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 (C(12) and C(14)) 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.

Cloning, expression, purification and preliminary X-ray analysis of a putative metagenome-derived lipase.

LipS is a novel thermostable putative lipase that was isolated from a metagenomic library using functional screening methods. The corresponding gene shows high similarity to that encoding a putative but uncharacterized esterase from Symbiobacterium thermophilum IAM14863 (99% nucleotide-sequence similarity). Two different constructs of the recombinant lipase were crystallized. Crystals belonging to space group P4(2)2(1)2 diffracted X-ray radiation to 2.8 Šresolution and crystals belonging to space group P4 diffracted to 2.0 Šresolution. The most probable content of their asymmetric units were two molecules (P4(2)2(1)2) and four or five molecules (P4), respectively.

Altered IgE epitope presentation: A model for hypoallergenic activity revealed for Bet v 1 trimer.

In order to reduce side effects in the course of allergen specific immunotherapy hypoallergenic allergen derivatives with reduced IgE reactivity have been made by genetic engineering. In contrast to other recombinant hypoallergenic allergen derivatives which showed reduced IgE reactivity, a recombinant trimer of the major birch pollen allergen Bet v 1 showed reduced allergenic activity despite preserved IgE reactivity. We studied rBet v 1 trimer by SDS-PAGE, mass spectrometry, circular dichroism and gel filtration. Furthermore we investigated IgE and IgG reactivity of the rBet v 1 trimer in solid and liquid phase assays and compared its allergenic activity with that of rBet v 1 wildtype using basophil activation assays. In solid phase immunoassays rBet v 1 trimer exhibited even stronger IgE reactivity than the rBet v 1 wildtype, whereas both proteins were equally well recognized by Bet v 1-specific IgG antibody probes. In fluid phase IgE experiments rBet v 1 trimer inhibited IgE reactivity to rBet v 1 wildtype but showed a more than 10-fold reduced allergenic activity compared to the rBet v 1 monomer. By analytical gel filtration it was demonstrated that, despite its monomeric appearance in SDS-PAGE the trimer occurred in fluid phase in the form of defined high molecular weight (>600 kDa) aggregates whereas rBet v 1 wildtype strictly appeared as monomeric protein. The results indicate that the hypoallergenic nature of the rBet v 1 trimer is due to formation of defined high molecular weight aggregates which may be responsible for an altered presentation of IgE epitopes in a form with reduced capacity to crosslink effector-cell bound IgE. We thus provide evidence for a novel mechanism for hypoallergenic activity.

Characterization of folded recombinant Der p 5, a potential diagnostic marker allergen for house dust mite allergy.

BACKGROUND: Der p 5 was reported as an important allergen in Dermatophagoides pteronyssinus, which is particularly recognized by patients suffering from asthma. The aim of this study was to produce, by recombinant DNA technology, a folded Der p 5 allergen for diagnostic, therapeutic and preventive purposes. METHODS: Der p 5-encoding cDNA was isolated from a lambda gt11 D. pteronyssinus expression cDNA library and expressed in Escherichia coli. rDer p 5 was purified to homogeneity and characterized by mass spectroscopy and circular dichroism. IgE reactivity was tested with sera from 117 mite-allergic patients and in a basophil histamine release experiment. Der p 5-specific rabbit IgG antibodies were produced for the ultrastructural localization of the allergen in mites by immunogold electron microscopy as well as for cross-reactivity studies. RESULTS: rDer p 5 is a heat-stable protein with predominantly alpha-helical secondary structure which reacted with IgE from 31% of mite-allergic patients' sera and showed no relevant cross-reactivity to group 5 allergens from storage mites and tropical mites. rDer p 5-specific rabbit IgG antibodies inhibited mite-allergic patients' IgE binding to Der p 5 and allowed to localize the allergen in secretory granules of midgut epithelial cells of house dust mites. CONCLUSIONS: The described rDer p 5 molecule may be useful for diagnosis and immunotherapy of house dust mite-allergic patients.

Crystallization and preliminary structure determination of the plant food allergen Pru av 2.

Thaumatin-like proteins (TLPs) have mostly been investigated in the context of their function as pathogenesis-related proteins and only in recent years have some of them been classified as allergens. Here, the purification and crystallization of the first allergenic TLP, Pru av 2, a 23.3 kDa protein isolated from ripe cherries, is reported. The crystals diffracted to 2.1 A resolution at a rotating-anode generator and were found to belong to space group P2(1), with unit-cell parameters a = 44.48, b = 41.04, c = 59.16 A, beta = 106.61 degrees and one molecule per asymmetric unit. In order to obtain high-resolution data, an annealing protocol was applied that improved the resolution limit from 1.6 to 1.3 A at a synchrotron.