Cross-linking, enzyme stabilization

Williams and coworkers have reported a DKR of ot-bromo [56a] and a-chloro esters [56b]. In the latter case, the KR is catalyzed by commerdally available cross-linked enzyme crystals derived from Candida cylindracea lipase. The racemization takes place through halide 5 2 displacement. The DKR is possible because the racemization of the substrate is faster than that of the produd (carboxylate). For the ester, the empty ii (C=0) orbital is able to stabilize the Sn2 transition state by accepting... 

Greenberg, C.S., Birckbichler, P.J. and Rice, R.H. (1991) Transglutaminases -multifunctional cross-linking enzymes that stabilize tissues. FASEB Journal 5, 3071-3077. 

The first cross-linked HNLs were reported by Costes et al. [72]. They compared MeHNL-CLECs with Celite -immobilized MeHNL. By cross-linking, the stability of the enzyme was improved, particularly in polar organic solvents. Furthermore, the cross-linked crystals could be reused without significant loss of activity. After six consecutive batches, 70% of the initial activity was retained, whereas the immobilized enzyme showed virtually no remaining activity (<1%). Nevertheless, crystallization and cross-linking cause a considerable loss of activity compared to the immobilization on Celite [72]. 

The first reported preparation of cross-linked enzyme crystals was by Quiocho and Richards in 1964 [1], They prepared crystals of carboxypeptidase-A and cross-linked them with glutaraldehyde. The material they prepared retained only about 5% of the activity of the soluble enzyme and showed a measurable increase in mechanical stability. The authors quite correctly predicted that cross-linked enzyme crystals, particularly ones of small size where the diffusion problem is not serious, may be useful as reagents which can be removed by sedimentation and filtration. Two years later the same authors reported a more detailed study of the enzymic behavior of CLCs of carboxypeptidase-A [2], In this study they reported that only the lysine residues in the protein were modified by the glutaraldehyde cross-linking. The CLCs were packed in a column for a flow-through assay and maintained activity after many uses over a period of 3 months. 

Little was done in the area of cross-linked enzyme crystals over the next 10 years. In 1977, the kinetic properties of CLCs of the protease subtilisin were reported by Tuchsen and Ottesen [3], They reported that cross-linked enzyme crystals of subtilisin were highly effective catalysts with increased thermal stability and increased stability toward acid compared to the soluble enzyme. They further reported that the CLCs of subtilisin showed essentially no autodigestion at 30°C. Like Quiocho and Richards before them, Tuchsen and Ottesen found... 

First and foremost, cross-linked enzyme crystals are crystals. Within the crystal lattice the concentration of protein approaches the theoretical limit. This is important to the process development chemist, who would much rather use a small quantity of a very active catalyst in a reactor than fill it with an immobilized enzyme. Typically an immobilized enzyme contains only 1-10% by weight enzyme, with the remaining carrier material simply occupying valuable reactor space. The crystallinity is absolutely required to achieve the stability exhibited by CLCs [8], Cross-linked soluble thermolysin and cross-linked precipitate of thermolysin are no more stable than the soluble enzyme. Crystals of proteins... 

Probably the most striking and valuable characteristic of cross-linked enzyme crystals is the remarkable stability they exhibit in comparison to soluble and immobilized enzymes. They can withstand exposure to organic solvents, high temperatures, mechanical stress such as shear, extremes of pH, and even exposure to proteases. 

Haring and Schreier have modified the active site of subtilisin cross-linked enzyme crystals by introducing selenium into it and thereby converting the enzyme into a peroxidase [36], The rigid CLC matrix allowed them to chemically modify subtilisin without loss of the tertiary structure. The kinetic resolution of racemic 2-hydroxy- 1-phenylethyl hydroperoxide was demonstrated using the semisynthetic CLC (Fig. 12). The reaction time was 25-30 min with an ee of 97%. The authors demonstrated the stability of these semisynthetic CLCs by cycling their enzyme 10 times. 

The true value of cross-linked enzyme crystals is that this technology minimizes many if not all of the problems which have limited the industrial use of enzymes to date. Issues of stability, purity, and cost are all addressed favorably by cross-linked enzyme crystal technology. 

However, the relatively high enzyme costs form an obstacle to commercialization. Inefficient laccase use is a result of its instability towards the oxidizing reaction conditions. We have recently shown that the stability of the laccase under reaction conditions can be improved by immobilization as a cross-linked enzyme aggregate (see Chapter 9). It has also been shown that a water-soluble iron complex of a sulfonated phthalocyanine ligand is an extremely effective catalyst for starch oxidation with hydrogen peroxide in an aqueous medium [11]. 

Stabilization oT NLases as Cross-linked Enzyme A regates... 

The immobilization of the enzyme, the redox catalyst, and sometimes also the cofactor can also take place at a solid support different from the electrode so that the components can be recovered within a solid-bed reactor (a column filled with the enzyme-containing particles) or by a filter plate or membrane. The immobilization of enzymes at solid supports or by the foraiation of cross-linked enzyme crystals can sometimes also enhance the enzyme stability. This concept has the advantage of the ease of separation but the disadvantage of diffusional limitations due to the heterogeneity of the reactions between the enzyme and the substrate and the cofactor or the redox catalyst. Additionally, the number of available redox centers is usually limited. 

L-Methionine has been prepared from fV-acetyl-D, L-me-thionine by deacylation with an aminoacylase.37 The lipase was supported on an ion-exchange resin. The reaction was driven to completion by chromatographic ion-exchange removal of the by-product acetic acid and unreacted substrate, the racemic mixture being fed in pulses to the column. The cross-linked enzyme crystals marketed by Altus Biologies are very useful in enzymatic resolutions, because of their enhanced stability to heat and organic solvents and because they can be reused many times. (For more detail on enantiospecific reactions with enzymes, see Chap. 9.)... 

Immobilization by chemical cross-linking without the addition of an inert carrier or matrix can provide the means to stabilize and reuse a biocatalyst without dilution of volumetric activity. A major deficiency in all of the aforementioned immobilization methods is that a substantial amount of a catalytically inert carrier or matrix is used to bind or contain the biocatalyst. In many cases, the amount of carrier is two orders of magnitude higher than the protein catalyst. Unfortunately, direct cross-linking of the enzyme, followed by precipitation of an amorphous solid often results in low activity and poor mechanically properties and so this method is not often used. Recently, however, cross-linked enzyme crystals have been reported to give many of the desirable properties of immobilized enzymes without the need for a support material (Sect. 6.4.1). 

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