Enzyme processes, selectivity

Acetylsucrose [63648-81-7] has been prepared in 40% yield by direct acetylation of sucrose using acetic anhydride in pyridine at 40° C (36). The 6-ester has subsequently been obtained in greater than 90% yield, by way of 4,6-cycHc orthoacetate. Other selective methods for the 6-acylated derivatives include the use of alkyl tin reagents such as dibutyl tin oxide (37) and of dibutyl stannolane derivatives (38). Selective acetylation of sucrose by an enzymic process has also been described. Treatment of sucrose with isopropenyl acetate in pyridine in the presence of Lipase P Amano gave, after chromatography, 6-0-acetylsucrose (33%) and 4/6-di-O-acetylsucrose (8%). The latter compound has been obtained in 47% yield by the prolonged treatment (39). 

The resolution of racemic ethyl 2-chloropropionate with aliphatic and aromatic amines using Candida cylindracea lipase (CCL) [28] was one of the first examples that showed the possibilities of this kind of processes for the resolution of racemic esters or the preparation of chiral amides in benign conditions. Normally, in these enzymatic aminolysis reactions the enzyme is selective toward the (S)-isomer of the ester. Recently, the resolution ofthis ester has been carried out through a dynamic kinetic resolution (DKR) via aminolysis catalyzed by encapsulated CCL in the presence of triphenylphosphonium chloride immobilized on Merrifield resin (Scheme 7.13). This process has allowed the preparation of (S)-amides with high isolated yields and good enantiomeric excesses [29]. 

Compared with ketoreductases, the synthetic application of alcohol oxidases has been less explored. However, selective oxidation of primary alcohols to aldehydes is superior to the chemical methods in terms of conversion yields, selectivity, and environmental friendliness of reaction conditions. In addition, coupling of alcohol oxidase with other enzymes provides a tremendous opportunity to develop multi-enzyme processes for the production of complex molecules. Therefore, a growing impact of alcohol oxidases on synthetic organic chemistry is expected in the coming years. 

The outstanding feature of the active transport is the selectivity of the complexation process, between K+ and Na. The enzyme is selective first to Na+ and then to K+ in its different conformations. Selectivity is governed by coulombic forces and differences in ionic radii. 

Inside the cell, numerous chemical processes take place at the same time. The cell solves the problem of generating a large number of different molecules at the same time with a high selectivity by the use of enzymes. The selectivity of these proteins is largely determined by their geometry. Also the selectivity of another class of proteins, the receptors, is influenced by geometrical features. Receptors and enzymes have in common that they are equipped with concave structures such as clefts or cavities [1] in which substrate molecules are bound or chemically modified. 

Finally, in a concluding paper Dr. Jorg Thommes considers enzyme recovery in Fluidized Bed Adsorption as a Primary Recovery Step in Protein Purification . As important as it is to track down new enzymes and selectively modify them, it remains equally important to actually make them available in the flask on the bench in adequate quantities at low cost with sufficient purity. Recovery is of central significance in this respect. Fluidized bed adsorption combines the process steps of cell separation, concentration and primary cleaning in recovery work. The procedure can also be excellently transferred from the laboratory to the pilot scale. 

Although molybdenum and tungsten enzymes carry the name of a single substrate, they are often not as selective as this nomenclature suggests. Many of the enzymes process more than one substrate, both in vivo and in vitro. Several enzymes can function as both oxidases and reductases, for example, xanthine oxidases not only oxidize purines but can deoxygenate amine N-oxides [82]. There are also sets of enzymes that catalyze the same reaction but in opposite directions. These enzymes include aldehyde and formate oxidases/carboxylic acid reductase [31,75] and nitrate reductase/nitrite oxidase [83-87]. These complementary enzymes have considerable sequence homology, and the direction of the preferred catalytic reaction depends on the electrochemical reduction potentials of the redox partners that have evolved to couple the reactions to cellular redox systems and metabolic requirements. 

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). 

Common catalytic systems are characterized by the presence of reagent molecules only, whereas the enzymatic system is multicomponent and possesses low concentrations of the substrates in water. The interaction between a substrate with an oxidant or a reducer is most often considered. This makes unnecessary simulation of the enzyme selectivity. However, free contact of reagent molecules with active sites preserves the possibility of various mechanism realizations which is the reason for decrease of the process selectivity. Apparently, a compromise should be found in resolving the question of selectivity in biomimics development in suggesting that, though complex gap mechanism is the effective method for distance and mutual orientation control of reactive groups in the enzyme, it may hardly be implemented in synthetic systems. 

The light chain, when separated from the heavy chain, behaves as an enzyme that selectively cleaves a peptide associated with synaptic vesicles. This peptide, synapt-obrevin, is required for docking and fusion of the vesicle during acetylcholine release. By enzymatically cleaving it, botulinum toxin renders vesicle docking and fusion impossible, and cholinergic neurotransmission comes to a halt. Because the light chain has enzymatic activity, just a few molecules can catalyze the destruction of synaptobrevin on thousands of vesicles. In this way, extreme potency is achieved via amplification of the process. 

Milk fat is valued for its pleasant flavor but its melting and rheological properties often need to be modified to make it more suitable for many food applications. The uses of milk fat can be increased by the application of various processing interventions such as fractionation, selective blending and texturization, and chemical or enzymic processes to produce speciality milk fat ingredients (Kaylegian, 1999). Most of these modification procedures... 

BDF has attracted attention as an eco-friendly fuel, and its demand is expected to increase further. We have introduced enzymatic processes which eliminate the drawbacks of chemical process. Not only the enzymatic process, but also processes using supercritical MeOH, ion-exchange resins, and MeOH vapor will likely be used in the future, and a company will decide the most suitable process for its own production environment. We hope that an enzyme process will be selected as a candidate. 

The most widely used immobilized enzyme process involves the use of the enzyme glucose isomerase for the conversion of glucose to fructose in com syrup (Carasik and Carroll 1983). The organism Bacillus coagu-lans has been selected for the production of glucose isomerase. The development of the immobilized cell slurry has not proceeded to the point where half-lives of the enzyme are more than 75 days. A half-life is defined as the time taken for a 50 percent decrease in activity. Such immobilized enzyme columns can be operated for periods of over three half-lives. 

Figure 10.30 Evolution of a natural, RNA-cleaving ribozyme into a DNA-cleaving enzyme the selection/amplification process using the selection/amplification substrate 10.34.

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