

<p>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.</p

HPLC-MS measurement of LipS catalyzing (<i>R</i>)-selectively the hydrolysis of ibuprofen phenyl ester.

<p>The products of the reaction were converted to the corresponding methyl esters for measurement.</p

Temperature optimum (A) and thermal stability (B) of LipS and LipT.

<p>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 <i>p</i>NP-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 <i>p</i>NP-dodecanoate at 70°C (LipS) and 75°C (LipT).</p

Topology of the inserted domains of α/β-hydrolases.

<p>Superimposition of the inserted domain of LipS (in red) with <b>A)</b> Est1E (2WTM, orange) and LJ0536 (3PF8, turquoise), <b>B)</b> human MGL (3PE6, purple) and <b>C)</b> 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.</p

Protein structure of LipS.

<p><b>A)</b> 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>B)</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. <b>C)</b> The catalytic triad residues of LipS are properly placed to establish hydrogen bonds.</p

Substrate spectrum of LipS and LipT shown as relative activity on 4-nitrophenyl (<i>p</i>NP) esters with fatty acid chains of 4 to 18 C-atoms.

<p>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.</p