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2017

(34) R.Wardenga (2017)
Biocatalytic access to a novel class of Mannich catalysts
Manufacturing Chemist, October 2017, pp 52-53

Abstract

Abstract:
Enzymicals AG highlights the possibility to synthesize novel diastereomers of 5-benzyl-C-methyl--proline by stereoselective hydrolysis of branched malonate diesters. Application of recombinant pig liver esterase isoenzymes was the key to success to gain the (3S,5S)-diastereomer which shows activity as anti-Mannich catalyst.

(33) B. Schoenenberger, A. Wszolek, R. Meier, H. Brundiek, M. Obkircher,and R. Wohlgemuth (2017),
Biocatalytic asymmetric Michael addition reaction of L-arginine to fumarate for the green synthesis of N-(([(4S)-4-amino-4-carboxy-butyl]amino) iminomethyl)-L-aspartic acid lithium salt (L-argininosuccinic acid lithium salt)
RCS-Advances, 2017, 7, 48952; DOI: 10.1039/c7ra10236d

Abstract

Abstract:
The basic natural amino acid L-argininosuccinate containing two chiral centers occurs in L-alanine, L-arginine, L-aspartate, L-glutamate and L-proline metabolic pathways and plays a role in the biosynthesis of secondary metabolites and other amino acids. It is a precursor for arginine in the urea cycle or the citrulline–NO cycle as well as a precursor to fumarate in the citric acid cycle via argininosuccinate lyase. We aimed to run part of the urea cycle in reverse by catalyzing not the elimination but the addition reaction of L-arginine to fumarate in order to synthesize L-argininosuccinate. Argininosuccinate lyase (ASL) from Saccharomyces cerevisiae has been chosen as the catalyst for this addition reaction. The selected ARG4 gene was synthesized and homogeneously expressed in E. coli leading to a highly active argininosuccinate lyase. The ASL-catalyzed addition reaction of L-arginine to fumarate has been successfully developed at gram scale. After a standard workup procedure the pure final product L-argininosuccinate has been isolated in good yield and high purity.

(32) Kotapati H.K., Robinson J., Lawrence D., Fortner K., Stanford C., Powell D., Wardenga R., Bornscheuer U. (2017),
Diastereoselective hydrolysis of branched malonate diesters by Porcine Liver Esterase: Synthesis of 5-benzyl substituted Cα-methyl-β-proline and catalytic evaluation,
European Journal of Organic Chemistry, DOI: 10.1002/ejoc.201700605

Abstract

Abstract:
Malonate diesters with highly branched side chains containing a preexisting chiral center were prepared from optically pure amino alcohols and subjected to asymmetric enzymatic hydrolysis by Porcine Liver Esterase (PLE). Recombinant PLE isoenzymes have been utilized in this work to synthesize diastereomerically enriched malonate half-esters from enantiopure malonate diesters. The diastereomeric excess of the product half-esters was further improved in the later steps of synthesis either by simple recrystallization or flash column chromatography. The diastereomerically enriched half-ester was transformed into a novel 5-substituted Cα-methyl-β-proline analogue (3R, 5S)-1c, in high optical purity employing a stereoselective cyclization methodology. This β-proline analogue was tested for activity as a catalyst of the Mannich reaction. The β-proline analogue derived from the hydrolysis reaction by the crude PLE appeared to catalyze the Mannich reaction between an α-imino ester and an aldehyde providing decent to good diastereoselectivities. However, the enantioselectivities in the reaction was low. The second diastereomer of the 5-benzyl substituted Cα-methyl-β-proline, (3S, 5S)-1c was prepared by enzymatic hydrolysis using PLE isoenzyme 3 and tested for its catalytic activity in the Mannich reaction. Amino acid, (3S, 5S)-1c catalyzed the Mannich reaction between isovaleraldehyde and an α-imino ester yielding the "anti" selective product with an optical purity of 99%ee.

(31) Wardenga R., Rother D. (2017)
Efficient Chiral Chemistry by Application of Stereoselective Biocatalysts in Micro-Aqueous Reaction Systems, Speciality Chemicals Magazine, volume 31, issue 01, February 2017, pages 16-17

Abstract

Abstract:
Dr Rainer Wardenga of Enzymicals and Dr Dörte Rother of Forschungszentrum Jülich discuss the applicability of diverse enzymes in micro-aqueous reaction systems, enabling the conversion of hydrophobic or water-unstable substrates while maintaining the stereoselectivity of the biocatalysts.
2016

(30) Schoenenberger B., Wszolek A., Milesi T., Obkircher M., Brundiek H., Wohlgemuth R. (2016),
Synthesis of Nω-Phospho-L-arginine by Biocatalytic Phosphorylation of L-Arginine, Chemcatchem, 10.1002/cctc.201601080, Volume 9, Issue 1, January 9, 2017, Pages 121–126

Abstract

Abstract:
The Nω-Phospho-L-arginine energy-buffering system is mainly present in invertebrates for regulating energy requirements when it is highly needed, as in the flight muscle of an insect or when energy supply fluctuates, as in the medically important protozoa Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. The lacking availability of this important metabolite was due to a tedious chemical procedure, by which Nω-phospho-L-arginine was prepared up to now over 5 reaction steps in a low yield. Therefore, we aimed at improving the synthetic methodology for the preparation of this important metabolite. As site- and enantioselective kinases have been very useful catalysts for biocatalytic phosphorylations in straightforward syntheses of phosphorylated metabolites, a stable and selective arginine kinase has been selected for the selective phosphorylation of L-arginine. The arg gene has been cloned and expressed in E.coli and a highly active arginine kinase has been prepared. A simple synthesis of Nω-phospho-L-arginine has been developed by arginine kinase-catalyzed phosphorylation of L-arginine combined with the recycling of the phosphorylating agent ATP using the phosphoenolpyruvate/pyruvate kinase system. After standard workup the desired product Nω-Phospho-L-arginine has been obtained in gram quantities and in one step.

(29) Sehl T., Bock S., Marx L., Maugeri Z., Walter L., Westphal R., Vogel C., Menyes U., Erhardt M., Muller M., Pohl M., Rother D. (2016),
Asymmetric synthesis of (S) phenylacetylcarbinol – closing a gap in C-C bond-formation, Green Chemistry, Green Chem., 2017,19, 380-384, DOI: 10.1039/C6GC01803C

Abstract

Abstract:
(S)-Phenylacetylcarbinol [(S)-PAC] and its derivatives are valuable intermediates for the synthesis of various APIs (active pharmaceutical ingredients), however their selective synthesis is challenging. As no highly selective enzymes or chemical catalysts for the stereoselective synthesis of (S)-PAC were available, we tailored a potent biocatalyst by semi-rational enzyme engineering to a stereoselectivity of >97 % for the (S)-PAC synthesis by enzyme design. Together with reaction- and process optimisation industrially relevant product concentrations >48 g/L (up to 320 mM) could be gained. In addition, the best enzyme variant gave access to a broad range of ring-substituted (S)-PAC derivatives with high stereoselectivity, especially for meta-substituted products.

(28) Hinze J., Süss P., Strohmaier S., Bornscheuer U. T., Wardenga R., v. Langermann J. (2016)
Recombinant Pig Liver Esterase-Catalyzed Synthesis of (1S,4R)-4-Hydroxy-2-cyclopentenyl Acetate Combined with Subsequent Enantioselective Crystallization, DOI: 10.1021/acs.oprd.6b00093, Org. Process Res. Dev. 2016, 20, 1258−1264

Abstract

Abstract:
Recombinant Pig Liver Esterase-Catalyzed Synthesis of (1S,4R)-4-Hydroxy-2-cyclopentenyl Acetate Combined with Subsequent Enantioselective Crystallization

The recombinant pig liv­er esterase cat­alyzed hydrol­ysis of cis-1,4-diacetoxy-2-cyclopentene form­ing (1S,4R)-4-hydroxy-2-cyclopentenyl ac­etate was inves­tigated and re­alized at prepar­ative scale. Rel­evant re­action con­ditions were exam­ined and opti­mized to achieve full conver­sion with an enantiomeric excess of about 86% ee. Enantiopure prod­uct was then obtained af­ter enantios­e­lective crystallization, which required fur­ther stud­ies of the sol­id phase behav­ior, including its bina­ry melting point phase diagram.

(27) Zorna K., Oroz-Guinea I., Brundiek H., Bornscheuer U. T. (2016)
Engineering and Application of Enzymes for Lipid Modification, an Update, Progress in Lipid Research, 2016 Jul;63:153-64. DOI:10.1016/j.plipres.2016.06.001

Abstract

Abstract:
This review first pro­vides a brief introduction into the most important tools and strate­gies for pro­tein en­g­i­neering (i.e. di­rected evolution and ratio­nal pro­tein design combined with high-throughput screening meth­ods) fol­lowed by examples from lit­era­ture, in which enzymes have been opti­mized for biocat­alyt­ic applications. This covers en­g­i­neered lipases with al­tered fatty acid chain length se­lectiv­ity, fatty acid specificity and improved performance in es­terification re­actions. Fur­thermore, re­cent achieve­ments reported for phos­pho­lipases, lipoxygenases, P450 monooxygenases, decarboxylating enzymes, fatty acid hydratases and the use of enzymes in cascade re­actions are treated.

(26) Brundiek H.., Höhne M. (2016) "Transaminases–A Biosynthetic Route for Chiral Amines." in: Applied Biocatalysis: From Fundamental Science to Industrial Applications, Wiley-VCH, ISBN 9783527336692

Abstract

Abstract:
No abstract available


(25) Gand M., Thöle Ch., Müller H., Brundiek H., Bashiric G., Höhne M. (2016)
A NADH-accepting imine reductase variant - im¬mobi¬lization and cofactor regeneration by oxidative deam¬ination, Journal of Biotechnology, Volume 230, 20 July 2016, Pages 11–18.

Abstract

Abstract:
En­g­i­neering cofactor specificity of enzymes is a promis­ing approach that can expand the application of enzymes for biocat­alyt­ic production of indus­trially rel­evant chem­icals. Until now, only NADPH-dependent imine reductases (IREDs) are known. This lim­its their applications to re­actions employing whole cells as a cost-ef­ficient cofactor regeneration system. For applications of IREDs as cell-free cat­a­lysts, (i) we cre­ated an IRED variant showing an improved activ­ity for NADH. With ratio­nal design we were able to identi­fy four residues in the (R)-se­lective IRED from Strepto­myces GF3587 (IR-Sgf3587), which co­or­dinate the 2′-phos­phate moiety of the NADPH cofactor. From a set of 15 variants, the high­est NADH activ­ity was caused by the single am­ino acid exchange K40A resul­ting in a 3-fold increased acceptance of NADH. (ii) We showed its applicability us­ing an im­mobilisate obtained ei­ther from pu­ri­fied enzyme or from lysate us­ing the EziG™ carri­ers. Applying the variant and NADH, we reached 88% conver­sion in a prepar­ative scale biotransformation when employing 4% (w/v) 2-methylpyrro­line. (iii) We demonstrated a one-enzyme cofactor regeneration approach us­ing the achiral am­ine N-methyl-3-am­inopentanone as a hydrogen donor co-substrate.
2015

(24) Süss P., Borchert S., Hinze J., Illner S., v.Langermann J., Kragl U.m Bornscheuer U.T., Wardenga R. (2015) Chemoenzymatic Sequential Multistep One-Pot Reaction for the Synthesis of (1S,2R)-1-(Methoxycarbonyl)cyclohex-4-ene-2-carboxylic Acid with Recombinant Pig Liver Esterase, Org. Process Res. Dev., 2015, 19 (12), pp 2034–2038, DOI: 10.1021/acs.oprd.5b00294

Abstract

Abstract:

Sequential Multistep One-pot Reaction

In this work, the devel­op­ment of a chemoenzymat­ic process for the production of (1S,2R)-1-(methoxycarbonyl)cyclohex-4-ene-2-carboxylic acid by ECS-PLE06 (recombinant pig liv­er esterase) is presented. Here­in an opti­mized es­terification pro­tocol, starting from the eco­nom­ically fa­vored meso-anhydride 1b, was estab­lished and combined with the high­ly se­lective ECS-PLE06-cat­alyzed desymmetrization re­action. Both re­actions proceed under mild re­action con­ditions, and the ex­cel­lent se­lectiv­ity of ECS-PLE06 results in an enantiomeric excess of ≥99.5% ee. Fur­thermore, the presented one-pot synthesis fa­cili­tates high­er isolated yields of up to 84% in compari­son to the classical two-step synthesis route (70% isolated yield), which requires more expensive starting ma­te­rials and two sep­a­rate workup steps.

(23) Kohls H., Sowa M., Anderson M., Dickerhoff J., Weisz K., Córdova A.; Berglund P., Bornscheuer U.T., Brundiek H., Höhne M. (2015) Selective Access to All Four Diastereomers of a 1,3-Amino Alcohol by Combination of a Keto Reductase- and an Amine Transaminase-Catalysed Reaction, Advanced Synthesis & Catalysis, 357 (8), 1808–1814

Abstract

Abstract:
Keywords: amine transaminase; amino alcohols; enzyme catalysis; keto reductase

Synthesis strategy

The biocat­alyt­ic synthesis of chiral am­ines has become a val­uable addition to the chemists’ toolbox. Howev­er, the ef­ficient asym­met­ric synthesis of functionalised am­ines bearing more than one stereo­centre, such as 1,3-am­ino alcohols, remains chal­leng­ing. By employing a keto reductase (KRED) and two enanti­ocomple­mentary am­ine transam­inases (ATA), we devel­oped a biocat­alyt­ic route to­wards all four diastereomers of 4-am­ino-1-phenylpentane-2-ol as a rep­resentative molecule bearing the 1,3-am­ino alcohol functionality. Starting from a racemic hydroxy ketone, a ki­net­ic res­olution us­ing an (S)-se­lective KRED pro­vided optically active hydroxy ketone (86% ee) and the corresponding diketone. Fur­ther transam­ination of the hydroxy ketone was performed by ei­ther an (R)- or an (S)-se­lective ATA, yield­ing the (2R,4R)- and (2R,4S)-1,3-am­ino alcohol diastereomers. The remain­ing two diastereomers were ac­cessible in two subsequent asym­met­ric steps: the diketone was reduced regio- and enantios­e­lectively by the same KRED, which yield­ed the (S)-config­ured hydroxy ketone. Eventually, the subsequent transam­ination of the crude prod­uct with (R)- and (S)-se­lective ATAs yield­ed the remain­ing (2S,4R)- and (2S,4S)-diastereomers, respectively.

(22) Müller J., Sowa M., Fredrich B., Brundiek H., Bornscheuer U.T., (2015) Enhancing the Acyltransferase Activity of Candida antarctica Lipase A by Rational Design, ChemBioChem, DOI: 10.1002/cbic.201500187

Abstract

Abstract:
Keywords: Acyltransferase; CAL-A; enzyme catalysis; ester synthesis; immobilization

Few lipases like the Can­dida antarctica lipase A (CAL-A) are known to pos­sess an acyltransferase activ­ity. This activ­ity enables the enzyme to synthesize fatty acid esters from nat­ural oils and alcohols even in the pres­ence of bulk wa­ter. Unfortunately, still fatty acids are formed in these re­actions as undesired side prod­uct. 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 inves­tigated concerning their acyltransferase/hydrolase activ­ity via var­ious biocat­alyt­ic approaches. Among the inves­tigated variants, the single mu­tant As­p122Leu showed a signif­icant decrease in the hydrolyt­ic activ­ity, reduc­ing the side prod­uct yield dur­ing acylation re­actions. As desired, this variant maintained process rel­evant fea­tures like pH-pro­file or thermostability similar to the wild-type.

(21) Schmidt S., Scherkus C., Muschiol J., Menyes U., Winkler T., Hummel W., Gröger H., Liese A., Herz H.G., Bornscheuer U.T.,
An Enzyme Cascade Synthesis of ε-Caprolactone and its Oligomers, Angew. Chem. Int. Ed. 2015, 54, 2784-2787

Abstract

Abstract:
Poly-ε-caprolactone (PCL) is currently produced only chemically on industrial scale in spite of the need for hazardous peracetic acid as oxidation reagent. Although Baeyer-Villiger monooxygenases (BVMO) allow in principle the enzymatic synthesis of ε-caprolactone (ε-CL) directly from cyclohexanone with molecular oxygen, current systems suffer from low productivity and entail substrate and product inhibition. In this work, we overcame major limitations for such a biocatalytic route to produce this bulk chemical by combining an alcohol dehydrogenase with a BVMO to enable an efficient oxidation of cyclohexanol to ε-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ-formed ε-CL in an aqueous phase using lipase A from Candida antarctica, thus solving efficiently the product inhibition problem and leading to formation of oligo-ε-CL at >20 g/L when starting from 200 mm cyclohexanol. This oligomer could easily be polymerized chemically to PCL.
2014
(20) Scherkus C., Liese A., Gröger H., Kragl U., Bornscheuer U. T., Menyes U., (2014) Prozessentwicklung zur enzymatischen Synthese eines biologisch abbaubaren Polymers, Chemie Ingenieur Technik, 86 (9), 1424–1425

Abstract

Abstract:
No abstract available


(19) Gand M., Müller H., Wardenga R., Höhne M.,(2014)
Characterization of three novel enzymes with imine reductase activity, Journal of Molecular Catalysis B: Enzymatic, 110, 126-132.

Abstract

Abstract:
Imine reductases (IRED) are promising catalysts for the synthesis of optically pure secondary cyclic amines. Three novel IREDs from Paenibacillus elgii B69, Streptomyces ipomoeae 91-03 and Pseudomonas putida KT2440 were identified by amino acid or structural similarity search, cloned and recombinantly expressed in E. coli and their substrate scope was investigated. Beside the acceptance of cyclic amines, also acyclic amines could be identified as substrates for all IREDs. For the IRED from Pseudomonas putida, a crystal structure (PDB-code 3L6D) is available in the database, but the function of the protein was not investigated so far. This enzyme showed the highest apparent E-value of approximately Eapp=52 for (R) methylpyrrolidine of the IREDs investigated in this study. Thus, an excellent enantiomeric purity of >99%eeP and 97% conversion was reached in a biocatalytic reaction using resting cells after 24h. Interestingly, a histidine residue could be confirmed as a catalytic residue by mutagenesis, but the residue is placed one turn aside compared to the formally known position of the catalytic Asp187 of Streptomyces kanamyceticus IRED.


(18) Schallmey, M., Koopmeiners, J., Wells, E., Wardenga, R., Schallmey, A. (2014)
Expanding the halohydrin dehalogenase enzyme family: Identification of novel enzymes by database mining. Applied and environmental microbiology, 80(23), 7303-7315.

Abstract

Abstract:
Halohydrin dehalogenases are very rare enzymes which are naturally involved in the mineralization of halogenated xenobiotics. Due to their catalytic potential and promiscuity, many biocatalytic reactions have been described which have led to several interesting and also industrially important applications. Nevertheless, only a handful of these enzymes have been made available through recombinant techniques and hence it is of general interest to expand the repertoire of these enzymes to enable novel biocatalytic applications. After identification of specific sequence motifs, 37 novel enzyme sequences were readily identified in public sequence databases. All enzymes which 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 to previously known phylogenetic subtypes. Consequently, four additional phylogenetic subtypes were defined which expand the halohydrin dehalogenase enzyme family at large. We show that the enormous wealth of environmental and genome sequences present in public databases can be tapped for the in silico identification of very rare but nonetheless biotechnologically important biocatalysts. Our findings help to readily identify halohydrin dehalogenases in ever growing sequence databases and, in consequence, make even more members of this interesting enzyme family available to the scientific and industrial community.


(17) Süss, P., Illner, S., v. Langermann, J., Borchert, S., Bornscheuer, U.T., Wardenga, R., Kragl, U. (2014)
Scale-Up of a Recombinant Pig Liver Esterase-Catalyzed Desymmetrization of Dimethyl Cyclohex-4-ene-cis-1,2-dicarboxylate. Org. Process Res. Dev., 18, 897-903

Abstract

Abstract:
Synthesis strategy

A recombinant isoenzyme of pig liver esterase was used for the highly enantioselective desymmetrization of dimethyl cyclohex-4-ene-cis-1,2-dicarboxylate. The selected recombinant esterase showed a significant advantage in enantioselectivity over the commonly used esterase from the mammalian source. The process was scaled up to yield 265 g of product with a simplified pH control, and the target molecule was obtained with an enantiopurity of >99.5% ee.


(16) Sehl, T., Hailes, C. H., Ward, J. M., Menyes, U., Pohl, M., Rother, D. (2014)
Efficient 2-step biocatalytic strategies for the synthesis of all nor(pseudo)ephedrine isomers. Green Chem., 2014, 16, 3341-3348

Abstract

Abstract:
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.

Synthesis strategy
2013
(15) Schallmey, A., Schallmey, M., Wardenga R. (2013)
Identifikation neuartiger Halohydrin-Dehalogenasen. Biospektrum, 19 (7), 816-817.

Abstract

Abstract:
Halohydrin dehalogenases (HHDHs) are biotechnologically relevant enzymes that can be applied as biocatalysts for the selective synthesis of various β-substituted alcohols. Despite that fact, only very few HHDHs are currently available. In an attempt to identify novel ones, database mining of publicly available sequence databases was performed using HHDH-specific sequence information. As a result, 19 novel HHDHs were obtained all exhibiting true HHDH activity.
(14) Wardenga, R., Bednarczyk, A., Höhne, M. (2013),
Asymmetric synthesis of chiral amines from ketones. How to apply biocatalysis and find a suitable enzyme. PharmaChem, 12, 22-25.

Abstract

Abstract:
Optically active amines play an important role in the pharmaceutical, agrochemical, and chemical industries. They are frequently used as synthons for the preparation of various pharmaceutically active substances. Consequently, there is a need for efficient methods to obtain the desired enantiomer of a given target structure in an optically pure form. Beside a range of chemical methods using for example, asymmetric synthesis with transition metal catalysts, enzymes represent a useful alternative to access this important class of compounds. This article focusses on the biocatalytic transaminase approach with emphasis on how to screen for suitable catalysts for the asymmetric synthesis starting from prostereogenic ketones.

(13) Sehl, T., Hailes, H. C., Ward, J. M., Wardenga, R., von Lieres, E., Offermann, H., Westphal, R., Pohl, M., Rother, D., (2013)
Two Steps in One Pot: Enzyme Cascade for the Synthesis of Nor(pseudo)ephedrine from Inexpensive Starting Materials. Angewandte Chemie International Edition vol. 52 (26) 6772-6775.

Abstract

Keywords:
  • asymmetric synthesis;
  • biocatalysis;
  • enzyme cascades;
  • phenylpropanolamine;
  • ω-transaminase
Two steps in one pot

Two steps in one pot: An enzyme cascade consisting of a lyase and an (R)- or (S)-selective ω-transaminase (TA) provides (1R,2R)-norpseudoephedrine and (1R,2S)-norephedrine in only two steps. The intermediate is not isolated in this one-pot reaction and the products are obtained in high enantio- and diastereomeric purity. Moreover, the by-product from the second reaction can be recycled to serve as the substrate for the first reaction.

(12) Staudt, S., Bornscheuer, U.T., Menyes, U., Hummel, W., Gröger, H. (2013),
Direct biocatalytic one-pot-transformation of cyclohexanol with molecular oxygen into ε-caprolactone, Enzyme Microb. Technol., 53, 288-292.

Abstract

Abstract:
biocatalytic process concept for ɛ-caprolactone

The development of a biocatalytic process concept for ɛ-caprolactone, which directly converts cyclohexanol as an easily available industrial raw material into the desired ɛ-caprolactone in a one-pot fashion while only requiring air as sole reagent, is reported. The desired product ɛ-caprolactone was obtained with 94–97% conversion when operating at a substrate concentration in the range of 20–60 mM. At higher substrate concentrations, however, a significant drop of conversion was found. Subsequent detailed studies on the impact of the starting material, intermediate and product components revealed a significant inhibition and partial deactivation of the BVMO by the product ɛ-caprolactone (in particular at higher concentrations) as well as an inhibition of the BVMO by cyclohexanol and cyclohexanone.

(11) Mallin, H., Menyes, U., Vorhaben, T., Höhne, M., Bornscheuer, U.T. (2013),
Immobilization of two (R)-amine transaminases on an optimized chitosan support for the enzymatic synthesis of optically pure amines, ChemCatChem, 5, 588-593.

Abstract

Abstract:
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.
2012

(10) Smith, M.E., Banerjee, S., Shi, Y., Schmidt, M., Bornscheuer, U.T. Masterson, D.S. (2012),
Investigation of the cosolvent effect on six isoenzymes of PLE in the enantioselective hydrolysis of selected a,a-disubstituted malonate esters, ChemCatChem, 4, 472-475

Abstract

Abstract:
Keywords:
  • cosolvent effects;
  • enantioselectivity;
  • enzymes;
  • inversion of chirality;
  • synthesis
biocatalytic process concept for ɛ-caprolactone

Six pigs in a pot: Pig liver esterase (PLE) is among the most widely studied esterase enzymes utilized in organic synthesis. Here we illustrate that the six recombinantly produced isoenzymes of PLE exhibit varying enantioselectivity during the hydrolysis of α,α-disubstituted malonate esters in cosolvent mixtures. We have observed a rare cosolvent-induced reversal of enantioselectivity for isoenzyme PLE 6 in the hydrolysis of a phthalimide-containing α,α-disubstituted malonate ester.

(9) Brundiek, H., Padhi, S.K., Evitt, A., Kourist, R., Bornscheuer, U.T. (2012),
Altering the scissile fatty acid binding site of Candida antarctica lipase A by protein engineering for the selective hydrolysis of medium chain fatty acids, Eur. J. Lipid Sci. Technol., 114, 1148-1153.

Abstract

Abstract:
Candida antarctica lipase A (CAL-A) is the first representative of a new subclass of lipases because of its unique cap domain. The acyl-binding tunnel – having a short alternative binding region – is mainly formed by this domain. In order to create CAL-A variants with a high specificity for medium chain length (MCL) fatty acids (C6–C12), we used rational protein design to block the primary acyl-binding tunnel of CAL-A at position G237, which is near the junction to the alternative binding pocket. By closing the junction to the main tunnel, CAL-A variants (G237A/L/V/Y) have been created, which are highly specific for medium chain fatty acids (MCFAs) as determined by chain length profiles with p-nitrophenyl esters and triacylglycerides. Especially the CAL-A variants G237L/V/Y, in which the junction to the primary tunnel is completely closed, show a distinct preference for the hydrolysis of hexanoate esters. Hydrolytic activity for substrates with a chain length >C6 is suppressed extensively in mutants G237L/V/Y. Therefore, these highly MCL specific CAL-A variants may represent interesting biocatalysts for the production of MCL-derived esters for the food, flavor, and fragrance industry.

Practical application: Since medium chain fatty acids (MCFAs: C6-C10) and their corresponding triacylglycerides (MCTs) provide quick access to energy and have been considered to be less implicated in the accumulation of body fat than long chain fatty acids, they represent interesting food additives. As functional oils, they are part of weight loss diets or are used in clinical nutrition. In the food industry MCTs are also utilized as storage stabilizing agents in cooking products, as release agents in food processing or as flavor diluent. Another interesting field of application of MCFA derived compounds, especially of C6 esters and alcohols, is as ingredients in flavors and fragrances. The CAL-A variants described in this study can be used for the biocatalytic synthesis of these compounds.

(8) Brundiek, H., Sass, S., Evitt, A., Kourist, R., Bornscheuer, U.T. (2012),
The short form of the recombinant CAL-A-type lipase UM03410 from the smut fungus Ustilago maydis exhibits an inherent trans fatty acid selectivity, Appl. Microb. Biotechnol., 94, 141-150; erratum: 94, 285.

Abstract

Abstract:
The Ustilago maydis lipase UM03410 belongs to the mostly unexplored Candida antarctica lipase (CAL-A) subfamily. The two lipases with […] the highest identity are a lipase from Sporisorium reilianum and the prototypic CAL-A. In contrast to the other CAL-A-type lipases, this hypothetical U. maydis lipase is annotated to possess a prolonged N-terminus of unknown function. Here, we show for the first time the recombinant expression of two versions of lipase UM03410: the full-length form (lipUMf) and an N-terminally truncated form (lipUMs). For comparison to the prototype, the expression of recombinant CAL-A in E. coli was investigated. Although both forms of lipase UM03410 could be expressed functionally in E. coli, the N-terminally truncated form (lipUMs) demonstrated significantly higher activities towards p-nitrophenyl esters. The functional expression of the N-terminally truncated lipase was further optimized by the appropriate choice of the E. coli strain, lowering the cultivation temperature to 20 °C and enrichment of the cultivation medium with glucose. Primary characteristics of the recombinant lipase are its pH optimum in the range of 6.5–7.0 and its temperature optimum at 55 °C. As is typical for lipases, lipUM03410 shows preference for long chain fatty acid esters with myristic acid ester (C14:0 ester) being the most preferred one. More importantly, lipUMs exhibits an inherent preference for C18:1Δ9 trans and C18:1Δ11 trans-fatty acid esters similar to CAL-A. Therefore, the short form of this U. maydis lipase is the only other currently known lipase with a distinct trans-fatty acid selectivity.

(7) Brundiek, H.B., Evitt, A.S., Kourist, R., Bornscheuer, U.T. (2012),
Creation of a lipase highly selective for trans fatty acid by protein engineering, Angew. Chem. Int. Ed., 51, 412-414; Erzeugung einer für trans-Fettsäuren hochselektiven Lipase durch Protein-Engineering, Angew. Chem., 124, 425-428.

Abstract

Keywords:
  • enzyme catalysis;
  • fatty acids;
  • high-throughput screening;
  • lipases;
  • protein engineering
biocatalytic process concept for ɛ-caprolactone

Sorting out: Protein engineering of lipase CAL-A led to the discovery of mutants with excellent chemoselectivity for the removal of trans and saturated fatty acids from partially hydrogenated vegetable oil. These fatty acids, identified as a major risk factor for human health, can now be removed by enzyme catalysis.

(6) Leipold, F., Wardenga, R., Bornscheuer, U.T. (2012),
Cloning, expression and characterisation of an eukaryotic cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, Appl. Microb. Biotechnol., 94, 705-717.

Abstract

Abstract:
In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer–Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme.
2011-2008

(5) Vorhaben, T., Häckel, M., Bornscheuer, U.T., Menyes, U. (2011),
Plasmagestützte Immobilisierung von Enzymen, Nachrichten Chemie, 59, 1147-1149.

Abstract

Abstract:
Physikalische Plasmen modifizieren Oberflächen gezielt und umweltfreundlich. Mit funktionellen Gruppen entstehen so in wenigen Schritten Oberflächen mit maßgeschneiderten Eigenschaften, beispielsweise um Enzyme darauf zu fixieren.

(4) Vorhaben, T., Böttcher, D., Jasinski, D., Brüser, V., Menyes, U., Schröder, K., Bornscheuer, U.T. (2010),
Plasma-modified polypropylene as carrier for the immobilization of Candida antarctica lipase B and Pyrobaculum calidifontis esterase, ChemCatChem, 2, 992-996.

Abstract

Abstract:
In this work, the immobilization of two hydrolases on plasma-modified polypropylene carriers was investigated. Treating Accurel MP1001 with an oxygen plasma was found most suitable to increase the hydrophilicity and to allow for efficient immobilization. Thus, for lipase B from Candida antarctica and for an esterase from Pyrobaculum calidifontis esterase (PestE) a 5-fold and 14-fold increase, respectively, in immobilization yield resulted compared to untreated carrier. In contrast to the oxygen-modified support, modification of the polypropylene carrier with ammonia plasma showed no positive effect. Furthermore, it could be shown that immobilized PestE catalyzed enantioselective transesterification of α-phenylethanol in vinyl acetate, whereas the free enzyme showed no activity. Both hydrolases could be recycled five times without significant loss of activity.

(3) Wardenga, R., Lindner, H.A., Hollmann, F., Thum, O., Bornscheuer, U.T. (2010),
Increasing the synthesis/ hydrolysis ratio of aminoacylase-1 by site-directed mutagenesis, Biochimie, 92, 102-109.

Abstract

Abstract:
Aminoacylase-1 from pig kidney (pAcy1) catalyzes the highly stereoselective acylation of amino acids, a useful conversion for the preparation of optically pure N-acyl-l-amino acids. The kinetic of this thermodynamically controlled conversion is determined by maximal velocities for synthesis (VmS) and hydrolysis (VmH) of the N-acyl-l-amino acid. To investigate which parameter affects maximal velocities, we focused on the proton acceptor potential of the catalytic base, E146, and studied the influence of the active site architecture on its contribution to the pKa of residue E146. The modeled structure of pAcy1 identified residue D346 as having the strongest impact on the electrostatic features of the catalytic base. Substitutions of D346 generally decreased enzymatic activities but also altered both the pH-dependency of hydrolytic activity and the VmS/VmH ratio of pAcy1. A reduced theoretical pKa value and a lowered experimental pH optimum of hydrolytic rates for the D346A mutant were associated with a 9-fold increase in VmS/VmH. This supports the importance of electrostatic contributions of D346 to the acid-base properties of E146 and demonstrates for the first time the possibility of engineering the VmS/VmH ratio of pAcy1.

(2) Vahl, K., Kahlert, H., Böttcher, D., Wardenga, R., Komorsky-Lovric, S., Bornscheuer, U., Scholz, F. (2008),
A potential high-throughput method for the determination of lipase activity by potentiometric FIA titrations, Anal. Chem. Acta, 610, 44-49.

Abstract

Abstract:
Potentiometric FIA titrations were performed to determine enzyme activities of lipase type B from Candida antarctica, CAL-B. Two substrates, triacetin and tributyrin were hydrolyzed in phosphate buffer solutions, and the concentration change of the base component of the buffer was titrated in a carrier solution containing hydrochloric acid and potassium chloride. The system was calibrated with butyric acid and acetic acid, respectively. FIA titration peaks were evaluated with respect to peak height and peak area. Butyric acid and acetic acid could be titrated in the buffer solution from 3 × 10−3 mol L−1 to 0.1 mol L−1. The detection limit of enzyme activity was determined to be 0.07 U mL−1 (15 min reaction time) and the minimum activity was calculated to be 0.035 units corresponding to 35 nmol min−1. The specific activities of lipase B for the hydrolysis of tributyrin and triacetin were determined as 16 ± 2 U mg−1 and 2 ± 0.2 U mg−1 (per mg commercial lipase preparation), respectively.

(1) Wardenga, R., Hollmann, F., Thum, O., Bornscheuer, U.T. (2008),
Functional expression of porcine aminoacylase 1 in E. coli using a codon optimized synthetic gene and molecular chaperones, Appl. Microbiol. Biotechnol., 81, 721-729.

Abstract

Abstract:
Efficient recombinant expression of N-acyl-l-aminoacylase 1 from pig kidney (pAcy1) was achieved in the prokaryotic host Escherichia coli. An optimized nucleotide sequence (codon adaptation index 0.95 for E. coli), was cloned into vector pET-52(b) yielding an E. coli-expressible pAcy1 gene. Formation of inclusion bodies was alleviated by co-expression of molecular chaperones resulting in 2.7- and 4.2-fold increased recovery of active pAcy1 using trigger factor or GroEL–GroES, respectively. Facile purification was achieved via StrepTag affinity chromatography. Overall, more than 80 mg highly active pAcy1 (94 U/mg) was obtained per liter of cultivation broth. The protein was analyzed for structural and functional identity, and the performances of further described expression and purification systems for pAcy1 were compared.