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Greifswald, Germany

Muller J.,University of Greifswald | Sowa M.A.,University of Greifswald | Fredrich B.,University of Greifswald | Brundiek H.,Enzymicals AG | Bornscheuer U.T.,University of Greifswald

A few lipases, such as Candida antarctica lipase A (CAL-A), are known to possess acyltransferase activity. This enables the enzyme to synthesize fatty acid esters from natural oils and alcohols even in the presence of bulk water. Unfortunately, fatty acids are still formed in these reactions as undesired side-products. To reduce the amount of fatty acids, several CAL-A variants were rationally designed based on its crystal structure. These variants were expressed in Escherichia coli and Pichia pastoris, purified, and their acyltransferase/hydrolase activities were investigated by various biocatalytic approaches. Among the investigated variants, mutant Asp122Leu showed a significant decrease in the hydrolytic activity, thus reducing the side-product yield during acylation. As desired, this variant retained wild-type process-relevant features like pH profile and thermostability. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Schmidt S.,University of Greifswald | Scherkus C.,TU Hamburg - Harburg | Muschiol J.,University of Greifswald | Menyes U.,Enzymicals AG | And 6 more authors.
Angewandte Chemie - International Edition

Poly-ε-caprolactone (PCL) is chemically produced on an industrial scale in spite of the need for hazardous peracetic acid as an oxidation reagent. Although Baeyer-Villiger monooxygenases (BVMO) in principle enable the enzymatic synthesis of ε-caprolactone (ε-CL) directly from cyclohexanone with molecular oxygen, current systems suffer from low productivity and are subject to substrate and product inhibition. The major limitations for such a biocatalytic route to produce this bulk chemical were overcome by combining an alcohol dehydrogenase with a BVMO to enable the efficient oxidation of cyclohexanol to ε-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ formed ε-CL in the aqueous phase by using lipase A from Candida antarctica, thus efficiently solving the product inhibition problem and leading to the formation of oligo-ε-CL at more than 20 gL-1 when starting from 200 mM cyclohexanol. This oligomer is easily chemically polymerized to PCL. Let's polymerize! Oligo-ε-caprolactone was produced in a one-pot enzymatic cascade synthesis starting from cyclohexanol. In the first step, cyclohexanol is oxidized by an alcohol dehydrogenase (ADH) in combination with the cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus, followed by direct ring-opening oligomerization of ε-caprolactone in an exclusively aqueous phase by lipase A from Candida antarctica (CAL-A). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Schallmey M.,RWTH Aachen | Koopmeiners J.,RWTH Aachen | Wells E.,RWTH Aachen | Wardenga R.,Enzymicals AG | And 2 more authors.
Applied and Environmental Microbiology

Halohydrin dehalogenases are very rare enzymes that are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described that have led to several interesting and industrially important applications. Nevertheless, only a few of these enzymes have been made available through recombinant techniques; hence, it is of general interest to expand the repertoire of these enzymes so as to enable novel biocatalytic applications. After the identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes that could be heterologously expressed also catalyzed typical halohydrin dehalogenase reactions. Phylogenetic inference for enzymes of the halohydrin dehalogenase enzyme family confirmed that all enzymes form a distinct monophyletic clade within the short-chain dehydrogenase/reductase superfamily. In addition, the majority of novel enzymes are substantially different from previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined, greatly expanding the halohydrin dehalogenase enzyme family. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for in silico identification of very rare but biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever-growing sequence databases and, as a consequence, make even more members of this interesting enzyme family available to the scientific and industrial community. © 2014, American Society for Microbiology. Source

Sehl T.,Julich Research Center | Hailes H.C.,University College London | Ward J.M.,University College London | Menyes U.,Enzymicals AG | And 2 more authors.
Green Chemistry

Chiral 1,2-amino alcohols are important building blocks for chemistry and pharmacy. Here, we developed two different biocatalytic 2-step cascades for the synthesis of all four nor(pseudo)ephedrine (N(P)E) stereoisomers. In the first one, the combination of an (R)-selective thiamine diphosphate (ThDP)-dependent carboligase with an (S)- or (R)-selective ω-transaminase resulted in the formation of (1R,2S)-NE or (1R,2R)-NPE in excellent optical purities (ee >99% and de >98%). For the synthesis of (1R,2R)-NPE, space-time yields up to ∼26 g L-1 d-1 have been achieved. Since a highly (S)-selective carboligase is currently not available for this reaction, another strategy was followed to complement the nor(pseudo)ephedrine platform. Here, the combination of an (S)-selective transaminase with an (S)-selective alcohol dehydrogenase yielded (1S,2S)-NPE with an ee >98% and a de >99%. Although lyophilized whole cells are cheap to prepare and were shown to be appropriate for use as biocatalysts, higher optical purities were observed with purified enzymes. These synthetic enzyme cascade reactions render the N(P)E-products accessible from inexpensive, achiral starting materials in only two reaction steps and without the isolation of the reaction intermediates. This journal is © the Partner Organisations 2014. Source

Mallin H.,University of Greifswald | Menyes U.,Enzymicals AG | Vorhaben T.,Neoplas GmbH | Hohne M.,University of Greifswald | Bornscheuer U.T.,University of Greifswald

Two (R)-selective amine transaminases from Gibberella zeae (GibZea) and from Neosartorya fischeri (NeoFis) were immobilized on chitosan as a carrier to improve their application in the biocatalytic synthesis of chiral (R)-amines. An (S)-selective enzyme from Vibrio fluvialis (VfTA) was used for comparison. After improving the immobilization conditions, all enzymes could be efficiently immobilized. Additionally, the thermal stability of GibZea and NeoFis could be improved and also a slight shift of the pH optimum was observed for GibZea. All enzymes showed good activity in the conversion of α-methylbenzylamine. In the asymmetric synthesis of (R)-2-aminohexane from the corresponding ketone, a 13.4-fold higher conversion (>99%) was found for the immobilized GibZea compared to the free enzyme. Hence, the covalent binding with glutaraldehyde of these enzymes on chitosan beads resulted in a significant stabilization of the amine transaminases investigated. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

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