Directed evolution of cutinase using in vitro compartmentalization
Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was ca...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Hwang, Bum-Yeol [verfasserIn] |
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| Format: |
E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2012 |
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| Schlagwörter: |
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| Anmerkung: |
© The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 |
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| Übergeordnetes Werk: |
Enthalten in: Biotechnology and bioprocess engineering - Seoul : Society, 1996, 17(2012), 3 vom: 01. Juni, Seite 500-505 |
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| Übergeordnetes Werk: |
volume:17 ; year:2012 ; number:3 ; day:01 ; month:06 ; pages:500-505 |
| Links: |
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| DOI / URN: |
10.1007/s12257-012-0026-1 |
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| Katalog-ID: |
SPR024559040 |
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| 100 | 1 | |a Hwang, Bum-Yeol |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Directed evolution of cutinase using in vitro compartmentalization |
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| 520 | |a Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. | ||
| 650 | 4 | |a cutinase |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a directed evolution |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a compartmentalization |7 (dpeaa)DE-He213 | |
| 650 | 4 | |a flurbiprofen |7 (dpeaa)DE-He213 | |
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| 912 | |a GBV_ILN_2068 | ||
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| 912 | |a GBV_ILN_2086 | ||
| 912 | |a GBV_ILN_2088 | ||
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| 912 | |a GBV_ILN_2106 | ||
| 912 | |a GBV_ILN_2107 | ||
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| 912 | |a GBV_ILN_2110 | ||
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2012 |
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2012 |
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10.1007/s12257-012-0026-1 doi (DE-627)SPR024559040 (SPR)s12257-012-0026-1-e DE-627 ger DE-627 rakwb eng Hwang, Bum-Yeol verfasserin aut Directed evolution of cutinase using in vitro compartmentalization 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. cutinase (dpeaa)DE-He213 directed evolution (dpeaa)DE-He213 compartmentalization (dpeaa)DE-He213 flurbiprofen (dpeaa)DE-He213 Enthalten in Biotechnology and bioprocess engineering Seoul : Society, 1996 17(2012), 3 vom: 01. Juni, Seite 500-505 (DE-627)373321821 (DE-600)2125481-3 1976-3816 nnns volume:17 year:2012 number:3 day:01 month:06 pages:500-505 https://dx.doi.org/10.1007/s12257-012-0026-1 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_65 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_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 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_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2116 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 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_4325 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 17 2012 3 01 06 500-505 |
| spelling |
10.1007/s12257-012-0026-1 doi (DE-627)SPR024559040 (SPR)s12257-012-0026-1-e DE-627 ger DE-627 rakwb eng Hwang, Bum-Yeol verfasserin aut Directed evolution of cutinase using in vitro compartmentalization 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. cutinase (dpeaa)DE-He213 directed evolution (dpeaa)DE-He213 compartmentalization (dpeaa)DE-He213 flurbiprofen (dpeaa)DE-He213 Enthalten in Biotechnology and bioprocess engineering Seoul : Society, 1996 17(2012), 3 vom: 01. Juni, Seite 500-505 (DE-627)373321821 (DE-600)2125481-3 1976-3816 nnns volume:17 year:2012 number:3 day:01 month:06 pages:500-505 https://dx.doi.org/10.1007/s12257-012-0026-1 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_65 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_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 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_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2116 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 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_4325 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 17 2012 3 01 06 500-505 |
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10.1007/s12257-012-0026-1 doi (DE-627)SPR024559040 (SPR)s12257-012-0026-1-e DE-627 ger DE-627 rakwb eng Hwang, Bum-Yeol verfasserin aut Directed evolution of cutinase using in vitro compartmentalization 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. cutinase (dpeaa)DE-He213 directed evolution (dpeaa)DE-He213 compartmentalization (dpeaa)DE-He213 flurbiprofen (dpeaa)DE-He213 Enthalten in Biotechnology and bioprocess engineering Seoul : Society, 1996 17(2012), 3 vom: 01. Juni, Seite 500-505 (DE-627)373321821 (DE-600)2125481-3 1976-3816 nnns volume:17 year:2012 number:3 day:01 month:06 pages:500-505 https://dx.doi.org/10.1007/s12257-012-0026-1 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_65 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_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 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_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2116 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 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_4325 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 17 2012 3 01 06 500-505 |
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10.1007/s12257-012-0026-1 doi (DE-627)SPR024559040 (SPR)s12257-012-0026-1-e DE-627 ger DE-627 rakwb eng Hwang, Bum-Yeol verfasserin aut Directed evolution of cutinase using in vitro compartmentalization 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. cutinase (dpeaa)DE-He213 directed evolution (dpeaa)DE-He213 compartmentalization (dpeaa)DE-He213 flurbiprofen (dpeaa)DE-He213 Enthalten in Biotechnology and bioprocess engineering Seoul : Society, 1996 17(2012), 3 vom: 01. Juni, Seite 500-505 (DE-627)373321821 (DE-600)2125481-3 1976-3816 nnns volume:17 year:2012 number:3 day:01 month:06 pages:500-505 https://dx.doi.org/10.1007/s12257-012-0026-1 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_65 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_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 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_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2116 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 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_4325 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 17 2012 3 01 06 500-505 |
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10.1007/s12257-012-0026-1 doi (DE-627)SPR024559040 (SPR)s12257-012-0026-1-e DE-627 ger DE-627 rakwb eng Hwang, Bum-Yeol verfasserin aut Directed evolution of cutinase using in vitro compartmentalization 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. cutinase (dpeaa)DE-He213 directed evolution (dpeaa)DE-He213 compartmentalization (dpeaa)DE-He213 flurbiprofen (dpeaa)DE-He213 Enthalten in Biotechnology and bioprocess engineering Seoul : Society, 1996 17(2012), 3 vom: 01. Juni, Seite 500-505 (DE-627)373321821 (DE-600)2125481-3 1976-3816 nnns volume:17 year:2012 number:3 day:01 month:06 pages:500-505 https://dx.doi.org/10.1007/s12257-012-0026-1 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_65 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_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 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_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2070 GBV_ILN_2086 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2116 GBV_ILN_2118 GBV_ILN_2119 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 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_4325 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 17 2012 3 01 06 500-505 |
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Hwang, Bum-Yeol |
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Hwang, Bum-Yeol misc cutinase misc directed evolution misc compartmentalization misc flurbiprofen Directed evolution of cutinase using in vitro compartmentalization |
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Directed evolution of cutinase using in vitro compartmentalization cutinase (dpeaa)DE-He213 directed evolution (dpeaa)DE-He213 compartmentalization (dpeaa)DE-He213 flurbiprofen (dpeaa)DE-He213 |
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Directed evolution of cutinase using in vitro compartmentalization |
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Directed evolution of cutinase using in vitro compartmentalization |
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Hwang, Bum-Yeol |
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Biotechnology and bioprocess engineering |
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directed evolution of cutinase using in vitro compartmentalization |
| title_auth |
Directed evolution of cutinase using in vitro compartmentalization |
| abstract |
Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 |
| abstractGer |
Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 |
| abstract_unstemmed |
Abstract High-throughput screening (HTS) is a key step to the success of directed evolution of enzymes. Recently, fluorescence-activated cell sorting (FACS) has emerged as a promising tool for HTS. Directed evolution of Fusarium solani cutinase, which is partially released from E. coli cells, was carried out using a FACS-based screening technique to increase its activity for (R)-flurbiprofen. First, the ability of using the FACS-based screening technique to screen active cutinase using wild type cutinase (WT) and inactivated cutinase mutant (S42A) was examined. Although the FACS-based screening using E. coli cells did not work well due to the diffusion of fluorescent product and/or an interference by the partially released cutinase from the the cells, FACS could be used to effectively screen active wild type cutinase via in vitro compartmentalization using water/oil/water emulsion microcompartments. Cutinase variants showing higher activity for (R)-flurbiprofen could be screened after four screening steps. The mutants 2–95 and 0–5 showed 8.0- and 6.8-fold higher activities for fluorescein (R)-flurbiprofen diester compared to that of the wild type cutinase, respectively. The mutant 0–5 also showed 5.0-fold higher activities for fluorescein butyl diester compared to that of the wild type cutinase. © The Korean Society for Biotechnology and Bioengineering and Springer-Verlag Berlin Heidelberg 2012 |
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Directed evolution of cutinase using in vitro compartmentalization |
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https://dx.doi.org/10.1007/s12257-012-0026-1 |
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10.1007/s12257-012-0026-1 |
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2024-07-04T01:26:51.076Z |
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| score |
7.3996086 |