Christian Elend’s research while affiliated with BRAIN AG and other places

The present invention relates to a polynucleotide encoding an enzyme having carboxyl esterase [E.C. 2.1.1.1] activity.

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90 degrees C and between pH 6.5 and 10; the half-life of the enzymes at 75 degrees C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53-2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.

Bereits seit den Ursprüngen der Menschheit nutzt der Mensch Mikroorganismen in Produktionsprozessen, ohne sich deren Existenz bewusst gewesen zu sein; beispielsweise kommen in der Lebensmittelherstellung Hefen (z. B. Bier, Wein), Schimmelpilze (z. B. Käse) oder Bakterien (z. B. Joghurt, Kefir) zum Einsatz. Die Existenz der jeweiligen Mikroorganismen und die von ihnen realisierten Stoffwechselvorgänge wurden jedoch erst in den letzten 150 Jahren intensiv erforscht und bis auf die molekulare Ebene aufgeklärt. Als erster konnte Louis Pasteur 1860 die Rolle der Hefe für die alkoholische Gärung nachweisen und zeigen, dass die Gärung von lebenden Zellen abhängig ist. Im 20. Jahrhundert wurden Mikroorganismen dann vielfältig in industriellen Produktionsprozessen (z. B. zur Herstellung von Aminosäuren, Antibiotika oder Feinchemikalien) genutzt. Vor diesem Hintergrund ist es das Ziel der folgenden Ausführungen (vgl. /14-2/, /14-4/, /14-6/, /14-9/, /14-8/, /14-10/, /14-11/, /14-12/, /14-13/, /14-14/), einen Überblick über die Vielfalt der Mikroorganismen, deren Zellaufbau und Stoffwechsel sowie ihre vielfältigen Anwendungsmöglichkeiten u. a. zur Erzeugung energiereicher Verbindungen aus organischen Stoffen zu geben. Gleichzeitig wird aufgezeigt, wo dem Einsatz lebender Zellen und Enzyme als Biokatalysatoren Grenzen gesetzt sind und welche verfahrenstechnischen Konsequenzen daraus resultieren.

We report on the isolation and biochemical characterization of a novel, cold-active and metagenome-derived lipase with a high stereo-selectivity for pharmaceutically important substrates. The respective gene was isolated from a cosmid library derived from oil contaminated soil and designated lipCE. The deduced aa sequence indicates that the protein belongs to the lipase family l.3, with high similarity to Pseudomonas fluorescens lipases containing a C-terminal secretion signal for ABC dependent transport together with possible motifs for Ca(2+)-binding sites. The overexpressed protein revealed a molecular weight of 53.2kDa and was purified by refolding from inclusion bodies after expression in Escherichia coli. The optimum temperature of LipCE was determined to be 30 degrees C. However, the enzyme still displayed 28% residual activity at 0 degrees C and 16% at -5 degrees C. Calcium ions strongly increased activity and thermal stability of the protein. Further detailed biochemical characterization of the recombinant enzyme showed an optimum pH of 7 and that it retained activity in the presence of a range of metal ions and solvents. A detailed analysis of the enzyme's substrate spectrum with more than 34 different substrates indicated that the enzyme was able to hydrolyze a wide variety of substrates including the conversion of long chain fatty acid substrates with maximum activity for pNP-caprate (C(10)). Furthermore LipCE was able to hydrolyze stereo-selectively ibuprofen-pNP ester with a high preference for the (R) enantiomer of >91% ee and it demonstrated selectivity for esters of primary alcohols, whereas esters of secondary or tertiary alcohols were nearly not converted.

The metagenomes of uncultured microbial communities are rich sources for novel biocatalysts. In this study, esterase EstA3 was derived from a drinking water metagenome, and esterase EstCE1 was derived from a soil metagenome. Both esterases are approximately 380 amino acids in size and show similarity to β-lactamases, indicating that they belong to family VIII of the lipases/esterases. EstA3 had a temperature optimum at 50°C and a pH optimum at pH 9.0. It was remarkably active and very stable in the presence of solvents and over a wide temperature and pH range. It is active in a multimeric form and displayed a high level of activity against a wide range of substrates including one secondary ester, 7-[3-octylcarboxy-(3-hydroxy-3-methyl-butyloxy)]-coumarin, which is normally unreactive. EstCE1 was active in the monomeric form and had a temperature optimum at 47°C and a pH optimum at pH 10. It exhibited the same level of stability as EstA3 over wide temperature and pH ranges and in the presence of dimethyl sulfoxide, isopropanol, and methanol. EstCE1 was highly enantioselective for (+)-menthylacetate. These enzymes display remarkable characteristics that cannot be related to the original environment from which they were derived. The high level of stability of these enzymes together with their unique substrate specificities make them highly useful for biotechnological applications.