Cofactors biocatalytic reactions

The highly selective biocatalytic reactions afford a substantial reduction in waste. The overall isolated yield is greater than 90%, and the product is more than 98% chemically pure with an enantiomeric excess of >99.9%. All three evolved enzymes are highly active and are used at such low loadings that counter-current extraction can be used to minimize solvent volumes. Moreover, the butyl acetate solvent is recycled with an efficiency of 85%.The E factor (kgs waste per kg product) for the overall process is 5.8 if process water is excluded (2.3 for the reduction and 3.5 for the cyanation) [47]. If process water is included, the E factor for the whole process is 18 (6.6 for the reduction and 11.4 for the cyanation). The main contributors to the E factor are solvent losses which accounted for 51% of the waste, sodium gluconate (25%), NaCl and Na2SO4 (combined circa. 22%). The three enzymes and the NADP cofactor account for <1% of the waste. The main waste streams are aqueous and directly biodegradable. 

Whole-cell biocatalytic reactions are most often used when the biotransformation to be conducted requires the input of energy. In biological systems this usually takes the form of reduced pyrimidine nucleotides or ATP but can be many of a number of reduced cofactors or modified reaction components. Using the whole cell allows the technologist to take advantage of the intact, preformed cellular machinery to efficiently provide the required cofactors or components. In order to provide the energy to catalyze these reactions a source of reducing power is usually required. The cooxidation of an oxidizable substrate such as... 

Biocatalytic synthetic reactions also include carbon dioxide fixation with the production of methanol in artificial multi-enzyme systems [188]. Formate dehydrogenase (FDH, EC 1.2.1.2) can catalyze the reduction of carbon dioxide to formate, and methanol dehydrogenase (MDH, EC 1.1.99.8) can catalyze the reduction of formate to methanol. Both of these enzymes require NAD+-NADE1 cofactor, and in the presence of the reduced dimethyl viologen mediator (MV+), they can drive a sequence of enzymatic reactions. The cascade of biocatalytic reactions results in the reduction of CO2 to formate catalyzed by FDEI followed by the reduction of formate to methanol catalyzed by MDH. A more complex system composed of immobilized cells of Parococcus denitrificans has been demonstrated for the reduction of nitrate and nitrite [189]. 

The same factors discussed in the previous section as being important for biocatalytic reactions in general apply to biocatalytic oxidation reactions as well. In addition, however, another aspect of biocatalysts becomes important the use of cofactors or coenzymes. Many enzymes function with the help of a specific cofactor, a small non-protein organic or metallo-organic group that is capable of facilitating the reaction to be performed. Enzymes that catalyze oxidation re-... 

Together with enantioselective hydrolysis/acylation reactions, enantioselective ketone reductions dominate biocatalytic reactions in the pharma industry [10], In addition, oxidases [11] have found synthetic applications, such as in enantioselective Baeyer-Villiger reactions [12] catalyzed by, for example, cyclohexanone monooxygenase (EC 1.14.13) or in the TEMPO-mediated oxidation of primary alcohols to aldehydes, catalyzed by laccases [13]. Hence, the class of oxidoreductases is receiving increased attention in the field of biocatalysis. Traditionally they have been perceived as difficult due to cofactor requirements etc, but recent examples with immobilization and cofactor regeneration seem to prove the opposite. 

Biocatalytic reaction using whole cells is more stable than isolated enzyme reaction, and can reduce the cost of catalysts and cofactors [10,49], because there is no need to disrupt cells and purify enzyme. 

Due to the high price of nicotinamide cofactors, their stoichiometric use in preparative biocatalytic reactions is prohibitive. Hence, for preparative application, in situ regeneration of the catalytically active, reduced form has been the preferred approach for many decades now [2a,3] and many excellent reviews are available summarizing the state of the art here la,4]. The most relevant approaches are summarized in Table 8.1 according to the nature of the... 

The use of organic solvents as reaction media for biocatalytic reactions can not only overcome the substrate solubility issue, but also facilitate the recovery of products and biocatalysts as well. This technique has been widely employed in the case of lipases, but scarcely applied for biocatalytic reduction processes, due to the rapid inactivation and poor stability of redox enzymes in organic solvents. Furthermore, all the advantages for nonaqueous biocatalysis can take effect only if the problem of cofactor dependence is also solved. Thus, bioreductions in micro- or nonaqueous organic media are generally restricted to those with substrate-coupled cofactor regeneration. 

Cofactors Commonly Encountered in Biocatalytic Reactions Applied to Chemical Synthesis... 

Biocatalytic ledox reactions offer great synthetic utility to organic chemists. The majority of oxidase-catalyzed preparative bioconversions are still performed using a whole-ceU technique, despite the fact that the presence of more than one oxidoreductase in cells often leads to product degradation and lower selectivity. Fortunately, several efficient cofactor regeneration systems have been developed (160), making some cell-free enzymatic bioconversions economically feasible (161,162). 

Biocatalytic hydrolysis or transesterification of esters is one of the most widely used enzyme-catalyzed reactions. In addition to the kinetic resolution of common esters or amides, attention is also directed toward the reactions of other functional groups such as nitriles, epoxides, and glycosides. It is easy to run these reactions without the need for cofactors, and the commercial availability of many enzymes makes this area quite popular in the laboratory. 

Biocatalytic approaches to cofactor regeneration can be divided into coupled-enzyme methods and coupled-substrate methods.In the coupled-enzyme method, the oxidized cofactors (NAD+ and NADP+) are recycled in situ by performing an oxidation reaction using a second enzyme and an inexpensive auxiliary substrate. This second enzyme must employ the same cofactor, but neither enzyme should be able to accept the same substrate. 

The biocatalytic reduction of carboxylic acids to their respective aldehydes or alcohols is a relatively new biocatalytic process with the potential to replace conventional chemical processes that use toxic metal catalysts and noxious reagents. An enzyme known as carboxylic acid reductase (Car) from Nocardia sp. NRRL 5646 was cloned into Escherichia coli BL21(DE3). This E. coli based biocatalyst grows faster, expresses Car, and produces fewer side products than Nocardia. Although the enzyme itself can be used in small-scale reactions, whole E. coli cells containing Car and the natural cofactors ATP and NADPH, are easily used to reduce a wide range of carboxylic acids, conceivably at any scale. The biocatalytic reduction of vanillic acid to the commercially valuable product vanillin is used to illustrate the ease and efficiency of the recombinant Car E. coli reduction system." A comprehensive overview is given in Reference 6, and experimental details below are taken primarily from Reference 7. 

Applications of whole-cell biocatalytic membrane reactors, in the agro-food industry and in pharmaceutical and biomedical treatments are listed by Giorno and Drioli [3], Frazeres and Cabral [9] have reviewed the most important applications of enzyme membrane reactors such as hydrolysis of macromolecules, biotransformation of lipids, reactions with cofactors, synthesis of peptides, optical resolution of amino acids. Another widespread application of the membrane bioreactor is the wastewater treatment will be discussed in a separate section. 

For the asymmetric biocatalytic reduction of ketones with in situ cofactor regeneration an enzyme-compatible biphasic reaction medium has been developed (Fig. 34) [53],... 

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