Preparation of Biocatalysts

Transfer of a hydride between substrate and coenzyme and proton relay in active site of an NADH-dependent D-2-hydroxy acid dehydrogenase. 

Methods to find a unique and useful enzyme and to mutate enzymes are described in this section. Screening sources from which to search for new biocatalysts became drivers. Enzymes from culturable and nonculturable organisms can be used. Biocatalysts from autotrophs as well as heterotrophs are shown. 

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Figure 3. Process scheme for preparation of biocatalysts by cell entrapment in epoxy beads. Reprinted, with permission, from Ref. 14. Copyright 1982, Science...

A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

L-Phenylalanine can be synthesised from trims-cinnamic add (Figure A8.12) catalysed by a L-phenylalanine ammonia-lyase from Rhodococcus glutinis. The commercialisation of the process was limited by the low conversion (70%), low stability of the biocatalyst and die severe inhibition exerted by trims-cinnamic add. These problems were largely overcome by researchers at Genex. The process, commercialised for a short period by Gen ex, involves a cell-free preparation of phenylalanine-ammonia-lyase activity from Rhodotorula rubra. 

The simplest way to prepare a biocatalyst for use in organic solvents and, at the same time, to adjust key parameters, such as pH, is its lyophilization or precipitation from aqueous solutions. These preparations, however, can undergo substrate diffusion limitations or prevent enzyme-substrate interaction because of protein-protein stacking. Enzyme lyophilization in the presence of lyoprotectants (polyethylene glycol, various sugars), ligands, and salts have often yielded preparations that are markedly more active than those obtained in the absence of additives [19]. Besides that, the addition of these ligands can also affect enzyme selectivity as follows. 

Despite the still growing number of available methods for the preparation of enantiopure compounds by the use ofasymmetric catalysis, kinetic resolution (KR) is still the most employed method in the industry [4], and in most cases biocatalysts (enzymes) are used. 

In the last decade, biocatalysis in nonaqueous media, using hydrolases, has been widely used for organic chemists. The possibilities that these biocatalysts offer for the preparation of different types of organic compounds, depending upon the nucleophile... 

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

Kokufuta, E. Novel Applications for Stimulus-Sensitive Polymer Geb in the Preparation of Functional immobilized Biocatalysts, Vol, 110, pp. 157-178. 

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