Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed the mutant strain BL03 that was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active clones in the thermophilic bacterium than in the mesophilic E. coli. From all clones functionally screened in E. coli, only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus. Four open reading frames (ORFs) were found which did not share significant similarity to known esterase enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance

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

Kolorimetrische und fluorimetrische Assays – Auf der Jagd nach neuen Biokatalysatoren für die Synthesechemie –

Enzymes and their powerful catalytic properties are a basis of life as we know it. Due to their composition of L‐amino acids and the resulting chirality, they show often high enantioselectivity. In order to exploit the natural biological diversity of these biocatalysts and the man‐made diversity of mutant libraries, fast and reliable screening and fingerprinting assays are needed for identification and characterisation of new enzymes for synthetic applications. One quite commonly applied assay principle is the usage of chromo‐ and fluorogenic surrogate substrates. Since their conversion can be detected easily by photo‐ or fluorimetry this method is suitable for high‐throughput and ultra‐high‐throughput screenings. In the scope of this work, known assay principles[1, 2] were extended and/or applied in order to characterise different kinds of selectivities of P450 monooxygenases, lipases, and esterase

A novel thermoalkalostable esterase from Acidicaldus sp. strain USBA-GBX-499 with enantioselectivity isolated from an acidic hot springs of Colombian Andes

Several thermo- and mesoacidophilic bacterial strains that revealed high lipolytic activity were isolated from water samples derived from acidic hot springs in Los Nevados National Natural Park (Colombia). A novel lipolytic enzyme named 499EST was obtained from the thermoacidophilic alpha-Proteobacterium Acidicaldus USBA-GBX-499. The gene estA encoded a 313-amino-acid protein named 499EST. The deduced amino acid sequence showed the highest identity (58 %) with a putative α/β hydrolase from Acidiphilium sp. (ZP_08632277.1). Sequence alignments and phylogenetic analysis indicated that 499EST is a new member of the bacterial esterase/lipase family IV. The esterase reveals its optimum catalytic activity at 55 °C and pH 9.0. Kinetic studies showed that 499EST preferentially hydrolyzed middle-length acyl chains (C6-C8), especially p-nitrophenyl (p-NP) caproate (C6). Its thermostability and activity were strongly enhanced by adding 6 mM FeCl3. High stability in the presence of water-miscible solvents such as dimethyl sulfoxide and glycerol was observed. This enzyme also exhibits stability under harsh environmental conditions and enantioselectivity towards naproxen and ibuprofen esters, yielding the medically relevant (S)-enantiomers. In conclusion, according to our knowledge, 499EST is the first thermoalkalostable esterase derived from a Gram-negative thermoacidophilic bacterium

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

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

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