BIOCATALYSIS


and the intermediates are of the same nature. Therefore, reaction cascades catalysed by a single enzyme are first-generation processes. When following the logic of the chosen classification of enzymatic processes, the final or fourth-generation biotransformations would be defined as coupled


chemoenzymatic reactions in continuous- flow systems based on all three of the biological principles mentioned. From a technical point of view, biotransformations of the fourth generation are the most


sophisticated ones, most closely resembling the metabolic activity of a living cell. At the same time, however, due to their complexity,


“The most elegant and technically the most attractive way to perform coupled chemoenzymatic


cofactor was achieved by its covalent binding to polyethylene glycol (PEG) with a molecular weight of 10 kDa. The reactor was operated continuously for 48 days under sterile conditions. A maximal conversion of 99.7% and a space-time yield (STY) of 42.5 g/l per day were reached. Hummel and


coworkers similarly described the stereoselective


reactions is to bring all reactants and catalysts in contact in one vessel in one


reaction medium”


these biotransformations are not as widespread as the enzymatic processes of earlier generations and their development is still at an early stage. Nevertheless, they can already be considered as a complementary technology to multi-step continuous-flow organic synthesis: A chemical technology that has recently emerged on the frontier of organic chemistry and chemical engineering.8,21 This article presents achievements and challenges of fourth- generation biotransformations, and highlight existing trends in this field, which in the foreseeable future may lead to the next generation of enzymatic processes.


Single-reactor processes in vitro Certainly, the most elegant and technically the most attractive way to perform coupled chemoenzymatic reactions is to bring all reactants and catalysts in contact in one vessel filled with one reaction medium. In this case the requirements with respect to equipment are reduced to a minimum. The single-reactor or so-called ‘in-pot’17 coupled-reaction processes in continuous flow were established as early as 1981, most probably for the first time by Wichmann and Wandrey for the continuous production of L-leucine in an ultrafiltration membrane reactor using a coupled parallel enzymatic system.22 Here, L-leucine dehydrogenase was used for the reductive amination of α-ketoisocaproate to the amino acid. The required cofactor NADH was regenerated in a coupled parallel reaction by oxidation of formate catalysed by formate dehydrogenase. Retention of the


conversion of benzoyl formate to D-mandelic acid by a D-(-)-mandelic acid dehydrogenase from Lactobacillus curvatus in a sterilised enzyme membrane reactor, which was operated continuously.23 Again, formate dehydrogenase was used for cofactor regeneration, and


immobilisation of NADH on PEG prevented loss of the cofactor in the continuously operated reactor. Space-time yields of 700 g/l per day were achieved with the optimised reactor at 95% conversion. Deactivation of enzyme and PEG-NADH led to a decrease of conversion to 90%. However, by adjustment of the residence time, continuous operation for 20 days was demonstrated. Covalent grafting of cofactors on soluble polymers for retention in continuously operating membrane reactors suffers from the disadvantage that enzymes often do not accept the cofactor derivatives. To overcome this problem, Obón et al proposed a new concept to retain native NADP(H) without chemical modification inside an enzyme membrane reactor by adding charged soluble polymers, such as polyethyleneimine (PEI), which bind the cofactor


electrostatically.24 The concept was


successfully tested in the continuous


simultaneous synthesis of gluconic acid and glutamic acid by


When the process was carried out in a continuously operated enzyme membrane reactor loaded with 1 mM PEI, 80% conversion and 7.8 g/l per day space-time yield with respect to gluconic acid could be achieved at the retention time of 12 hours. Seelbach and Kragl showed that the retention of NADH in a continuous synthesis can also be achieved without immobilisation or addition of charged polymer, but by using nanofiltration membranes instead of ultrafiltration ones.25 Hecke and coworkers described the production of lactobionic acid from lactose in a coupled two-enzyme reaction both in discontinuous- and continuous-operation modes.26 The continuous enzymatic synthesis of N-acetylneuraminic acid from


“There is no established continuous


N-acetylglucosamine (GlcNAc) in an enzyme membrane reactor employing two enzymes was developed by Kragl and coworkers.27 Wiles and coworkers transferred a coupled chemoenzymatic reaction for the oxidation of alkenes from batch operation to an efficient continuous-flow process.28 The process involves the lipase-catalysed in-situ formation of peracetic acid from hydrogen peroxide and ethyl acetate, which oxidises the model substrate 1-methylcyclohexene to form the product 1-methylcyclohexene oxide. The coupled reaction is carried out in a continuously operated packed-bed microreactor. Compared with the batch-mode experiments, higher concentrations of hydrogen peroxide were applied without detectable catalyst deactivation after 24 hours. At 100% conversion, a space-time yield of 646 g/l per day was obtained. Lozano and coworkers reported on a continuous,


chemoenzymatic process catalysed by four or more catalysts in one


pot...nevertheless, in the future processes


involving more than


three catalysts will be realised in a single reactor”


coupling the NADP+-dependent glucose oxidation catalysed by glucose oxidase with the reductive amination of α-ketoglutaric acid catalysed by glutamate dehydrogenase.


chemoenzymatic dynamic kinetic resolution (DKR) process for the production of (R)-1- phenylethyl propionate from


(rac)-1-phenylethanol (22) and vinyl propionate .29-31 In a multiphase packed-bed reactor, commercially available immobilised Candida antarctica lipase B (Novozym 435) was used as a


heterogeneous catalyst in the kinetic


resolution30 of the alcohol. Racemisation of unreacted (S)-1-phenylethanol was achieved with acidic zeolite catalysts. Both heterogenous catalysts were coated with ionic


September/October 2012 sp2 Inter-Active 29


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