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PUBLICATIONS
2015
Schmidt, Sandy; Scherkus, Christian; Muschiol, Jan; Menyes, Ulf; Winkler, Till; Hummel, Werner; Gröger, Harald; Liese, Andreas; Herz, Hans Georg; Bornscheuer, Uwe T.
An enzyme cascade synthesis of epsilon-caprolactone and its oligomers Journal Article
In: Angewandte Chemie - International Edition, vol. 54, no. 9, pp. 2784–2787, 2015, ISSN: 15213773.
@article{Schmidt2015cb,
title = {An enzyme cascade synthesis of epsilon-caprolactone and its oligomers},
author = {Sandy Schmidt and Christian Scherkus and Jan Muschiol and Ulf Menyes and Till Winkler and Werner Hummel and Harald Gröger and Andreas Liese and Hans Georg Herz and Uwe T. Bornscheuer},
url = {http://doi.wiley.com/10.1002/anie.201410633},
doi = {10.1002/anie.201410633},
issn = {15213773},
year = {2015},
date = {2015-02-01},
urldate = {2015-02-01},
journal = {Angewandte Chemie - International Edition},
volume = {54},
number = {9},
pages = {2784--2787},
abstract = {Poly-$epsilon$-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 $epsilon$-caprolactone ($epsilon$-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 $epsilon$-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ formed $epsilon$-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-$epsilon$-CL at more than 20 g L-1 when starting from 200 mM cyclohexanol. This oligomer is easily chemically polymerized to PCL.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Poly-$epsilon$-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 $epsilon$-caprolactone ($epsilon$-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 $epsilon$-CL. Key to success was a subsequent direct ring-opening oligomerization of in situ formed $epsilon$-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-$epsilon$-CL at more than 20 g L-1 when starting from 200 mM cyclohexanol. This oligomer is easily chemically polymerized to PCL.
Schmidt, Sandy; Scherkus, Christian; Muschiol, Jan; Menyes, Ulf; Winkler, Till; Hummel, Werner; Gröger, Harald; Liese, Andreas; Herz, Hans-Georg; Bornscheuer, Uwe T
Eine Enzymkaskade zur Synthese von epsilon-Caprolacton und dessen Oligomeren Journal Article
In: Angewandte Chemie, vol. 127, no. 9, pp. 2825–2828, 2015, ISSN: 1521-3757.
@article{Schmidt2015,
title = {Eine Enzymkaskade zur Synthese von epsilon-Caprolacton und dessen Oligomeren},
author = {Sandy Schmidt and Christian Scherkus and Jan Muschiol and Ulf Menyes and Till Winkler and Werner Hummel and Harald Gröger and Andreas Liese and Hans-Georg Herz and Uwe T Bornscheuer},
doi = {10.1002/ange.201410633},
issn = {1521-3757},
year = {2015},
date = {2015-01-01},
journal = {Angewandte Chemie},
volume = {127},
number = {9},
pages = {2825--2828},
abstract = {In industriellem Maßstab wird Poly-epsilon-caprolacton (PCL) gegenwärtig nur chemisch produziert, wobei mit Peressigsäure ein gefährliches Reagens als Oxidationsmittel genutzt wird. Baeyer-Villiger-Monooxygenasen (BVMOs) ermöglichen im Prinzip die enzymatische Synthese von epsilon-Caprolacton (epsilon-CL) direkt ausgehend von Cyclohexanon mit molekularem Sauerstoff, doch gegenwärtige Systeme leiden unter niedriger Produktivität sowie Substrat- und Produktinhibierung. Wir überwanden wesentliche Limitationen eines solchen biokatalytischen Wegs durch die Kombination einer Alkoholdehydrogenase mit einer BVMO für die effiziente Oxidation von Cyclohexanol zu epsilon-CL. Entscheidend war die direkte Ringöffnungs-Oligomerisierung des in situ gebildeten epsilon-CL in wässriger Phase unter Nutzung der Lipase A aus Candida antarctica. So wurde das Problem der Produktinhibierung gelöst, und Oligo-epsilon-CL wurde mit >20 g L−1 ausgehend von 200 mM Cyclohexanol erhalten. Dieses Oligomer konnte chemisch leicht zu PCL polymerisiert werden.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In industriellem Maßstab wird Poly-epsilon-caprolacton (PCL) gegenwärtig nur chemisch produziert, wobei mit Peressigsäure ein gefährliches Reagens als Oxidationsmittel genutzt wird. Baeyer-Villiger-Monooxygenasen (BVMOs) ermöglichen im Prinzip die enzymatische Synthese von epsilon-Caprolacton (epsilon-CL) direkt ausgehend von Cyclohexanon mit molekularem Sauerstoff, doch gegenwärtige Systeme leiden unter niedriger Produktivität sowie Substrat- und Produktinhibierung. Wir überwanden wesentliche Limitationen eines solchen biokatalytischen Wegs durch die Kombination einer Alkoholdehydrogenase mit einer BVMO für die effiziente Oxidation von Cyclohexanol zu epsilon-CL. Entscheidend war die direkte Ringöffnungs-Oligomerisierung des in situ gebildeten epsilon-CL in wässriger Phase unter Nutzung der Lipase A aus Candida antarctica. So wurde das Problem der Produktinhibierung gelöst, und Oligo-epsilon-CL wurde mit >20 g L−1 ausgehend von 200 mM Cyclohexanol erhalten. Dieses Oligomer konnte chemisch leicht zu PCL polymerisiert werden.
Schmidt, Sandy; Genz, Maika; Balke, Kathleen; Bornscheuer, Uwe T.
The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase Journal Article
In: Journal of Biotechnology, vol. 214, pp. 199–211, 2015, ISSN: 18734863.
@article{Schmidt2015b,
title = {The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase},
author = {Sandy Schmidt and Maika Genz and Kathleen Balke and Uwe T. Bornscheuer},
doi = {10.1016/j.jbiotec.2015.09.026},
issn = {18734863},
year = {2015},
date = {2015-01-01},
journal = {Journal of Biotechnology},
volume = {214},
pages = {199--211},
abstract = {Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24 h incubation at 25. °C) showcasing a great achievement for practical applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24 h incubation at 25. °C) showcasing a great achievement for practical applications.
Schmidt, Sandy; Büchsenschütz, Hanna C.; Scherkus, Christian; Liese, Andreas; Gröger, Harald; Bornscheuer, Uwe T.
Biocatalytic Access to Chiral Polyesters by an Artificial Enzyme Cascade Synthesis Journal Article
In: ChemCatChem, vol. 7, no. 23, pp. 3951–3955, 2015, ISSN: 18673899.
@article{Schmidt2015ab,
title = {Biocatalytic Access to Chiral Polyesters by an Artificial Enzyme Cascade Synthesis},
author = {Sandy Schmidt and Hanna C. Büchsenschütz and Christian Scherkus and Andreas Liese and Harald Gröger and Uwe T. Bornscheuer},
doi = {10.1002/cctc.201500823},
issn = {18673899},
year = {2015},
date = {2015-01-01},
journal = {ChemCatChem},
volume = {7},
number = {23},
pages = {3951--3955},
abstract = {Chiral polyesters in general can be employed for versatile biomedical purposes, but in vitro enzyme catalyzed biocatalytic routes by a multiple-step cascade to make these functional biodegradable chiral polyesters have been hardly investigated. Recently, we developed an artificial three-step enzymatic cascade synthesis by combining an alcohol dehydrogenase (ADH), a Baeyer-Villiger monooxygenase (BVMO) and a lipase (CAL-A). Here, we extended this cascade for the synthesis of chiral methyl-substituted oligo-$epsilon$-caprolactone derivatives to achieve both, the generation of chirality in a monomer and the subsequent polymerization. Several substrates were examined and provided access to functionalized chiral compounds in high yields (up to >99 %) and optical purities (up to >99 % ee). By subsequent enzymatic enantioselective ring opening of the enantiopure monomers, oligomeric lactones were successfully synthesized. Rotate me! By combining three enzymes (LK-ADH, CHMO, CAL-A), which are evolutionary not connected, to an artificial cascade, optically active chiral oligomers can be synthesized from nonchiral or racemic methyl-substituted cyclohexanol derivatives.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chiral polyesters in general can be employed for versatile biomedical purposes, but in vitro enzyme catalyzed biocatalytic routes by a multiple-step cascade to make these functional biodegradable chiral polyesters have been hardly investigated. Recently, we developed an artificial three-step enzymatic cascade synthesis by combining an alcohol dehydrogenase (ADH), a Baeyer-Villiger monooxygenase (BVMO) and a lipase (CAL-A). Here, we extended this cascade for the synthesis of chiral methyl-substituted oligo-$epsilon$-caprolactone derivatives to achieve both, the generation of chirality in a monomer and the subsequent polymerization. Several substrates were examined and provided access to functionalized chiral compounds in high yields (up to >99 %) and optical purities (up to >99 % ee). By subsequent enzymatic enantioselective ring opening of the enantiopure monomers, oligomeric lactones were successfully synthesized. Rotate me! By combining three enzymes (LK-ADH, CHMO, CAL-A), which are evolutionary not connected, to an artificial cascade, optically active chiral oligomers can be synthesized from nonchiral or racemic methyl-substituted cyclohexanol derivatives.