Temperature directed evolution

This 580 to 280 K decrease in reaction temperature for trimethylsilane formation corresponds to a decrease in the reaction activation energy from 33 kcal/mol to -16 kcal/mol. Alternatively, if the rates of these processes could be measured at a common temperature, they would differ by more than 5 orders of magnitude. The fact that trimethylsilane is evolved at either 580 K or 280 K and not at temperatures in between suggests that there are distinctly different active sites for forming this product. The ratio of these active sites is a fimction of the temperature at which die surface is ion bombarded, and the transition from high to low temperature product evolution correlates directly with a factor of 1.5 to 2 increase in the Cu/Si... 

Saalfrank JW, Maier WF. 2004. Directed evolution of noble-metal-free catalysts for the oxidation of CO at room temperature. Angew Chem Int Ed 43 2028-2031. 

Since most synthetic applications require enzymes catalyzing nonnatural substrates, their properties often have to be improved. One way to achieve this is to optimize reaction conditions such as pH, temperature, solvents, additives, etc. [6-9]. Another way is to modulate the substrates without compromising the synthetic efficiency of the overall reaction [10]. In most cases for commercial manufacturing, however, the protein sequences have to be altered to enhance reactivity, stereoselectivity and stability. It was estimated that over 30 commercial enzymes worldwide have been engineered for industrial applications [11]. Precise prediction of which amino acids to mutate is difficult to achieve. Since the mid 1990s, directed evolution... 

Well logs from Hydrate Ridge indicated acceptably consistent estimates of hydrate occurrence, particularly water saturation, and RAB. Hydrate concentration data from logging tools have been confirmed and quantified by more direct core methods of IR sensing of temperature, gas evolution, and chlorinity decrease. 

With the example of p-nitrophenyl esterase the Arnold group demonstrated that even the thermostability of an enzyme is amenable to directed evolution (Giver, 1998). As a test of thermostability the activity in standard conditions (pH 7.5, 0.25 mM p-nitrophenyl acetate) at 30 °C (AJ was compared with the activity in the standard assay at 30 °C (Ar) but after heating to Tm for 10 min (with subsequent cooling on ice and incubation for 30 min at room temperature). The results are shown in Figure 11.10. This work demonstrates that enhanced activity and enhanced stability need not be mutually exclusive. However, the improvements along one of the two axes sometimes tend to be minor, i.e., by a factor between 3 and 5. 

Directed evolution is an iterative process that mimics the natural evolution process in vitro, by generating a diverse library of enzymes and selecting those with the desired features. Natural evolution is very effective in the long term (bacteria adapt to every environment, living even in so-called black smokers, deep-ocean vents where temperatures can reach 350°C and the pressure is 200bar [93]). Unfortunately, it typically takes millions of years. Happily, directed evolution can be carried out within weeks or months and with an unlimited number of parents. Importantly, and unlike rational design, directed evolution is a stochastic method. It does not require any structural or mechanistic information on the enzyme of interest (although such information can help). 

We chose subtilisin E to test our prediction that directed evolution makes mutations at uncoupled positions (Voigt et al., 2000b). Directed evolution increased the temperature optimum for activity, T pt, of Bacillus subtilis subtilisin E from 59° to 76°C, with eight mutations (Zhao and Arnold, 1999). In an independent study, thirteen mutations improved the activity toward the hydrolysis of su c c i iivI-A 1 a-A1 a-Pro-Phe- >-nitroanilide (s-AAPF-j Na) in the organic solvent dimethylformamide (DMF). The mutants were found by screening 2000 to 5000 clones from... 

In this directed evolution experiment, increased thermostability was accompanied by an increase in catalytic activity at all temperatures (Fig. 6), as measured on the substrate used during screening, jfr-nitrophenyl acetate. Variant 8G8 is 3.7 times more active than wild type at the wild-type Topt the improvement is 4.5-fold at 8G8 s new Topt of 60°C. Activity was allowed to decrease in the first generation (variant 1A5D1 was an intermediate product of a separate evolution experiment to increase p-nitrobenzyl esterase activity in aqueous organic solvent and differs from wild type at five positions), but was recovered in later generations. 

Taguchi et al. (1998) used directed evolution to increase the low-temperature activity of the mesophilic subtilisin BPN. Random muta-... 

The number of thermally adaptive mutations resulting from directed evolution studies is too small at present to support a detailed statistical analysis. Here we summarize some properties of the mutations discovered in the studies reviewed above, and compare them to the amino-acid differences seen among naturally occurring enzymes that have adapted to different temperatures. Lists of the amino-acid substitutions discovered... 

Amino-acid Substitutions Discovered During the Directed Evolution of Low Temperature Activity... 

To date, only a small number of directed evolution studies of temperature adaptation have been carried out. From this small data set we can nonetheless draw several interesting conclusions. 

Directed evolution as a tool to probe the basis of protein structure, stability, and function is in its infancy, and many fruitful avenues of research remain to be explored. Studies so far have focused on proteins that unfold irreversibly, making detailed thermodynamic analysis impossible. The application of these methods to reversibly folding proteins could provide a wealth of information on the thermodynamic basis of high temperature stability. A small number of studies on natural thermophilic proteins have identified various thermodynamic strategies for stabilization. Laboratory evolution makes it possible to ask, for example, whether proteins have adopted these different strategies by chance, or whether certain protein architectures favor specific thermodynamic mechanisms. It will also be possible to determine how other selective pressures, such as the requirement for efficient low temperature activity, influence stabilization mechanisms. The combination of directed evolu-... 

The most extensive studies on directed evolution have been carried out with Thermus aquaticus DNA Pol I (Taq Pol I). Motifs A and B have both been targeted for random mutagenesis by complementing the recA718polA12 temperature-sensitive Pol I muta-... 

A common aim of directed evolution is to increase the stability of an enzyme to conditions of practical use that may be very different from those the enzyme naturally functions in. Factors such as heat, altered pH, and the presence of oxidants or organic solvents can lead to denaturation or loss of enzyme function. Many researchers have successfully increased the stability of an enzyme to thermal denaturation (41, 42). Work with p-nitrobenzyl esterase uicreased the melting temperature 14°C after six rounds of EP-PCR and recombination without forfeiting enzyme activity (41). As another example, phosphite dehydrogenase catalyzes the formation of phosphate from phosphite, by reducing NAD+ to NADH. However, the usefulness... 

Much like temperature, many industrial applications of enzymes require activity in the presence of organic solvents. Interestingly, Burton et al.118 have pointed out that thermostability and tolerance of organic solvents are characteristics that are often linked sequence and structural modifications that improve activity to one denaturing influence often also improve tolerance to others. Unfortunately, enzymes have generally evolved to function in aqueous media solvents are believed to denature and inactivate enzymes by exclusion of water from critical structural or functional components. Thus, many enzymes cannot tolerate common bioprocess conditions and techniques such as directed evolution must be used to improve their stability. 

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