Numerous processes utilizing biocatalysts for the production of fine and bulk chemicals, pharmaceuticals or agrochemicals are established in industry today [72]. An ever in-creasing availability of novel biocatalysts, improvements in bioengineering tools for the optimization and modification of enzymes as well as safety, health and environmental issues still boost the number of industrial application of enzymes [143, 148]. Examples of implemented biocatalytic processes include the use of a racemase/d-hydantoinase/d -carbamoylase enzyme system for the production of enantiomerically pureα-amino acids at Evonik shown in Figure 1.3 [84], the use of pig liver esterase for the production of various pharmaceutical intermediates at manufacturing scale at DSM [39] or the bulk production of acrylamide from acrylonitrile using nitrile hydratases in whole cells of Rhodococcus sp. at Nitto Chemical Ltd. [72].
HN NH
Figure 1.3: Reaction scheme of the racemase/d-hydantoinase/d-carbamoylase system for the enantioselective production of α-amino acids [84].
1.2.1 (Bio)process development
The development and optimization of bioprocesses needs to consider the properties of the biocatalyst and the chemical reaction it catalyzes on the one hand, and principles of chemical engineering on the other hand [43]. Biocatalyst and reaction characteristics are traditionally mainly addressed by the natural scientist as they require comprehen-sive (bio)-chemical know-how, whereas reactor design, optimization and scale-up are classically performed by the process engineer. As depicted in Figure 1.4, all disciplines are highly interconnected and require interdisciplinary thinking from an early stage of development on. Some general aspects are discussed in the following. An extensive discussion of strategic approaches in bioprocess development can be found in literature [22, 43, 66].
With regard to the biocatalyst, two major questions need to be answered: (1) is there a need for optimization of the enzyme itself via protein engineering? (2) Should whole cell catalysts, immobilized enzymes or free, isolated enzymes be used? Nature provides
1.2 Industrial biocatalysis
Figure 1.4: (Bio)process development: aspects for optimization.
a good toolbox of diverse catalysts, but often these require tailoring in order to adapt the catalyst to non-natural substrates and industrial process conditions [92]. The use of whole-cell biocatalysts has many advantages such as low cost enzyme supply and good stability and are therefore often preferred industrially. However, side reactions and limitations with regard to mass transfer may occur and limit the applicability of whole-cell preparations. One or more additional steps are necessary to obtain isolated enzymes, which usually goes in hand with loss of activity yield. On the other hand, side reactions may be completely suppressed and stability issues improved by immobilization.
The type of enzyme preparation has immediate consequences on the choice of a suited reactor type. Since enzyme costs usually add significantly to the overall cost of the process, enzyme retention and reuse is of major concern. For reactions carried out in batch mode, enzyme recovery can be achieved by (ultra)filtration, centrifugation and other physical methods such as magnetic forces when magnetic carriers are used. In continuous processes, membranes of a suited pore size can be used for the retention of whole-cells and isolated enzymes. Packed bed reactors are often used in the case of immobilized enzymes.
Once a suitable biocatalyst has been chosen, a detailed investigation of suited physico-chemical reaction conditions (T, pH, solvent etc.) and their influence on enzyme stability as well as kinetic and thermodynamic properties of the reaction is usually carried out.
Based on the obtained results a suited reactor type may be chosen. A number of opti-mization targets may be defined. These include aspects such as facility of downstream-processing and reducing general process costs (energy consumption, hardware), as well as specific process parameters such as the achievement of a high space-time yield (STY), conversion X, (enantio)selectivity, a low E-factor2 and maximal total turnover number (ttn) of catalyst and cofactor.3
2The E-factor introduced by Sheldon [114] measures the ”greeness” of a reaction and is defined as the weight of waste produced per weight of product.
3Important definitions frequently used in this study are summarized in Appendix B on page 109.
1 Introduction
1.2.2 Highly concentrated and solvent-free reactions
In industrial application it is generally highly desired to use high substrate concentrations in biocatalytic processes. The use of solvent-free reactions as the extreme is therefore especially interesting. Higher yields and reaction rates can usually be achieved. Addi-tionally, energy costs are reduced and a largely improved E-factor can be obtained. The necessity for smaller reactor sizes leads to decreased capital investments [135]. While a number of solvent-free chemical and biocatalytic reactions including e.g. polymer-izations and esterifications have been described in literature up to date, only relatively few solvent-free enantioselective biocatalytic processes can be found of which some are summarized here. Von Langemann et al. used an (S)-selective hydroxynitrile lyase from Manihot esculenta for the conversion of acetophenone and derivatives to the correspond-ing cyanohydrins accordcorrespond-ing to Figure 1.5 [134]. Excellent ee’s of> 99 % were obtained at a very good yield of up to 78 %.
Figure 1.5: Hydroxynitrile lyase-catalyzed conversion of acetophenone to corresponding cyanohydrin [134].
Li et al. observed an interesting unnatural ability of nuclease p1 to catalyze the solvent-free aldol addition of aromatic aldehydes and cyclic ketones according to Figure 1.6 in a solvent-free system [71]. However, the obtained yields of 17-55 % atee 49-99 % were low to satisfactory.
Figure 1.6: Nuclease p1-catalyzed aldol addition in solvent-free system [71].
The Candida antarctica lipase A (CALA)-catalyzed enantioselective transesterifica-tion of methyl 2-chloromandelate via transesterificatransesterifica-tion with vinylpropionate in a solvent-free system was described bei Uhm et al. [131]. The resulting chiral (S)-chloromandelic acid ester is a precursor for pharmaceutical products such as the antiplatelet agent (S )-clopidogrel. A highee of >99 % was reached at a yield of 41 %.
Xiong and coworkers described the enantioselective lipase-catalyzed transesterification of mandelonitrile and vinyl acetate [145]. Depending on the lipase used,>99 %ee were