Biocatalytic reaction biotransformation

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... 

In recent years biotransformations have also shown their potential when applied to nucleoside chemistry [7]. This chapter will give several examples that cover the different possibiUties using biocatalysts, especially lipases, in order to synthesize new nucleoside analogs. The chapter will demonstrate some applications of enzymatic acylations and alkoxycarbonylations for the synthesis of new analogs. The utQity of these biocatalytic reactions for selective transformations in nucleosides is noteworthy. In addition, some of these biocatalytic processes can be used not only for protection or activation of hydroxyl groups, but also for enzymatic resolution of racemic mixtures of nucleosides. Moreover, some possibilities with other biocatalysts that can modify bases, such as deaminases [8] or enzymes that catalyze the synthesis of new nucleoside analogs via transglycosylation [9] are also discussed. 

The following examples illustrate Lonza s activities in large-scale biocatalysis for asymmetric synthesis. There are more examples of resolutions than true asymmetric syntheses, and this reflects the general current situation in the field of large-scale biotransformations. However, considerable research work is underway, both in universities and industry, to broaden the types of biocatalytic reactions that can be applied on a commercial scale. 

Hydrolases were in the first catalogue after the company was founded in 1950 but, not surprisingly, the chiral molecules originated mainly from the chiral pool. The first biocatalytic reactions were developed with kidney acylases and later with esterases and lipases, in the beginning mainly animal-derived biocatalysts [10], The set-up of in-house biocatalyst production from microbial and plant sources as well as the construction of a new biotechnology laboratory with ten fermenters of up to 300 L total volume, allow the development and production of improved biocatalysts and for them to be applied in the asymmetric synthesis of laboratory chemicals. There are today more than 100 biocatalytic processes in routine production and a project management team is handling custom biotransformations. 

For recent extensive reviews on biotransformations with lipases, see Kazlauskas and Bom-scheuer [77], Johnson [78], Rubin and Dennis [79], Itoh et al. [80], and Boland et al. [81]. The most widespread and frequently used biocatalytic reaction involving chiral compounds is kinetic resolution of racemates. Other biocatalytic stereoselective methods, although less frequently used, are asymmetrization of prochiral and meso compounds. These will be briefly discussed in Secs. C and D, respectively. 

The anticoagulant fondaparinux, a synthetic analogue of the terminal fragment of heparin, is synthesized using multiple protection/deprotection steps that result in a route of up to 50 steps. There is, as yet, no enzymatic system that approaches the capability to make such a molecule." As this modified pentasaccharide is a natural product, it should, in theory, be accessible through a series of biotransformations, but we currently lack the biocatalytic tools to achieve more than a few steps and would stiU need to use some protection steps to avoid multiple products. Enzymatic synthesis in vivo depends largely on the levels and selectivities of glycosylating enzymes to achieve multistep reactions, a situation that has been mimicked in vitro for simpler systems." ... 

In this section, enzymes in the EC 2.4. class are presented that catalyze valuable and interesting reactions in the field of polymer chemistry. The Enzyme Commission (EC) classification scheme organizes enzymes according to their biochemical function in living systems. Enzymes can, however, also catalyze the reverse reaction, which is very often used in biocatalytic synthesis. Therefore, newer classification systems were developed based on the three-dimensional structure and function of the enzyme, the property of the enzyme, the biotransformation the enzyme catalyzes etc. [88-93]. The Carbohydrate-Active enZYmes Database (CAZy), which is currently the best database/classification system for carbohydrate-active enzymes uses an amino-acid-sequence-based classification and would classify some of the enzymes presented in the following as hydrolases rather than transferases (e.g. branching enzyme, sucrases, and amylomaltase) [91]. Nevertheless, we present these enzymes here because they are transferases according to the EC classification. 

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. 

Enzyme-catalyzed reactions based on such biphasic systems have been shown to be promising alternatives for developing green chemical processes because of their physical and chemical characteristics [9]. By combining these media with enzymes, the possibilities of carrying out integral green biocatalytic processes has been already demonstrated [10-12]. Such biphasic systems can be used for both the biotransformation and extraction of products simultaneously, even under extremely harsh conditions, because of the different miscibilities of ILs and SC-CO2. 

Since the mid-1970s biotransformations have become a very well-established tool in the fine chemicals industry. Biocatalytic systems, including crude and purified enzymes as well as whole-cell systems performing highly selective reactions under mild conditions, are widely used, especially in synthesis and production of biologically active compounds in the agrochemical and pharmaceutical sectors. 

The biocatalytic addition of ammonia to trans-cinnamic acid, 28, proceeds enantio-selectively in the presence of whole-cells containing L-phenylalanine ammonia lyase. Of the suitable strains, wild-type strains of Rhodococcus rubra as well as Rhodotorula rubra were found to be very efficient. The biotransformation is carried out in basic media at pH 10.6. In addition, analogous reactions furnishing non-natural substituted derivatives of L-phenylalanine starting from substituted trans-cinnamic acid derivatives were reported by Mitsui, and Great Lakes, respectively [38 c]. 

When a low-water biotransformation is planned, a variety of choices must be made about the precise reaction conditions to be used. As in all biocatalytic systems, the rate and yield obtained can be greatly affected by the choices made. Some of the factors that must be considered are the same as in conventional aqueous media temperature, reactant concentrations, the form in which the enzyme is added. New factors must be taken into account such as solvent selection and the level of residual water in the system. Other factors become somewhat modified acid-base conditions remain important, but, usually, pH is no longer a useful parameter to characterize them. 

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