Enzyme cascades, immobilized

Analogously, Van Pelt et al. (2009) have realized a one-pot bienzymatic cascade combining the (5 )-selective oxynitrilase from M. esculenta and a purified nitrile hydratase from Nitriliruptor alkaliphilus for the synthesis of aliphatic S)-a-hydroxycarboxylic amides (Table 17.6) both enzymes were immobilized as CLEAs to enhance their stability (Van Pelt et al., 2009). 

Multi-enzymatic catalysts are recognized as the future generation of biocatalysts. They were particularly designed as enzymes encapsulated into hybrid capsules [129]. It has already been proved that the multi-enzymes organization in a single capsule with separated space enhanced the catalytic capacity of the system. Such an example is the dual biocatalyst of formaldehyde and formate dehydrogenases encapsulated in the capsule lumen/wall. It catalyzed the cascade transformation of CO into formaldehyde [132]. Enzymes co-immobilization in separated spaces of the same cavity exhibited higher formaldehyde yields compared to the free enzyme. Also, the recyclability of the biocatalytic system confirmed the superior mechanical stability [132]. 

Biocatalysis. Chemical conversion of a substance with the aid of an isolated or immobilized enzyme. One or several enzymes (enzyme cascade) can be used in a one-pot reaction. Some enzymes allow the reaction to be run in organic solvents... 

Synthesis of 2-amino-1,3,4-butanetriol by a transketolase/ transaminase enzyme cascade via continuous flow dual reactor. The enzymes were immobilized inside the silica microreactors separately. 

Although the catalysts do not interfere with each other, the immobilization process resulted in higher yields and ees than with the raw enzyme the initial activity was over five times greater. Additionally, the biocomposite with enzyme was able to be recycled and maintained its activity, which demonstrates the utility of such an immobilization system for potential cascades with mutually interfering catalysts. 

Effective and simple immobilization of enzymes can be obtained by the cross-linking of enzyme aggregates, so-called CLEAs [55]. In this way, essentially any enzyme, including crude preparations, can be transformed into a heterogeneous type of material, insoluble in both water and organic solvents, that is stable and recyclable with high retention of the enzyme s original activity [56], These enzyme preparations are, therefore, of special value for both bio-bio and bio-chemo cascade processes. 

As noted in the preceding section, successful compartmentahzation by immobilization may be the key to the compatibility of different enzymes in a cascade... 

Chemoenzymatic polymerizations have the potential to further increase macro-molecular complexity by overcoming these limitations. Their combination with other polymerization techniques can give access to such structures. Depending on the mutual compatibility, multistep reactions as well as cascade reactions have been reported for the synthesis of polymer architectures and will be reviewed in the first part of this article. A unique feature of enzymes is their selectivity, such as regio-, chemo-, and in particular enantioselectivity. This offers oppormnities to synthesize novel chiral polymers and polymer architectures when combined with chemical catalysis. This will be discussed in the second part of this article. Generally, we will focus on the developments of the last 5-8 years. Unless otherwise noted, the term enzyme or lipase in this chapter refers to Candida antarctica Lipase B (CALB) or Novozym 435 (CALB immobilized on macroporous resin). 

Biocatalytic synthetic reactions also include carbon dioxide fixation with the production of methanol in artificial multi-enzyme systems [188]. Formate dehydrogenase (FDH, EC 1.2.1.2) can catalyze the reduction of carbon dioxide to formate, and methanol dehydrogenase (MDH, EC 1.1.99.8) can catalyze the reduction of formate to methanol. Both of these enzymes require NAD+-NADE1 cofactor, and in the presence of the reduced dimethyl viologen mediator (MV+), they can drive a sequence of enzymatic reactions. The cascade of biocatalytic reactions results in the reduction of CO2 to formate catalyzed by FDEI followed by the reduction of formate to methanol catalyzed by MDH. A more complex system composed of immobilized cells of Parococcus denitrificans has been demonstrated for the reduction of nitrate and nitrite [189]. 

A plug flow reactor may be realized using immobilized enzymes within a column reactor or using soluble enzymes within a cascade of membrane reactors. A batch or a repetitive batch process with soluble enzymes (see below) has the same productivity as the plug flow reactor. 

The CSTR cascade is often used as a substitute for CPFR behavior, and tubular reactors themselves are rarely found in bioprocessing. Exceptions are waste water treatment in oxidation ponds, river analysis (Metcalf and Eddy, 1972), sterilization technology, and fixed-bed reactors filled with immobilized enzymes for bioconversions (Lilly and Dunnill, 1972). 

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. 

In certain cases, two successive enzymatic reactions can be carried out on a substrate solution by reacting it with two different immobilized enzymes. Thus, the isolation of the intermediate product may not be necessary. This is equivalent to events in a cell where successive chemical transformations of a substrate take place via a cascade of different enzymes present in the cell system. 

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