Crosslinked Enzymes And Novel Catalytic Materials

Enzymes by and large are not readily accepted in chemical industry due to

High cost

Limited substrate specificity and

Low enantioselectivity for unnatural synthetic substrates.

Apart, from these, the main reason often being the lack of enzyme stability at elevated temperatures and rigorous process conditions. One approach by which this limitation is overcome is by cross-linking the enzymes and crystallizing these cross-linked enzyme systems. These are termed cross-linked enzyme systems, or CLECs. Cross-linking ''locks'' the protein in a particular conformation. Historically, glutaraldehyde (1) has been by far the most popular cross-linking agent. However, a number of other reagents are now known (Fig. 6.1).

Chemically, CLECs are significantly more stable against denaturation by heat, organic solvents, and proteolysis than the corresponding soluble proteins. In certain cases the enzyme crystal may even be more active than the same enzyme in solution. For example, CLECs of lipases and subtilisin formulated with surfactants exhibited specific activity in organic solvents higher than that of native enzymes. Crystals of Candida Rugosa Lipase (CRL)

DI:DNB

Traill's re age: ut

-CHO

CUm 3 r'd Idii hyde

-CHO

DI:DNB

Traill's re age: ut

CUm 3 r'd Idii hyde

Sulto-ir-SMPT

Sulto-ir-SMPT

Sulfo-HSAB

Figure 6.1 Reagents for cross-linking proteins.

Sulfo-HSAB

Figure 6.1 Reagents for cross-linking proteins.

cross-linked with sulpho-LC-SMPT were five to seven times more active than pure native CRL in the olive oil assay.

Apart from this, the CLECs remain insoluble throughout the process and can be recycled many times, thereby, increasing the productivity of the catalyst. A good example of the high productivity of CLEC catalysts in organic solvents is the resolution of 1-phenyl ethanol with vinyl acetate in toulene catalyzed by CLECs of Pseudomonas cepacia lipase (LPS-CLECs). In this reaction a substrate to catalyst ratio of 4600:1 was achieved.

Various enzymes, such as lipases, esterases, and dehydogenases, have been cross-linked and all of them showed higher activity and greater stability. In the case of dehydrogenases, the cocrystallization with the cofactor was adopted. Hence, this nascent science of ''crystalomics'' provides us with a general strategy for putting proteins to work rapidly and efficiently in the harsh conditions typical of most practical industrial processes.

One of the requirements of enzymatic organic synthesis is to develop protein catalysts with tailored activities and selectivities. The various approaches toward the attainment of this goal are as follows:

1. Construct (synthesize) a protein with a designed catalytic activity

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