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See for example a) D. J. Berrisford, C. Bolm, K. B. Sharpless, Angew. Chem. 1995, 107, 1159-1171 ; Angew. Chem., Int. Ed. Engl. 1995, 34, 1059-1070; b) R. Noyori, Asymmetric Catalysis in Organic Synthesis, Wiley, New York, 1994; c) A. Pfaltz, Acc. Chem. Res. 1993, 26, 339-345.
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Noyori, R.1
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a) K.-E. Jaeger, B. Schneidinger, K. Liebeton, D. Haas, M. T. Reetz, S. Philippou, G. Gerritse, S. Ransac, B. W. Dijkstra in Molecular Biology of Pseudomonads (Eds.: T. Nakazawa, K. Furukawa, D. Haas, S. Silver), ASM Press. Washington, 1996, pp. 319-330;
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35
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85033149827
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note
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[6a,9] using the following primers: A: 5′-GCG CAA TTA ACC CTC ACT AAA GGG AAC AAA-3′ and B: 5′-GCG TAA TAC GAC TCA CTA TAG GGC GAA-3′ PCR was performed under the following conditions: 2 min at 98°C(1 × ): 1 min at 94°C,2 min at 64°C, 1 min at 72°C (25 ×); 7 min 72°C (1 × );
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36
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0029947989
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b) PCR products were ligated into plasmid pUCPL6A derived from pUCPKS (A. A. Watson, R. A. Alm, J. S. Mattick, Gene 1996, 172, 163-164). Plasmids were propagated in E. coli JM 109, isolated, and used to transform P. aeruginosa PABST7.1 ( K.-E. Jaeger, B. Schneidinger, F. Rosenau, M. Werner, D. Lang, B. W. Dijkstra, K. Schimossek, A. Zonta, M. T. Reetz, J. Mol. Catal. B: Enzym. 1997, 3, 3-12). For each new cycle of mutagenesis the lipase gene of the best perfoming clone for a given mutant generation was isolated and recloned into pBluescript II KS.
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Watson, A.A.1
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37
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0343765715
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b) PCR products were ligated into plasmid pUCPL6A derived from pUCPKS (A. A. Watson, R. A. Alm, J. S. Mattick, Gene 1996, 172, 163-164). Plasmids were propagated in E. coli JM 109, isolated, and used to transform P. aeruginosa PABST7.1 ( K.-E. Jaeger, B. Schneidinger, F. Rosenau, M. Werner, D. Lang, B. W. Dijkstra, K. Schimossek, A. Zonta, M. T. Reetz, J. Mol. Catal. B: Enzym. 1997, 3, 3-12). For each new cycle of mutagenesis the lipase gene of the best perfoming clone for a given mutant generation was isolated and recloned into pBluescript II KS.
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J. Mol. Catal. B: Enzym.
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Jaeger, K.-E.1
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40
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85033134400
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note
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Our current strategy of using the best mutant of a given generation is not necessarily optimal. It is conceivable that a less selective mutant may lead to a better result in the subsequent mutagenesis round.
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42
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b) F. Theil, Chem. Rev. 1995, 95, 2203-2227;
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50
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85033137877
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note
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In our studies this also occurred to some extent, since the mutants having a low activity (which in some cases may lead to higher enantioselectivities) are "sorted out" upon screening. Only those mutants are considered which induce a notable conversion within ten minutes under the reaction conditions. Indeed, many mutants show practically no activity, which is not surprising.
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