Directed Evolution of Enantioselective Enzymes: Iterative Cycles of CASTing for Probing Protein‐Sequence Space

Reetz, Manfred T.; Wang, Liwen; Bocola, Marco

doi:10.1002/anie.200502746

Cited by 305 publications

(154 citation statements)

References 91 publications

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“…7. Beneficial positions in KeySIDE are identified through random mutagenesis in directed evolution experiments throughout the protein and in contrast to the CASTing (Reetz et al 2006b) or iterative saturation mutagenesis (Reetz et al 2006a) approaches, not selected through rational design or not restricted to the substrate binding site. The uphill walk of increasing fitness of the Ymphytase evolutionary pathway is shown in red-colored arrow (Fig.…”

Section: Discussionmentioning

confidence: 99%

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

Shivange

Roccatano

Schwaneberg

2015

Appl Microbiol Biotechnol

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Bacterial phytases have attracted industrial interest as animal feed supplement due to their high activity and sufficient thermostability (required for feed pelleting). We devised an approach named KeySIDE, an iterative Key-residues interrogation of the wild type with Substitutions Identified in Directed Evolution for improving Yersinia mollaretii phytase (Ymphytase) thermostability by combining key beneficial substitutions and elucidating their individual roles. Directed evolution yielded in a discovery of nine positions in Ymphytase and combined iteratively to identify key positions. The "best" combination (M6: T77K, Q154H, G187S, and K289Q) resulted in significantly improved thermal resistance; the residual activity improved from 35 % (wild type) to 89 % (M6) at 58 °C and 20-min incubation. Melting temperature increased by 3 °C in M6 without a loss of specific activity. Molecular dynamics simulation studies revealed reduced flexibility in the loops located next to helices (B, F, and K) which possess substitutions (Helix-B: T77K, Helix-F: G187S, and Helix-K: K289E/Q). Reduced flexibility in the loops might be caused by strengthened hydrogen bonding network (e.g., G187S and K289E/K289Q) and a salt bridge (T77K). Our results demonstrate a promising approach to design phytases in food research, and we hope that the KeySIDE might become an attractive approach for understanding of structure-function relationships of enzymes.

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Section: Discussionmentioning

confidence: 99%

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

Shivange

Roccatano

Schwaneberg

2015

Appl Microbiol Biotechnol

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“…The organic phase acts as substrate reservoir and also as extraction solvent for product isolation. For example, inhibition of a PAMO mutant during whole-cell BV oxidation was overcome by utilization of buffer/dioctyl phthalate in a ratio 1:1 (Reetz et al 2006). Yang et al used recombinant E. coli with cyclopentadecanone monooxygenase for the improved BV oxidation of cyclododecanone to lauryl lactone exploiting two-phase semicontinuous reactor with hexadecane as organic solvent Scheme 1 Encapsulation of recombinant cells E. coli with overexpressed Baeyer-Villiger monooxygenases in polyelectrolyte complex (PEC) capsules with semipermeable membrane and their utilization as biocatalysts of Baeyer-Villiger oxidations.…”

Section: Two-phase System Strategiesmentioning

confidence: 99%

Baeyer-Villiger oxidations: biotechnological approach

Bučko

Gemeiner

Schenkmayerová

et al. 2016

Appl Microbiol Biotechnol

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Baeyer-Villiger monooxygenases (BVMOs) are a very well-known and intensively studied class of flavin-dependent enzymes. Their substrate promiscuity, high chemo-, regio-, and enantioselectivity are prerequisites for the use in synthetic chemistry and should pave the way for successful industrial processes. Nonetheless, only a very limited number of industrial relevant transformations are known, mainly due to the lack of BVMOs stability and cofactor dependency. In this review, we focus on novel BVMO-mediated transformations, BVMOs in cascade type reactions, potential industrial applications, and how limitations have been tackled by the community. Special attention will be put on whole-cell immobilization strategies. We emphasize to bridge recent developments in fundamental research to industrial applications.

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“…A QuikChange experiment would be sufficient to generate 400 (20×20; two positions) or 8000 (three positions) different protein variants. Due to primer length limitations, saturation of more than three positions has to our best knowledge not been reported for the CASTing strategy 31,32,[76][77][78][79] . QuikChange based mutagenesis methods were further advanced to saturate 4 (PFunkel 80 ) to 6 (PFLF-MSDM 81 ) positions simultaneously.…”

Section: Comparison Of the Methods For Recombining Amino Acid Substitmentioning

confidence: 89%

“…31 A further highlight is the epoxide hydrolase from Aspergillus niger (ANEH) with in increase E value (10.8 to 115 for hydrolysis of glycidyl phenyl ether) after five rounds of site saturation mutagenesis. 32 …”

Section: Structure Analysis Based Protein Evolution Strategymentioning

confidence: 97%

Directed evolution 2.0: improving and deciphering enzyme properties

2015

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Directed evolution has matured to a routinely applied algorithm to tailor enzyme properties to meet the demands in various applications. In order to free directed enzyme evolution from methodological restraints and to efficiently explore its potential, many different strategies have been used in directed evolution campaigns. Analysis of directed evolution campaigns reveals that traditional approaches, in which several iterative rounds of diversity generation and screening are performed, are gradually replaced by strategies which require less time, less screening efforts, and generate a molecular understanding of the targeted properties. In this review, conceptual advances in knowledge generating directed evolution strategies are summarized, compared to each other and to traditional directed evolution strategies. Finally, a 'KnowVolution' (knowledge gaining directed evolution) termed strategy is proposed.

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Directed Evolution of Enantioselective Enzymes: Iterative Cycles of CASTing for Probing Protein‐Sequence Space

Abstract: ReCASTing for success: The recently introduced method of combinatorial active‐site saturation test (CAST) has been applied in iterative cycles in the directed evolution of enantioselective wild‐type epoxide hydrolases (WT‐EH).

Cited by 305 publications

References 91 publications

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

Baeyer-Villiger oxidations: biotechnological approach

Directed evolution 2.0: improving and deciphering enzyme properties

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