Biocatalyst reaction conditions

Biocatalysts usually require mild reaction conditions for an optimal activity (physiologic temperature and pH) and, in general, they show high activity, chemo- and enantioselectivity. Furthermore, when using enzymes, many functional group protections and/or activations can be avoided, allowing shorter synthetic transformations. The use of enzymes is therefore very attractive from an environmental and economic point of view. 

For most applications, enzymes are purified after isolation from various types of organisms and microorganisms. Unfortunately, for process application, they are then usually quite unstable and highly sensitive to reaction conditions, which results in their short operational hfetimes. Moreover, while used in chemical transformations performed in water, most enzymes operate under homogeneous catalysis conditions and, as a rule, cannot be recovered in the active form from reaction mixtures for reuse. A common approach to overcome these limitations is based on immobilization of enzymes on solid supports. As a result of such an operation, heterogeneous biocatalysts, both for the aqueous and nonaqueous procedures, are obtained. 

Over the years of evolution, Nature has developed enzymes which are able to catalyze a multitude of different transformations with amazing enhancements in rate [1]. Moreover, these enzyme proteins show a high specificity in most cases, allowing the enantioselective formation of chiral compounds. Therefore, it is not surprising that they have been used for decades as biocatalysts in the chemical synthesis in a flask. Besides their synthetic advantages, enzymes are also beneficial from an economical - and especially ecological - point of view, as they stand for renewable resources and biocompatible reaction conditions in most cases, which corresponds with the conception of Green Chemistry [2]. 

Biodesulfurization (BDS) is the excision (liberation or removal) of sulfur from organosul-fur compounds, including sulfur-bearing heterocycles, as a result of the selective cleavage of carbon-sulfur bonds in those compounds by the action of a biocatalyst. Biocatalysts capable of selective sulfur removal, without significant conversion of other components in the fuel are desirable. BDS can either be an oxidative or a reductive process, resulting in conversion of sulfur to sulfate in an oxidative process and conversion to hydrogen sulfide in a reductive process. However, the reductive processes have been rare and mostly remained elusive to development due to lack of reproducibility of the results. Moderate reaction conditions are employed, in both processes, such as ambient temperature (about 30°C) and pressure. 

Up to the late 1990s, combined multi-step chemo-chemo conversions were restricted to a few catalytic examples. Apparently, there has been little effort or interest in developing a toolkit of chemocatalytic reactions that are mutually compatible with respect to reaction conditions. Consequently, chemocatalysts have not yet reached the same level of mutual compatibility as biocatalysts. Some recent examples prove, however, the potential power of chemo-chemo catalytic cascades. 

The use of CLEA preparations of commercially available HNLs allowed for the enantiocomplementary production of cyanohydrins from a pyridinecarboxaldehyde at a much higher chiral purity than had previously been demonstrated with any chemical catalyst. The key to the success of this process was the use of the CLEA -immobilized biocatalysts that allowed reaction conditions to be chosen to minimize the negative effects of the nonspecific background reaction. 

Cycloaddition of azodicarboxylates to 2-vinylpyridine in the presence of a biocatalyst (Saccharomyces cerevisiae) proceeded in a highly selective fashion to afford only pyrido[3,2-r ]pyridazine derivatives 259 in >80% yield compared to that found in the literature when the reaction was carried out in an organic medium to give a mixture of 259 in <20% yield and its isomer pyrido[l,2-r ]triazine derivatives 260 <1979T2027, 1993BCJ2429>. Similarly, 1,2,3,4-tetrahydropyrido[3,4-r ]pyridazine-l,2-dicarboxylate 261 was obtained under the same reaction conditions in >60% yield by using 4-vinylpyridine. 

Unlike the syntheses known hitherto, by applying these new biocatalysts it is possible to produce chiral alcohols with optical purities of >99% under moderate reaction conditions in an aqueous milieu. 

When choosing reaction conditions, such as water activity and solvent, for an enzymatic reaction, possible effects on the equilibrium position of the reaction should be considered. When the aim is to produce an equilibrium mixture as the final product, the position of this equilibrium is of course of vital importance. It is, however, also important that the biocatalyst expresses sufficient catalytic activity under the conditions used, so that equilibrium is reached within a reasonable time. In practice, it often happens that a compromise must be made between high reaction rate and high equilibrium conversion. 

The desire for a sustainable development in chemistry lays the foundation for environmentally benign processes. From the view point of organic chemistry, the construction of carbon skeletons plays the pivotal role. The extraordinarily mild reaction conditions in addition to the non-toxic and non-bumable properties and ubiquitous availability of water as the reaction medium make enzyme-catalyzed C-C-bond formation the first choice even for industrial production. Thanks to subtle selectivity features of the corresponding enzymes a rather broad range in substrate specificity meets with a highly conserved stereospecificity at the newly connected carbon centers. In addition, these features and the availability of the respective biocatalysts are open to intervention by recombinant genetechnological techniques. 

Biocatalysts do not operate by different scientific principles from organic catalysts. The existence of a multitude of enzyme models including oligopeptidic or polypeptidic catalysts proves that all enzyme action can be explained by rational chemical and physical principles. However, enzymes can create unusual and superior reaction conditions such as extremely low pfCa values or a high positive potential for a redox metal ion. Enzymes increasingly have been found to catalyze almost any reaction of organic chemistry. 

The catalytic action of biocatalysts (enzymes, abzymes, antibodies, cells) is extremely efficient and selective compared to conventional chemical catalysts. They demonstrate higher reaction rates, milder reaction conditions and greater stereospecificity. Most of these properties come from the high molecular flexibility biocatalysts exhibit. On the other hand, this is also the origin of their major limit that holds back their application at the large scale, that is, the molecular stability, and then the catalyst lifetime. 

One of the reactions catalyzed by esterases and lipases is the reversible hydrolysis of esters (Figure 19.1, Reaction 2). These enzymes also catalyze transesterilications and the desymmetrization of mew-substrates (vide infra). Many esterases and lipases are commercially available, making them easy to use for screening desired biotransformations without the need for culture collections and/or fermentation capabilities.160 In addition, they have enhanced stability in organic solvents, require no co-factors, and have a broad substrate specificity, which make them some of the most ideal industrial biocatalysts. Alteration of reaction conditions with additives has enabled enhancement and control of enantioselectivity and reactivity with a wide variety of substrate structures.159161164... 

The rapid development of biotechnology during the 1980s provided new opportunities for the application of reaction engineering principles. In biochemical systems, reactions are catalyzed by enzymes. These biocatalysts may be dispersed in an aqueous phase or in a reverse micelle, supported on a polymeric carrier, or contained within whole cells. The reactors used are most often stirred tanks, bubble columns, or hollow fibers. If the kinetics for the enzymatic process is known, then the effects of reaction conditions and mass transfer phenomena can be analyzed quite successfully using classical reactor models. Where living cells are present, the growth of the cell mass as well as the kinetics of the desired reaction must be modeled [16, 17]. 

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