Combi-CLEAs

The major advantage offered by the use of CLEAs is the possibility of bienzy-matic reactions by the application of combi-CLEAs, as shown by Mateo et al. [78]. The utilization of apparently incompatible enzymes with respect to the reaction conditions is thereby possible. 

Fig. 9.28 One-pot conversion of benzaldehyde to S-mandelic acid with a combi CLEA.

Accordingly, we conducted experiments with CLEAs of MeHnL and PfNLase in tandem in a 90 10 DlPE-bulFer pH 5.5 medium [19]. The reaction proceeded to nearly full conversion (see Figure 16.4a) and the product ee was 94%. Combining both enzymes in a bienzymatic catalyst (combi-CLEA, Figure 16.4b) resulted in further improvement and 98% enantiomerically pure (S)-3a was obtained. It would seem that the nitrile intermediate is immediately hydrolysed in the combiCLEA particles, which supresses dififiision into the water phase and possible racemisa-tion. The amount of (S)-mandeUc amide ((S)-4a, see Figures 16.3 and 16.4, approx. 40%) that accompanied the formation of (S)-3a was more than would be expected from the data in Table 16.1 and made us aware of possible stereochemical effects on the acid/amide mechanistic switch [5] in PfNLase as will be discussed later. 

Figure 16.6 Bienzymatic synthesis of (R)-4-phenyl-2-hydroxy-( )-but-3-enoic acid from cinnamic aldehyde (0.1 M) and HCN (0.5 M) in the presence of a combi-CLEA of PaHnL and NIT-104, in 50 50 DIPE-buffer pH 5.5 at r.t legend 1c 2c 3c , 4c ...

Combi-CLEAs, enzyme pathways and or cofactor regeneration. 96... 

CLEAs are made by the traditional protein methods of precipitation (e.g., solvents, salting out, etc.), but not full purification is required.The main features of CLEAs are the combination of easy procedures, low cost of protein processing, and robustness of the biocatalyst, which is required for the development of a biocatalyzed industrial process. This strategy has been demonstrated useful for multimeric enzymes. An interesting approach is to develop an enzymatic cascade based on many steps, i.e.,multienzymatic biotransformation process in just single CLEAs, or in noncascade type, which are named as combi-CLEAs. The vast potential of CLEAs at industrial level allows to explore this technolc for many appHcations in the food industries such as vinification, or citric juice clarification, or for medical purposes Hke scar debriding or cystic fibrosis appHcations. ... 

Cao L. Carrier-bound immobilized enzymes. Principles, application and ilesi .Weinheim WDey-VCH 2005. 95. Dalai S, Kapoor M, Gupta MN. Preparation and characterization of combi-CLEAs catalyzing multiple non-cascade reactions.J Mol Catal B Enzym 2007 44 128-32. 

Chmura, A., Rustler, S., Paravidino, M., Rantwijk, F. V., Stolz, A., and Sheldon, R. A. (2013) The combi-CLEA approach cascade synthesis of enantiomerically pure (S)-mandelic acid. Tetrahedron Asymmetry, 24, 1225-1232. 

Such CEEAs can also be produced that contain two or more different enzymes in the same aggregates. These so-called combi-CLEAs allow multiple reactions (either cascade or noncascade) to be carried out in the same reaction space (Figure 4.2). This preparation strategy also has some advantages in promoting high specific-enzyme activity and catalytic productivity since reaction rates can be improved by close spatial proximity between the different enzymes and substrates. 

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