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PUBLICATIONS
2017
Scherkus, Christian; Schmidt, Sandy; Bornscheuer, Uwe T.; Gröger, Harald; Kara, Selin; Liese, Andreas
Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction Journal Article
In: Biotechnology and Bioengineering, vol. 114, no. 6, pp. 1215–1221, 2017, ISSN: 10970290.
@article{Scherkus2017b,
title = {Kinetic insights into ϵ-caprolactone synthesis: Improvement of an enzymatic cascade reaction},
author = {Christian Scherkus and Sandy Schmidt and Uwe T. Bornscheuer and Harald Gröger and Selin Kara and Andreas Liese},
doi = {10.1002/bit.26258},
issn = {10970290},
year = {2017},
date = {2017-01-01},
journal = {Biotechnology and Bioengineering},
volume = {114},
number = {6},
pages = {1215--1221},
abstract = {A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215–1221. textcopyright 2017 Wiley Periodicals, Inc.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A computational approach for the simulation and prediction of a linear three-step enzymatic cascade for the synthesis of ϵ-caprolactone (ECL) coupling an alcohol dehydrogenase (ADH), a cyclohexanone monooxygenase (CHMO), and a lipase for the subsequent hydrolysis of ECL to 6-hydroxyhexanoic acid (6-HHA). A kinetic model was developed with an accuracy of prediction for a fed-batch mode of 37% for substrate cyclohexanol (CHL), 90% for ECL, and >99% for the final product 6-HHA. Due to a severe inhibition of the CHMO by CHL, a batch synthesis was shown to be less efficient than a fed-batch approach. In the fed-batch synthesis, full conversion of 100 mM CHL was 28% faster with an analytical yield of 98% compared to 49% in case of the batch synthesis. The lipase-catalyzed hydrolysis of ECL to 6-HHA circumvents the inhibition of the CHMO by ECL enabling a 24% higher product concentration of 6-HHA compared to ECL in case of the fed-batch synthesis without lipase. Biotechnol. Bioeng. 2017;114: 1215–1221. textcopyright 2017 Wiley Periodicals, Inc.
2016
Beier, Andy; Bordewick, Sven; Genz, Maika; Schmidt, Sandy; Bergh, Tom; Peters, Christin; Joosten, Henk Jan; Bornscheuer, Uwe T.
Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase Journal Article
In: ChemBioChem, vol. 17, no. 24, pp. 2312–2315, 2016, ISSN: 14397633.
@article{Beier2016bb,
title = {Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase},
author = {Andy Beier and Sven Bordewick and Maika Genz and Sandy Schmidt and Tom Bergh and Christin Peters and Henk Jan Joosten and Uwe T. Bornscheuer},
url = {https://doi.org/10.1002/cbic.201600484},
doi = {10.1002/cbic.201600484},
issn = {14397633},
year = {2016},
date = {2016-12-01},
journal = {ChemBioChem},
volume = {17},
number = {24},
pages = {2312--2315},
publisher = {John Wiley & Sons, Ltd},
abstract = {Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMOAcineto); this used NADH 4200-fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen-bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.
Genz, Maika; Melse, Okke; Schmidt, Sandy; Vickers, Clare; Dörr, Mark; Bergh, Tom; Joosten, Henk Jan; Bornscheuer, Uwe T.
Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates Journal Article
In: ChemCatChem, vol. 8, no. 20, pp. 3199–3202, 2016, ISSN: 18673899.
@article{Genz2016b,
title = {Engineering the Amine Transaminase from Vibrio fluvialis towards Branched-Chain Substrates},
author = {Maika Genz and Okke Melse and Sandy Schmidt and Clare Vickers and Mark Dörr and Tom Bergh and Henk Jan Joosten and Uwe T. Bornscheuer},
doi = {10.1002/cctc.201601007},
issn = {18673899},
year = {2016},
date = {2016-01-01},
journal = {ChemCatChem},
volume = {8},
number = {20},
pages = {3199--3202},
abstract = {Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chiral amines are important building blocks, especially for the pharmaceutical industry. Although amine transaminases (ATAs) are versatile enzymes to synthesize chiral amines, the wildtype enzymes do not accept ketones with two large substituents next to the carbonyl functionality. Using bioinformatic tools to design a seven-site mutant library followed by high-throughput screening, we were able to identify variants of the enzyme from Vibrio fluvialis (VF-ATA) with a widened binding pocket, as exemplified for a range of ketones. Three variants allowed the asymmetric synthesis of 2,2-dimethyl-1-phenylpropan-1-amine—not accessible by any wildtype ATA described so far. The best variant containing four mutations (L56V, W57C, F85V, V153A) gave 100 % conversion of the ketone to yield the amine with an enantiomeric excess value >99 %, notably with preference for the (R)-enantiomer. In silico modeling enabled the reconstruction of the substrate binding mode to the newly evolved pocket and, hence, allowed explanation of the experimental results.
Scherkus, Christian; Schmidt, Sandy; Bornscheuer, Uwe T.; Gröger, Harald; Kara, Selin; Liese, Andreas
A Fed-Batch Synthetic Strategy for a Three-Step Enzymatic Synthesis of Poly-ϵ-caprolactone Journal Article
In: ChemCatChem, vol. 8, no. 22, pp. 3446–3452, 2016, ISSN: 18673899.
@article{Scherkus2016b,
title = {A Fed-Batch Synthetic Strategy for a Three-Step Enzymatic Synthesis of Poly-ϵ-caprolactone},
author = {Christian Scherkus and Sandy Schmidt and Uwe T. Bornscheuer and Harald Gröger and Selin Kara and Andreas Liese},
doi = {10.1002/cctc.201600806},
issn = {18673899},
year = {2016},
date = {2016-01-01},
journal = {ChemCatChem},
volume = {8},
number = {22},
pages = {3446--3452},
abstract = {A three-step enzymatic reaction sequence for the synthesis of poly-ϵ-caprolactone (PCL) was designed running in a fed-batch operation. The first part of the cascade consisted of two oxidation steps starting with alcohol dehydrogenase catalyzed oxidation from cyclohexanol to cyclohexanone and further oxidation to ϵ-caprolactone (ECL) by means of a Baeyer–Villiger monooxygenase. As a third step, lipase-catalyzed hydrolysis of the lactone to 6-hydroxyhexanoic acid (6-HHA) was designed. With this biocatalytic multistep process reported herein, severe substrate surplus and product inhibition could be circumvented by the fed-batch operation by adding the cyclohexanol substrate and by in situ product removal of ECL by hydrolysis, respectively. Up to 283 mm product concentration of 6-HHA was reached in the fed-batch operated process without loss in productivity within 20 h. After extraction and subsequent polymerization catalyzed by Candida antarctica lipase B, analysis of the unfractionated polymer revealed a bimodal distribution of the polymer population, which reached a mass average molar mass (Mw) value of approximately 63 000 g mol−1 and a dispersity (Mw/Mn) of 1.1 for the higher molecular weight population, which thus revealed an alternative route to the conventional synthesis of PCL.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A three-step enzymatic reaction sequence for the synthesis of poly-ϵ-caprolactone (PCL) was designed running in a fed-batch operation. The first part of the cascade consisted of two oxidation steps starting with alcohol dehydrogenase catalyzed oxidation from cyclohexanol to cyclohexanone and further oxidation to ϵ-caprolactone (ECL) by means of a Baeyer–Villiger monooxygenase. As a third step, lipase-catalyzed hydrolysis of the lactone to 6-hydroxyhexanoic acid (6-HHA) was designed. With this biocatalytic multistep process reported herein, severe substrate surplus and product inhibition could be circumvented by the fed-batch operation by adding the cyclohexanol substrate and by in situ product removal of ECL by hydrolysis, respectively. Up to 283 mm product concentration of 6-HHA was reached in the fed-batch operated process without loss in productivity within 20 h. After extraction and subsequent polymerization catalyzed by Candida antarctica lipase B, analysis of the unfractionated polymer revealed a bimodal distribution of the polymer population, which reached a mass average molar mass (Mw) value of approximately 63 000 g mol−1 and a dispersity (Mw/Mn) of 1.1 for the higher molecular weight population, which thus revealed an alternative route to the conventional synthesis of PCL.
Balke, Kathleen; Schmidt, Sandy; Genz, Maika; Bornscheuer, Uwe T.
Switching the Regioselectivity of a Cyclohexanone Monooxygenase toward (+)-trans-Dihydrocarvone by Rational Protein Design Journal Article
In: ACS Chemical Biology, vol. 11, no. 1, pp. 38–43, 2016, ISSN: 15548937.
@article{Balke2016b,
title = {Switching the Regioselectivity of a Cyclohexanone Monooxygenase toward (+)-trans-Dihydrocarvone by Rational Protein Design},
author = {Kathleen Balke and Sandy Schmidt and Maika Genz and Uwe T. Bornscheuer},
doi = {10.1021/acschembio.5b00723},
issn = {15548937},
year = {2016},
date = {2016-01-01},
journal = {ACS Chemical Biology},
volume = {11},
number = {1},
pages = {38--43},
abstract = {The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.
Dörr, Mark; Fibinger, Michael P. C.; Last, Daniel; Schmidt, Sandy; Santos-Aberturas, Javier; Böttcher, Dominique; Hummel, Anke; Vickers, Clare; Voss, Moritz; Bornscheuer, Uwe T.
Fully automatized high-throughput enzyme library screening using a robotic platform Journal Article
In: Biotechnology and Bioengineering, vol. 113, no. 7, pp. 1421–1432, 2016, ISSN: 10970290.
@article{Dorr2016,
title = {Fully automatized high-throughput enzyme library screening using a robotic platform},
author = {Mark Dörr and Michael P. C. Fibinger and Daniel Last and Sandy Schmidt and Javier Santos-Aberturas and Dominique Böttcher and Anke Hummel and Clare Vickers and Moritz Voss and Uwe T. Bornscheuer},
doi = {10.1002/bit.25925},
issn = {10970290},
year = {2016},
date = {2016-01-01},
journal = {Biotechnology and Bioengineering},
volume = {113},
number = {7},
pages = {1421--1432},
abstract = {A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A fully automatized robotic platform has been established to facilitate high-throughput screening for protein engineering purposes. This platform enables proper monitoring and control of growth conditions in the microtiter plate format to ensure precise enzyme production for the interrogation of enzyme mutant libraries, protein stability tests and multiple assay screenings. The performance of this system has been exemplified for four enzyme classes important for biocatalysis such as Baeyer-Villiger monooxygenase, transaminase, dehalogenase and acylase in the high-throughput screening of various mutant libraries. This allowed the identification of novel enzyme variants in a sophisticated and highly reliable manner. Furthermore, the detailed optimization protocols should enable other researchers to adapt and improve their methods.
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; 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.
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.