Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries
Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Heuson, Egon [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2015 |
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| Schlagwörter: |
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| Anmerkung: |
© Springer-Verlag Berlin Heidelberg 2015 |
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| Übergeordnetes Werk: |
Enthalten in: Applied microbiology and biotechnology - Berlin : Springer, 1975, 100(2015), 1 vom: 09. Okt., Seite 397-408 |
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| Übergeordnetes Werk: |
volume:100 ; year:2015 ; number:1 ; day:09 ; month:10 ; pages:397-408 |
| Links: |
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| DOI / URN: |
10.1007/s00253-015-6988-0 |
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| Katalog-ID: |
SPR003008533 |
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| 100 | 1 | |a Heuson, Egon |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries |
| 264 | 1 | |c 2015 | |
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| 500 | |a © Springer-Verlag Berlin Heidelberg 2015 | ||
| 520 | |a Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. | ||
| 650 | 4 | |a Aminotransferase |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Transaminase |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Cysteine sulfinic acid |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a High-throughput screening |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Enzyme library |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a Colorimetric assay |7 (dpeaa)DE-He213 | |
| 700 | 1 | |a Petit, Jean-Louis |4 aut | |
| 700 | 1 | |a Debard, Adrien |4 aut | |
| 700 | 1 | |a Job, Aurélie |4 aut | |
| 700 | 1 | |a Charmantray, Franck |4 aut | |
| 700 | 1 | |a de Berardinis, Véronique |4 aut | |
| 700 | 1 | |a Gefflaut, Thierry |4 aut | |
| 773 | 0 | 8 | |i Enthalten in |t Applied microbiology and biotechnology |d Berlin : Springer, 1975 |g 100(2015), 1 vom: 09. Okt., Seite 397-408 |w (DE-627)265509564 |w (DE-600)1464336-4 |x 1432-0614 |7 nnns |
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| 912 | |a GBV_ILN_32 | ||
| 912 | |a GBV_ILN_39 | ||
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| 912 | |a GBV_ILN_62 | ||
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| 912 | |a GBV_ILN_165 | ||
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| 912 | |a GBV_ILN_281 | ||
| 912 | |a GBV_ILN_285 | ||
| 912 | |a GBV_ILN_293 | ||
| 912 | |a GBV_ILN_370 | ||
| 912 | |a GBV_ILN_381 | ||
| 912 | |a GBV_ILN_602 | ||
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| 912 | |a GBV_ILN_702 | ||
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| 912 | |a GBV_ILN_2011 | ||
| 912 | |a GBV_ILN_2014 | ||
| 912 | |a GBV_ILN_2015 | ||
| 912 | |a GBV_ILN_2020 | ||
| 912 | |a GBV_ILN_2021 | ||
| 912 | |a GBV_ILN_2025 | ||
| 912 | |a GBV_ILN_2026 | ||
| 912 | |a GBV_ILN_2027 | ||
| 912 | |a GBV_ILN_2031 | ||
| 912 | |a GBV_ILN_2034 | ||
| 912 | |a GBV_ILN_2037 | ||
| 912 | |a GBV_ILN_2038 | ||
| 912 | |a GBV_ILN_2039 | ||
| 912 | |a GBV_ILN_2044 | ||
| 912 | |a GBV_ILN_2048 | ||
| 912 | |a GBV_ILN_2049 | ||
| 912 | |a GBV_ILN_2050 | ||
| 912 | |a GBV_ILN_2055 | ||
| 912 | |a GBV_ILN_2056 | ||
| 912 | |a GBV_ILN_2057 | ||
| 912 | |a GBV_ILN_2059 | ||
| 912 | |a GBV_ILN_2061 | ||
| 912 | |a GBV_ILN_2064 | ||
| 912 | |a GBV_ILN_2068 | ||
| 912 | |a GBV_ILN_2070 | ||
| 912 | |a GBV_ILN_2086 | ||
| 912 | |a GBV_ILN_2093 | ||
| 912 | |a GBV_ILN_2106 | ||
| 912 | |a GBV_ILN_2107 | ||
| 912 | |a GBV_ILN_2110 | ||
| 912 | |a GBV_ILN_2113 | ||
| 912 | |a GBV_ILN_2118 | ||
| 912 | |a GBV_ILN_2119 | ||
| 912 | |a GBV_ILN_2129 | ||
| 912 | |a GBV_ILN_2143 | ||
| 912 | |a GBV_ILN_2144 | ||
| 912 | |a GBV_ILN_2147 | ||
| 912 | |a GBV_ILN_2153 | ||
| 912 | |a GBV_ILN_2188 | ||
| 912 | |a GBV_ILN_2232 | ||
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| 912 | |a GBV_ILN_4037 | ||
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| 912 | |a GBV_ILN_4125 | ||
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| 912 | |a GBV_ILN_4246 | ||
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| 912 | |a GBV_ILN_4251 | ||
| 912 | |a GBV_ILN_4305 | ||
| 912 | |a GBV_ILN_4306 | ||
| 912 | |a GBV_ILN_4307 | ||
| 912 | |a GBV_ILN_4313 | ||
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| 912 | |a GBV_ILN_4393 | ||
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| 951 | |a AR | ||
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10.1007/s00253-015-6988-0 doi (DE-627)SPR003008533 (SPR)s00253-015-6988-0-e DE-627 ger DE-627 rakwb eng Heuson, Egon verfasserin aut Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Springer-Verlag Berlin Heidelberg 2015 Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. Aminotransferase (dpeaa)DE-He213 Transaminase (dpeaa)DE-He213 Cysteine sulfinic acid (dpeaa)DE-He213 High-throughput screening (dpeaa)DE-He213 Enzyme library (dpeaa)DE-He213 Colorimetric assay (dpeaa)DE-He213 Petit, Jean-Louis aut Debard, Adrien aut Job, Aurélie aut Charmantray, Franck aut de Berardinis, Véronique aut Gefflaut, Thierry aut Enthalten in Applied microbiology and biotechnology Berlin : Springer, 1975 100(2015), 1 vom: 09. Okt., Seite 397-408 (DE-627)265509564 (DE-600)1464336-4 1432-0614 nnns volume:100 year:2015 number:1 day:09 month:10 pages:397-408 https://dx.doi.org/10.1007/s00253-015-6988-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_267 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_381 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2110 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2360 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4393 GBV_ILN_4700 AR 100 2015 1 09 10 397-408 |
| spelling |
10.1007/s00253-015-6988-0 doi (DE-627)SPR003008533 (SPR)s00253-015-6988-0-e DE-627 ger DE-627 rakwb eng Heuson, Egon verfasserin aut Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Springer-Verlag Berlin Heidelberg 2015 Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. Aminotransferase (dpeaa)DE-He213 Transaminase (dpeaa)DE-He213 Cysteine sulfinic acid (dpeaa)DE-He213 High-throughput screening (dpeaa)DE-He213 Enzyme library (dpeaa)DE-He213 Colorimetric assay (dpeaa)DE-He213 Petit, Jean-Louis aut Debard, Adrien aut Job, Aurélie aut Charmantray, Franck aut de Berardinis, Véronique aut Gefflaut, Thierry aut Enthalten in Applied microbiology and biotechnology Berlin : Springer, 1975 100(2015), 1 vom: 09. Okt., Seite 397-408 (DE-627)265509564 (DE-600)1464336-4 1432-0614 nnns volume:100 year:2015 number:1 day:09 month:10 pages:397-408 https://dx.doi.org/10.1007/s00253-015-6988-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_267 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_381 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2110 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2360 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4393 GBV_ILN_4700 AR 100 2015 1 09 10 397-408 |
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10.1007/s00253-015-6988-0 doi (DE-627)SPR003008533 (SPR)s00253-015-6988-0-e DE-627 ger DE-627 rakwb eng Heuson, Egon verfasserin aut Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Springer-Verlag Berlin Heidelberg 2015 Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. Aminotransferase (dpeaa)DE-He213 Transaminase (dpeaa)DE-He213 Cysteine sulfinic acid (dpeaa)DE-He213 High-throughput screening (dpeaa)DE-He213 Enzyme library (dpeaa)DE-He213 Colorimetric assay (dpeaa)DE-He213 Petit, Jean-Louis aut Debard, Adrien aut Job, Aurélie aut Charmantray, Franck aut de Berardinis, Véronique aut Gefflaut, Thierry aut Enthalten in Applied microbiology and biotechnology Berlin : Springer, 1975 100(2015), 1 vom: 09. Okt., Seite 397-408 (DE-627)265509564 (DE-600)1464336-4 1432-0614 nnns volume:100 year:2015 number:1 day:09 month:10 pages:397-408 https://dx.doi.org/10.1007/s00253-015-6988-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_267 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_381 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2110 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2360 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4393 GBV_ILN_4700 AR 100 2015 1 09 10 397-408 |
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10.1007/s00253-015-6988-0 doi (DE-627)SPR003008533 (SPR)s00253-015-6988-0-e DE-627 ger DE-627 rakwb eng Heuson, Egon verfasserin aut Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Springer-Verlag Berlin Heidelberg 2015 Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. Aminotransferase (dpeaa)DE-He213 Transaminase (dpeaa)DE-He213 Cysteine sulfinic acid (dpeaa)DE-He213 High-throughput screening (dpeaa)DE-He213 Enzyme library (dpeaa)DE-He213 Colorimetric assay (dpeaa)DE-He213 Petit, Jean-Louis aut Debard, Adrien aut Job, Aurélie aut Charmantray, Franck aut de Berardinis, Véronique aut Gefflaut, Thierry aut Enthalten in Applied microbiology and biotechnology Berlin : Springer, 1975 100(2015), 1 vom: 09. Okt., Seite 397-408 (DE-627)265509564 (DE-600)1464336-4 1432-0614 nnns volume:100 year:2015 number:1 day:09 month:10 pages:397-408 https://dx.doi.org/10.1007/s00253-015-6988-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_267 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_381 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2110 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2360 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4393 GBV_ILN_4700 AR 100 2015 1 09 10 397-408 |
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10.1007/s00253-015-6988-0 doi (DE-627)SPR003008533 (SPR)s00253-015-6988-0-e DE-627 ger DE-627 rakwb eng Heuson, Egon verfasserin aut Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries 2015 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © Springer-Verlag Berlin Heidelberg 2015 Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. Aminotransferase (dpeaa)DE-He213 Transaminase (dpeaa)DE-He213 Cysteine sulfinic acid (dpeaa)DE-He213 High-throughput screening (dpeaa)DE-He213 Enzyme library (dpeaa)DE-He213 Colorimetric assay (dpeaa)DE-He213 Petit, Jean-Louis aut Debard, Adrien aut Job, Aurélie aut Charmantray, Franck aut de Berardinis, Véronique aut Gefflaut, Thierry aut Enthalten in Applied microbiology and biotechnology Berlin : Springer, 1975 100(2015), 1 vom: 09. Okt., Seite 397-408 (DE-627)265509564 (DE-600)1464336-4 1432-0614 nnns volume:100 year:2015 number:1 day:09 month:10 pages:397-408 https://dx.doi.org/10.1007/s00253-015-6988-0 lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_165 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_267 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_381 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2110 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2360 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4326 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4393 GBV_ILN_4700 AR 100 2015 1 09 10 397-408 |
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Enthalten in Applied microbiology and biotechnology 100(2015), 1 vom: 09. Okt., Seite 397-408 volume:100 year:2015 number:1 day:09 month:10 pages:397-408 |
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Enthalten in Applied microbiology and biotechnology 100(2015), 1 vom: 09. Okt., Seite 397-408 volume:100 year:2015 number:1 day:09 month:10 pages:397-408 |
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Aminotransferase Transaminase Cysteine sulfinic acid High-throughput screening Enzyme library Colorimetric assay |
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Applied microbiology and biotechnology |
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Heuson, Egon @@aut@@ Petit, Jean-Louis @@aut@@ Debard, Adrien @@aut@@ Job, Aurélie @@aut@@ Charmantray, Franck @@aut@@ de Berardinis, Véronique @@aut@@ Gefflaut, Thierry @@aut@@ |
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These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. 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Heuson, Egon |
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Heuson, Egon misc Aminotransferase misc Transaminase misc Cysteine sulfinic acid misc High-throughput screening misc Enzyme library misc Colorimetric assay Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries |
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Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries Aminotransferase (dpeaa)DE-He213 Transaminase (dpeaa)DE-He213 Cysteine sulfinic acid (dpeaa)DE-He213 High-throughput screening (dpeaa)DE-He213 Enzyme library (dpeaa)DE-He213 Colorimetric assay (dpeaa)DE-He213 |
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misc Aminotransferase misc Transaminase misc Cysteine sulfinic acid misc High-throughput screening misc Enzyme library misc Colorimetric assay |
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continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries |
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Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries |
| abstract |
Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. © Springer-Verlag Berlin Heidelberg 2015 |
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Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. © Springer-Verlag Berlin Heidelberg 2015 |
| abstract_unstemmed |
Abstract In the course of a project devoted to the stereoselective synthesis of non-proteinogenic α-amino acids using α-transaminases (α-TA), we report the design and optimization of generic high-throughput continuous assays for the screening of α-TA libraries. These assays are based on the use of l- or d-cysteine sulfinic acid (CSA) as irreversible amino donor and subsequent sulfite titration by colorimetry. The assays’ quality was assessed under screening conditions. Hit selection thresholds were accurately determined for every couple of substrates and a library of 232 putative transaminases expressed in Escherichia coli host cells was screened. The reported high throughput screening assays proved very sensitive allowing the detection with high confidence of activities as low as 10 μU (i.e., 0.01 nmol substrate converted per min). The assays were also evidenced to be stereochemically discriminant since l-CSA and d-CSA allowed the exclusive detection of l-TA and d-TA, respectively. These generic assays thus allow testing the stereoselective conversion of a wide range of α-keto acids into α-amino acids of interest. As a proof of principle, the use of 2-oxo-4-phenylbutyric acid as acceptor substrate led to the identification of 54 new α-TA offering an access to valuable l- or d-homophenylalanine. © Springer-Verlag Berlin Heidelberg 2015 |
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Continuous colorimetric screening assays for the detection of specific l- or d-α-amino acid transaminases in enzyme libraries |
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Petit, Jean-Louis Debard, Adrien Job, Aurélie Charmantray, Franck de Berardinis, Véronique Gefflaut, Thierry |
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Petit, Jean-Louis Debard, Adrien Job, Aurélie Charmantray, Franck de Berardinis, Véronique Gefflaut, Thierry |
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10.1007/s00253-015-6988-0 |
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| score |
7.401272 |