Natural evolution thermostability

Creating enzymes that are both more thermostable and more active is particularly exciting for industrial applications. In addition, these studies nicely demonstrate that behaviors of natural enzymes may not necessarily be due to physical limitations intrinsic to proteins themselves, as is often assumed. Instead they reflect what is both relevant to the organism and accessible to natural evolution 184l... 

P450s are generally less stable than peroxidases. Recently a naturally thermostable P450 (stable up to 85 °C) was identified and characterized [25, 100 -103], Both P450s and peroxidases are inactivated during catalysis, via heme alkylation by terminal olefins (see Section 10.8.3) and oxidative damage by peroxides. Eukaryotic P450s are associated with cell membranes and are therefore insoluble and difficult to use outside the cell. We believe that many of these limitations can be addressed by directed evolution. 

Figure 4-10. Enzymes isolated from organisms growing at different temperatures often exhibit a tradeoff between thermostability and catalytic activity measured at low temperature. Enzymes that are both highly thermostable and highly active at low temperatures are rare in nature but highly desired for various applications and can be obtained by directed evolution with relatively few mutations I 84. ...

For 3.8 billion years, enzyme evolution has occurred primarily in microbes exposed to novel environmental conditions. However, in the last two decades, new methods have been developed for laboratory evolution of enzymes for production of chemicals, pharmaceuticals, and biofuels. Directed evolution has been widely used to improve thermostability and alter substrate specificity. Current efforts aim to improve the catalytic abilities of evolved enzymes, which are usually considerably poorer than those of naturally occurring enzymes, and to evolve novel pathways using promiscuous activities of existing enzymes. These efforts will provide new insights into the adaptation of protein scaffolds for new functions that will both help us to understand the evolutionary history of modern enzymes and provide the basis for a wide range of applications in biotechnology. 

Studies on Thermotoga maritima have been focusing mainly on evolution, metabolism, and the correlation of protein structure and stability. It has been established that viability of the hyperthermophile in its hostile natural habitats is based on the inherent thermostability of its whole cell inventory. In some cases this is assisted by extrinsic components such as the cell membrane or specific ligands and compatible solutes. As a response to short-term temperature... 

Concerning the biocatalyst development by directed evolution, some limitations of naturally occurring enzymes such as thermostability, activity, and tolerance towards organic solvents have been successfully overcome. It is also important to mention that this methodology is highly effective in altering, or improving almost all kinds of protein properties for different classes of enzymes. 

In nature, enzymes play an important role in the survival and reproduction of their source organism. Therefore, many enzymes in their natural form are not suitable for application directly as biocatalysts in bioprocessing. For example, most enzymes are active at relatively mild conditions, thus may not be viable under the harsher conditions encountered in most indnstrial production systems. Various methods have been used to improve the properties of enzymes, including selectivity, activity and thermostability, in order to enable them to function as efficient industrial biocatalysts. Enzyme engineering, which encompasses rational design and directed evolution, is an efficient method to improve enzyme properties. Rational design seeks for beneficial mutations or protein sequences by applying empirically derived rules or theoretical models. Meanwhile, directed evolution uses a combinatorial approach to create libraries of enzymes from which enhanced variants can be identified... 

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