Gerichtete Evolution stereoselektiver Enzyme: Eine ergiebige Katalysator‐Quelle für asymmetrische Reaktionen

Abstract

Asymmetrische Katalyse spielt eine Schlüsselrolle in der modernen synthetischen organischen Chemie, wobei künstliche Katalysatoren und Enzyme die zwei wichtigsten Optionen darstellen. Die Verwendung von Enzymen in der organischen Chemie und Biotechnologie hat in der zweiten Hälfte des vergangenen Jahrhunderts stark zugenommen, oft blieb jedoch der Anwendungsbereich aus mehreren Gründen eingeschränkt. Dazu gehören begrenzte Substratakzeptanz, geringe oder fehlende Stereoselektivität, unzureichende Stabilität und manchmal Produktinhibierung. Während der vergangenen 15 Jahre wurde die genetische Technik der gerichteten Evolution so weit entwickelt und verfeinert, dass all diese Probleme angegangen und in der Regel gelöst werden können. Sie basiert auf wiederholende Zyklen von Genmutagenese, Expression und Screening (oder Selektion). Der vorliegende Aufsatz fokussiert auf gerichtete Evolution stereoselektiver Enzyme, ein fundamental neuer Ansatz zur asymmetrischen Katalyse. Der Schwerpunkt liegt auf Methodenentwicklung in dem Bestreben, diese Art der Katalysator‐Optimierung einfacher, schneller und effizienter als in der Vergangenheit zu gestalten. Die neueren Methoden bescheren dem Chemiker und dem Biotechnologen robuste und selektive Katalysatoren für eine Vielzahl von nützlichen Anwendungen.

Enhancing the Thermal Robustness of an Enzyme by Directed Evolution: Least Favorable Starting Points and Inferior Mutants Can Map Superior Evolutionary Pathways

“…These could include insufficient screening, inherent amino acid bias, imperfect PCR conditions or an actual local minimum on the theoretical fitness landscape. Contrary to conventional wisdom in the field of directed evolution, [3,6,9] application of the second strategy based on the use of non-improved or even inferior mutants in ISM led to superior mutants in the third generation (Figure 2). This interesting phenomenon might be related to the theory of neutral drift [17] and/or to Eigen's concept of quasi-species in natural evolution, [18] which Mannervik has invoked previously in several important directed evolution studies.…”

“…The respective sites were likewise not considered in iterative saturation mutagenesis (although past experience has shown that this might in fact be successful). [9] Such decisions need not cause unease, because past and present experience shows that a given ISM study can lead to many different hits, depending on how much time is invested in exploring different sites and pathways. Since the libraries are small, requiring a minimum of screening, such mini-explorations of protein sequence space generally prove to be rewarding.…”

“…Since the libraries are small, requiring a minimum of screening, such mini-explorations of protein sequence space generally prove to be rewarding. [9] In the first round of saturation mutagenesis at 12 sites with the creation of 12 mutant libraries, a total of 5495 transformants were assayed, which in each case guaranteed 95 % library coverage (again, due to the bias phenomenon, this is only a rough guide). Table 1 lists the respective hits.…”

“…The reason for the pronounced efficacy of ISM, which has been observed in numerous cases involving different enzymes, is due to strong cooperative effects occurring between mutations within a randomized site and between two or more sites as the evolutionary process proceeds (more than additivity). [9] The choice of the randomization sites is crucial, which in turn depends on the protein property to be evolved. For each catalytic property (e.g., thermostability, rate, stereoselectivity, etc.)…”

“…[8,9] It is a straightforward process that begins by identifying sites in an enzyme appropriate for saturation mutagenesis (amino acid randomization), a site being defined as comprising one, two or more amino acid positions in the protein. Following the formation of initial mutant libraries and a screening procedure specific for a given catalytic property, the best variants ("hits") are identified.…”