Crosslinked enzyme crystals

Crystals constitute the most concentrated form of enzyme and they can therefore be attractive as catalysts. In the crystallisation of a crude enzyme preparation a considerable purification of the enzyme can be achieved which is a further advantage. In order to stabilise the enzyme crystals to make them useful catalysts they are often crosslinked with bifunctional reagents such as glutaraldehyde. Very high catalytic activity and stability has been reported for these crosslinked enzyme crystals, some of which are commercially available (Margolin, 1996). 

Zelinski, T. and Waldmann, H. (1997) Crosslinked enzyme crystals (CLECs) efficient and stable biocatalysts for preparative organic chemistry. Angeiv. Chem. Int. Ed. Engl., 36, 722. 

Sobolov SB, Bartoszko-Malik A, Oeschger TR et al. (1994) Crosslinked enzyme crystals of fructose diphosphate aldolase development as a biocaUilyst for synthesis. Tetrahed Lett 35 7751-7754... 

CLEC Crosslinked enzyme crystal nadpV Nicotinamide adenine... 

Table 9 Advantages and Limitations of Crosslinked Enzyme Crystals... 

Crosslinking of enzyme crystals with e.g. glutaraldehyde results in the so-called CLECs produced by Altus Biologies inc. 

In this polymerization, the biofunctional component (enzyme) can be concentrated in an interfacial area between the frozen ice crystal and the supercooled monomer phase, and immobilized by molecular entanglement between the enzyme and polymer molecules. This is a different procedure for fixation from the usual entrapping method with a crosslinked structure in a gel. Therefore, we may call this procedure the adhesion-method to distinguish it from the usual entrapping. This term was extended to cover the use of the usual synthetic polymers including hydrophobic polymers as the supports. One of the characteristic properties of products obtained in this way was that there is a maximum activity at a certain monomer concentration. The maximum activity is observed when the increased inner surface area is balanced by the increased leakage of enzyme and these occur with a decrease of monomer concentration. Immobilization by physical entrapping was also studied by Rosiak [26], Carenza [27] and Ha [28]. 

Penicillin G amidase was immobilized on pre-fabricated carriers or insolubUized as crosslinked crystals. Eupergit-related value for R (mean particle radius of swelled carrier) was 80 pm [87]. V , (assuming maximum intrinsic activity per accessible catalyst volume, based on active enzyme molecules 1 unit=l pmol min at 28°C) was 90 and 170 U cm for Eupergit C and 250L, respectively [87]. D ff (effective diffusion coefficient) was taken from literature [87] or calculated as shown in the text. Km (intrinsic Michaelis constant) was uniformly taken as 13 mM [87] and S = 268 mM corresponds to the substrate concentration at catalyst surface of a 10 % solution of penicillin G salt, q was calculated according to Atkinson et al. for spherical particles [85]. For simplification, surface and pore related indices have been omitted. 

Galactose oxidase (GO) catalyses the two-electron oxidation of primary alcohols to aldehydes. It contains a single type II copper centre. The enzyme employs the metal and a protein radical cofactor to effect the chemistry. The crystal structure shows a square pyramidal five-coordinate copper site with the metal coordinated by two histidines, two tyrosines and a water or acetate ligand. The equatorial tyrosine, Tyr272, has an interesting crosslink to a cysteine group ortho to the tyrosine oxygen. 

Following the demonstration of the exceptional stability and activity of crosslinked crystals (CLECs) of thermolysin in aqueous and organic media, and its application in peptide synthesis [369], this novel technology has been rapidly commercialized and shown to be applicable to a variety of enzymes [370-383]. 

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