Optimization of enzymes

Immobilization. Enzymes, as individual water-soluble molecules, are generally efficient catalysts. In biological systems they are predorninandy intracellular or associated with cell membranes, ie, in a type of immobilized state. This enables them to perform their activity in a specific environment, be stored and protected in stable form, take part in multi-enzyme reactions, acquire cofactors, etc. Unfortunately, this optimization of enzyme use and performance in nature may not be directiy transferable to the laboratory. 

When selecting a suitable feed symp, the main criteria are optimization of enzyme productivity and minimization of the formation of by-products. Typical feed symp specifications are shown in Table 5. Higher symp concentration and higher viscosity results in a reduced isomerization rate due to diffusion resistance in the pores of the immobilized enzyme. A deaeration step is desirable to remove dissolved oxygen that would otherwise iacrease the formation of by-products. The pH is adjusted to the optimum level for the productivity of the enzyme. 

An intriguing influence of a cosolvent immiscible with water on the enantioselec-tivity of the enzyme-catalyzed hydrolysis was observed. It was proven that enzyme enantioselectivity is directly correlated with the cosolvent hydrophobicity. In the best example, for ethyl ether as cosolvent, the reaction proceeded with E = 55, and the target compound was obtained in 33% yield with 92.7% ee. This finding may be of great practical importance, particularly in industrial processes [24], since it will enable better optimization of enzyme-catalyzed processes. It is clear that, in future, immobilized enzymes, as heterogeneous catalysts, wiU be widely used in most industrial transformations, especially in the preparation of pharmaceuticals [25]. 

HCV NS5B polymerase is an RNA-dependent RNA polymerase that is essential for viral replication. Thus, the inhibition of this enzyme offers a potential treatment for hepatitis C infection. Beaulieu et al. [51] report on the parallel optimization of enzyme inhibition potency and physical properties. In the first stage of hit characteri-... 

In a second example of the identification of IKK(3 inhibitor leads (termed IKK2 in this paper), Baxter et al. [54] report on the optimization of enzyme and cellular potency, physicochemical properties, ADME properties, and PK. This group targets... 

CDK2 is involved with controlling normal cell proliferation. Disregulation in cancer makes this a good antitumor target. Pevarello et al. [62] describe the parallel optimization of enzyme inhibition potency, cellular activity, physicochemical properties, and PK. A low MW hit (MW = 201) was specifically selected with the... 

DPP-4 is a serine protease that inactivates GLP-1. GLP-1 stimulates insulin secretion and suppresses glucagon release. The inhibition of DPP-4 prolongs the half-life of GLP-1 and brings about beneficial effects on glucose levels and glucose tolerance in type 2 diabetics. Backes et al. [64] report on the parallel optimization of enzyme binding affinity and inhibition, selectivity, ADME properties, and PK (Scheme 19). 

McGeehan, and J. Wiseman, Rapid optimization of enzyme substrates using defined substrate mixtures, J. Biol. Chem. 267 1434 (1992). 

The optimization of enzymes strongly depends on the field of appHcation. For industrial applications, high reaction rates, stabiHty under process conditions, and regio- and enantioselectivity are the most important properties of a catalyst, whereas affinity or substrate selectivity are of second order interest for a distinct process to be catalyzed. On the other hand, enzymes with a wide range of activity can be used for the production of several products reducing... 

Physiological optimization of enzyme synthesis by variation of the culture parameters is usually required to enhance the catalytic activity of whole-cell biocatalysts to such a level that it can be apphed in a biocatalytic process. In addition, physiological conditions can influence the selectivity of the reaction, since enzymes with opposite selectivities can be differentially expressed. In some cases, genetic engineering is required to obtain biocatalysts with a desired selectivity that does not consume the product of choice (see 5.3.5). Alternatively, one may choose to isolate the desired activity from the culture in order to use the biocatalyst in an enzyme reactor. 

Locating minima is not always straightforward since a reaction surface is usually complex, and a geometry optimization calculation will only locate minima close to the starting point. It is usually not feasible to systematically explore all possible conformers, so chemical intuition and corroborative evidence from experiment play important roles. A nice example is the identification of the coordination geometry of oxo-iron(IV) intermediate in TauD (22). As mentioned above, during optimization of enzyme active sites, key atoms are sometimes fixed to mimic the constraints that the protein environment exerts on the active site (20). 

Hydrates have further applications in bioengineering through the research of John and coworkers (Rao et al., 1990 Nguyen, 1991 Nguyen et al., 1991, 1993 Phillips et al., 1991). These workers have used hydrates in reversed micelles (water-in-oil emulsions) to dehydrate protein solutions for recovery and for optimization of enzyme activity, at nondestructive and low-energy conditions. 

S. Kamat, J. Barrera, E. J. Beckman, and A. J. Russell, Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids I. Optimization of enzyme environment, Biotechnol. Bioeng. 1992a, 40, 158-166. 

X. Chen, A. Johnson, J. S. Dordick, and D. G. Rethwisch, Chemoenzymatic synthesis of linear poly(sucrose acrylate) Optimization of enzyme activity and polymerization conditions, Macromol. Chem. Phys., 195 (1994) 3567-3578. 

Going beyond product synthesis and just looking into the path of assembly or optimization of enzyme systems to exploit the synthetic potential of NRPS systems was pioneered by Wang and colleagues in the 1970s, mainly on gramicidin S, and later also attempted by Kleinkauf and co-workers with gramicidin S as well as with enniatins, cyclosporins, and ACV-type of tripeptides (reviewed in [87]). 

Recently, a controversial debate has arisen about whether the optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of tunneling and thereby accelerate reaction rates [7]. Kinetic isotope effect experiments have indicated that hydrogen tunneling plays an important role in many proton and hydride transfer reactions in enzymes [8, 9]. Enzyme catalysis of horse liver alcohol dehydrogenase may be understood by a model of vibrationally enhanced proton transfer tunneling [10]. Furthermore, the double proton transfer reaction in DNA base pairs has been studied in detail and even been hypothesized as a possible source of spontaneous mutation [11-13]. 

Temperature is a variable of paramount importance in any bioprocess. Temperature optimization of bioreactor operation is a complicated task since many variables and parameters are involved that are strongly dependent on temperature. Besides, temperature exerts opposite effects on enzyme activity and stability. Then, thermal optimization of enzyme reactor operation requires that temperature explicit functions for all parameters involved be determined and validated. Optimization wifi... 

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