Selection of the Enzyme

In contrast to most biotransformation projects, the work for the present biotransformation did not start with an enzyme screening. Only Subtilisin Carlsberg, by far the best enzyme obtained in the screening for the racemic resolution of nitrile 

As Subtilisin Carlsberg was, and still is, one of the cheapest enzymes available on the market, no further proteases were tested. Four Subtilisin Carlsberg preparations from Solvay Enzymes [9] and Novo Nordisk [10] were tested successfully Solvay-Protease M 440 (solid form) and L 660 (liquid form) as well as the Novo enzymes Alcalase 2.0 T (solid) and Alcalase 2.5 L (liquid). 

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It is possible to use an enzyme with hydrogen peroxide in a combined desize-bleach but great care is needed in selection of the enzyme and optimisation of the concentrations [319]. 

Lowe et al. investigated the substrate selectivity of the proteases papain and chy-motrypsin using PEGA-bound combinatorial peptide hbraries (32) (Fig. 10.3) [24]. The quahtative extent of enzymatic cleavage of the resin-bound peptide in case of an accepted substrate strand was rapidly visualized by a significant reduction in the fluorescence of the beads visuahzed with a fluorescence microscope. Furthermore it was proven that substrate selectivity of the enzyme remain the same on the solid phase and in solution. 

This is the definition of the enantiomeric ratio E, which indicates the selectivity of the enzyme for the fast reacting enantiomer (A ) relative to the slowly reacting enantiomer... 

Figure 2.8 shows the enantiomeric excess of the remaining substrate A as a function of the conversion, for several values of the enantiomeric ratio, according to the Chen equation. When the selectivity of the enzyme is high ( =100), the remaining substrate is enantiopure from 50% conversion on. For lower selectivities, the yield of enantiopure substrate is lower. But sooner or later an enantiomeric excess of virtually 100% will be reached for the remaining substrate. At the moment that a sufficiently high ee has been obtained, the reaction should be terminated, because otherwise this enantiopure substrate will be converted further until racemic product remains. 

Of course, the selection of the appropriate enzyme is fundamental because yield and selectivity of the enzymes vary extremely. For instance, Candida ru-gosa lipase will give high yields but has a low selectivity. In contrast, lipase from Aspergillus niger exhibits high selectivity [i3]. 

When water molecules interact with an enzyme, it is natural that conformational changes can occur, which in turn can cause changes in the selectivity of the enzyme. Since enantioselectivity of enzymes is of major importance for many applications, it is a common task to investigate how to choose reaction conditions providing the maximal enantioselectivity. As might be expected, because water can interact with enzymes in many ways, it is difficult to generalize the effects. In some studies of lipase-catalyzed esterification reactions, no effects of water activity on enantioselectivity were observed [30]. In a similar study, no effects were observed in most cases, while the enantioselectivity of one lipase-catalyzed reaction decreased... 

The competition between transferase and hydrolysis reactions can be described in terms of nucleophile (acceptor) selectivities of the enzymes, and selectivity constants can be defined. These constants are meant to quantify the intrinsic selectivity of the enzymes. Selectivity constants in combination with the concentrations (or thermodynamic activities) of the competing nucleophiles give the transferase/hydrolysis ratio. The selectivity constants are defined as follows [38, 39] ... 

Enzymes provide catalytic pathways which are often superior to non-enzymic routes. The advantage of enzymes can overcome their disadvantages as relatively unstable species which workbest in aqueous solution. In synthetic processes, the selectivity of the enzyme-catalysed process is not approached by chemical catalysis particularly regarding the enantioselective synthesis of chiral molecules. Furthermore, protective group chemistry is often not required in enzyme-catalysed reactions. 

The conventional production consisted of many process steps, typical for a fine chemical process. These process steps are given in Figure 6. The selectivity of the enzyme is rather high. The downstream processing is rather laborious,... 

Comparison of Figs. 3 and 4 reveals that alcohol chain length exerted more influence on ester yield than the acid carbon size. For alcohol size varying from C5 to C8, conversion was lower than 40%, and for acid size from C4 to Cl6, similar esterification yields (60-80%) were found. These results may reflect both the intrinsic selectivity of the enzyme and different accessibility of substrates to enzyme active site (22). 

The first step in the clinical development of rofecoxib was to determine the degree of selectivity when the drug was administered to humans. As outlined above, the assessment of selectivity of Cox inhibitors for the two isoforms is dependent on the systems used and in vitro systems may not reflect the degree of selectivity of the enzyme inhibitors in vivo. Two clinical models were used to assess the selectivity of rofecoxib in humans. For both tests, tissues were obtained from patients or volunteers receiving either rofecoxib or NSAIDs, and the ability of these tissues to synthesize prostanoids ex vivo was determined. The simplest of these models is the whole blood assay, which was mentioned earlier in this chapter as one of the tests than can be used for evaluation of Cox selectivity (Patrignani et al, 1994). To use this test in the clinical setting, two samples of blood are drawn from individuals receiving a Cox inhibitor. One sample is allowed... 

The addition of small molecules has been shown to change the enantioselectivity of certain enzyme-catalyzed reactions. It is believed that such molecules bind to a site in the protein different from the active site, which leads to a conformational change in the active site. Such enzymes are called allosteric enzymes, i.e. enzymes that comprise of multiple subunits and multiple active sites. Binding of a cosubstrate or small molecule may cause an increase or decrease in the activity or selectivity of the enzyme. 

As suggested in Example 29-4, urea can be determined by measuring the initial rate of production of the products of this reaction. The high selectivity of the enzyme permits the use of nonselective detection methods, such as electrical conductance, for initial rate measurements. There are commercial instruments that operate on this principle. The sample is mixed with a small amount of an enzyme-buffer solution in a conductivity cell. The maximum rate of increase in conductance is measured within 10 s of mixing, and the concentration of urea is determined from a calibration curve consisting of a plot of maximum initial rate as a function of urea concentration. The precision of the instrument is on the order of 2% to 5% for concentrations in the physiological range of 2 to 10 mM. 

Considerations of this kind led the author to undertake an investigation using enzymes to reveal the chemical nature of phosphorus bonds that may occur in phosphoproteins. This interest came through the accidental observation that a variety of phosphomonoesterases of mammalian origin and from plants will dephosphorylate ovalbumin, a protein with a low phosphorus content. Of course, a prerequisite in the selection of the enzymes for such work is that the dephosphorylation process should not be accompanied by any other enzymatic reactions that might result from the presence of small amounts of impurities in even highly purified phosphatase preparation. in particular, an extensive proteolysis has to be excluded. 

The reason why these enzymes have received considerable attention over the years is that they display a high degree of enantiotopic selectivity on the prochiral aldehyde and ketone substrates. The selectivity of these enzymes is in many instances masked by the rate of spontaneous racemization of the cyanohydrins, which are prone to racemization under non-acidic conditions. This balance of selectivity of the enzymes versus the competition with the spontaneous racemization reaction as a function of the pH was described as early as 1921 using the hydroxynitrile lyase enzyme from peach leaves [22], These early experiments describe one of the challenges of applying hydroxynitrile lyases on an industrial scale. 

To achieve true homogeneous catalysis, enzyme solubility may be increased by coupling polyethyleneglycol to its surface1871. The coupling may alter the stability, activity and selectivity of the enzyme. 

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