Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example
Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Yuan, Mei [verfasserIn] |
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| Format: |
Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2016 |
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| Rechteinformationen: |
Nutzungsrecht: © Springer Science+Business Media New York 2016 |
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| Schlagwörter: |
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| Übergeordnetes Werk: |
Enthalten in: Applied biochemistry and biotechnology / A - New York, NY : Springer, 1994, 179(2016), 4, Seite 545-557 |
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| Übergeordnetes Werk: |
volume:179 ; year:2016 ; number:4 ; pages:545-557 |
| Links: |
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| DOI / URN: |
10.1007/s12010-016-2012-0 |
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OLC1977666132 |
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| 520 | |a Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. | ||
| 540 | |a Nutzungsrecht: © Springer Science+Business Media New York 2016 | ||
| 650 | 4 | |a Positive mutants | |
| 650 | 4 | |a Biochemistry, general | |
| 650 | 4 | |a Directed evolution | |
| 650 | 4 | |a Chemistry | |
| 650 | 4 | |a High-throughput screening | |
| 650 | 4 | |a Pseudomonas aeruginosa arylsulfatase | |
| 650 | 4 | |a Alkaline lysis | |
| 650 | 4 | |a Biotechnology | |
| 700 | 1 | |a Yang, Xiaolan |4 oth | |
| 700 | 1 | |a Li, Yuwei |4 oth | |
| 700 | 1 | |a Liu, Hongbo |4 oth | |
| 700 | 1 | |a Pu, Jun |4 oth | |
| 700 | 1 | |a Zhan, Chang-guo |4 oth | |
| 700 | 1 | |a Liao, Fei |4 oth | |
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10.1007/s12010-016-2012-0 doi PQ20160719 (DE-627)OLC1977666132 (DE-599)GBVOLC1977666132 (PRQ)c1552-75bdbbb94f808627e4b9e56c82fe5dba04d24831e47c44b5d5544c119f6603160 (KEY)0068751020160000179000400545facilealkalinelysisofescherichiacolicellsinhighthr DE-627 ger DE-627 rakwb eng 570 540 660 DNB 42.00 bkl Yuan, Mei verfasserin aut Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example 2016 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. Nutzungsrecht: © Springer Science+Business Media New York 2016 Positive mutants Biochemistry, general Directed evolution Chemistry High-throughput screening Pseudomonas aeruginosa arylsulfatase Alkaline lysis Biotechnology Yang, Xiaolan oth Li, Yuwei oth Liu, Hongbo oth Pu, Jun oth Zhan, Chang-guo oth Liao, Fei oth Enthalten in Applied biochemistry and biotechnology / A New York, NY : Springer, 1994 179(2016), 4, Seite 545-557 (DE-627)182278573 (DE-600)1193054-8 (DE-576)043085105 0273-2289 nnns volume:179 year:2016 number:4 pages:545-557 http://dx.doi.org/10.1007/s12010-016-2012-0 Volltext http://www.ncbi.nlm.nih.gov/pubmed/26899233 http://search.proquest.com/docview/1799328556 GBV_USEFLAG_A SYSFLAG_A GBV_OLC SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_70 GBV_ILN_4012 42.00 AVZ AR 179 2016 4 545-557 |
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10.1007/s12010-016-2012-0 doi PQ20160719 (DE-627)OLC1977666132 (DE-599)GBVOLC1977666132 (PRQ)c1552-75bdbbb94f808627e4b9e56c82fe5dba04d24831e47c44b5d5544c119f6603160 (KEY)0068751020160000179000400545facilealkalinelysisofescherichiacolicellsinhighthr DE-627 ger DE-627 rakwb eng 570 540 660 DNB 42.00 bkl Yuan, Mei verfasserin aut Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example 2016 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. Nutzungsrecht: © Springer Science+Business Media New York 2016 Positive mutants Biochemistry, general Directed evolution Chemistry High-throughput screening Pseudomonas aeruginosa arylsulfatase Alkaline lysis Biotechnology Yang, Xiaolan oth Li, Yuwei oth Liu, Hongbo oth Pu, Jun oth Zhan, Chang-guo oth Liao, Fei oth Enthalten in Applied biochemistry and biotechnology / A New York, NY : Springer, 1994 179(2016), 4, Seite 545-557 (DE-627)182278573 (DE-600)1193054-8 (DE-576)043085105 0273-2289 nnns volume:179 year:2016 number:4 pages:545-557 http://dx.doi.org/10.1007/s12010-016-2012-0 Volltext http://www.ncbi.nlm.nih.gov/pubmed/26899233 http://search.proquest.com/docview/1799328556 GBV_USEFLAG_A SYSFLAG_A GBV_OLC SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_70 GBV_ILN_4012 42.00 AVZ AR 179 2016 4 545-557 |
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10.1007/s12010-016-2012-0 doi PQ20160719 (DE-627)OLC1977666132 (DE-599)GBVOLC1977666132 (PRQ)c1552-75bdbbb94f808627e4b9e56c82fe5dba04d24831e47c44b5d5544c119f6603160 (KEY)0068751020160000179000400545facilealkalinelysisofescherichiacolicellsinhighthr DE-627 ger DE-627 rakwb eng 570 540 660 DNB 42.00 bkl Yuan, Mei verfasserin aut Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example 2016 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. Nutzungsrecht: © Springer Science+Business Media New York 2016 Positive mutants Biochemistry, general Directed evolution Chemistry High-throughput screening Pseudomonas aeruginosa arylsulfatase Alkaline lysis Biotechnology Yang, Xiaolan oth Li, Yuwei oth Liu, Hongbo oth Pu, Jun oth Zhan, Chang-guo oth Liao, Fei oth Enthalten in Applied biochemistry and biotechnology / A New York, NY : Springer, 1994 179(2016), 4, Seite 545-557 (DE-627)182278573 (DE-600)1193054-8 (DE-576)043085105 0273-2289 nnns volume:179 year:2016 number:4 pages:545-557 http://dx.doi.org/10.1007/s12010-016-2012-0 Volltext http://www.ncbi.nlm.nih.gov/pubmed/26899233 http://search.proquest.com/docview/1799328556 GBV_USEFLAG_A SYSFLAG_A GBV_OLC SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_70 GBV_ILN_4012 42.00 AVZ AR 179 2016 4 545-557 |
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10.1007/s12010-016-2012-0 doi PQ20160719 (DE-627)OLC1977666132 (DE-599)GBVOLC1977666132 (PRQ)c1552-75bdbbb94f808627e4b9e56c82fe5dba04d24831e47c44b5d5544c119f6603160 (KEY)0068751020160000179000400545facilealkalinelysisofescherichiacolicellsinhighthr DE-627 ger DE-627 rakwb eng 570 540 660 DNB 42.00 bkl Yuan, Mei verfasserin aut Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example 2016 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. Nutzungsrecht: © Springer Science+Business Media New York 2016 Positive mutants Biochemistry, general Directed evolution Chemistry High-throughput screening Pseudomonas aeruginosa arylsulfatase Alkaline lysis Biotechnology Yang, Xiaolan oth Li, Yuwei oth Liu, Hongbo oth Pu, Jun oth Zhan, Chang-guo oth Liao, Fei oth Enthalten in Applied biochemistry and biotechnology / A New York, NY : Springer, 1994 179(2016), 4, Seite 545-557 (DE-627)182278573 (DE-600)1193054-8 (DE-576)043085105 0273-2289 nnns volume:179 year:2016 number:4 pages:545-557 http://dx.doi.org/10.1007/s12010-016-2012-0 Volltext http://www.ncbi.nlm.nih.gov/pubmed/26899233 http://search.proquest.com/docview/1799328556 GBV_USEFLAG_A SYSFLAG_A GBV_OLC SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_70 GBV_ILN_4012 42.00 AVZ AR 179 2016 4 545-557 |
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10.1007/s12010-016-2012-0 doi PQ20160719 (DE-627)OLC1977666132 (DE-599)GBVOLC1977666132 (PRQ)c1552-75bdbbb94f808627e4b9e56c82fe5dba04d24831e47c44b5d5544c119f6603160 (KEY)0068751020160000179000400545facilealkalinelysisofescherichiacolicellsinhighthr DE-627 ger DE-627 rakwb eng 570 540 660 DNB 42.00 bkl Yuan, Mei verfasserin aut Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example 2016 Text txt rdacontent ohne Hilfsmittel zu benutzen n rdamedia Band nc rdacarrier Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. Nutzungsrecht: © Springer Science+Business Media New York 2016 Positive mutants Biochemistry, general Directed evolution Chemistry High-throughput screening Pseudomonas aeruginosa arylsulfatase Alkaline lysis Biotechnology Yang, Xiaolan oth Li, Yuwei oth Liu, Hongbo oth Pu, Jun oth Zhan, Chang-guo oth Liao, Fei oth Enthalten in Applied biochemistry and biotechnology / A New York, NY : Springer, 1994 179(2016), 4, Seite 545-557 (DE-627)182278573 (DE-600)1193054-8 (DE-576)043085105 0273-2289 nnns volume:179 year:2016 number:4 pages:545-557 http://dx.doi.org/10.1007/s12010-016-2012-0 Volltext http://www.ncbi.nlm.nih.gov/pubmed/26899233 http://search.proquest.com/docview/1799328556 GBV_USEFLAG_A SYSFLAG_A GBV_OLC SSG-OLC-TEC SSG-OLC-CHE SSG-OLC-PHA SSG-OLC-DE-84 GBV_ILN_70 GBV_ILN_4012 42.00 AVZ AR 179 2016 4 545-557 |
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Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example |
| abstract |
Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. |
| abstractGer |
Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. |
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Facile alkaline lysis of Escherichia coli cells in high-throughput (HTP) mode for screening enzyme mutants was tested with Pseudomonas aeruginosa arylsulfatase (PAAS). The alkaline lysis buffer was 1.0 M Tris-HCl at pH 9.0 plus 0.1 % Tween-20 and 2.0 mM 4-aminobenzamidine, mixed with cell suspension at 8:1 to 12:1 ratio for continuous agitation of mixtures in 96-well plates under room temperature; enzymatic activity in lysates was measured with 96-well microplate. PAAS activity tolerated final 0.1 % Tween-20. Individual clones were amplified for 12 h in 0.50 mL TB medium with 48-well plates to enhance the repeatability of induced expression. During continuous agitation of the mixture of cells and the lysis buffer, PAAS activities in lysates were steady from 3 to 9 h and comparable to sonication treatment but better than freezing-thawing. Coefficients of variation of activities of PAAS/mutants in lysates after treatment for 7 h reached ∼22 %. The mutant M72Q had specific activity 2-fold of G138S. By HTP lysis of cells, M72Q was recognized as a positive mutant over G138S with the area under the curve of 0.873. Therefore, for enzymes tolerating concentrated alkaline buffers, the proposed alkaline lysis approach may be generally applicable for HTP lysis of host cells during directed evolution. |
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Facile Alkaline Lysis of Escherichia coli Cells in High-Throughput Mode for Screening Enzyme Mutants: Arylsulfatase as an Example |
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