Enzyme crystals, cross-linked

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... 

The most effective of these include immobilization [80], lipid coating [81], surfactant coating [82], use of cross-linked enzyme crystals [83], cross-linked enzyme aggregates [84], and membrane reactors [85]. 

Like many other useful discoveries, enzyme immobilization by cross-linking was actually an unintended by-product of another research project. In 1964, Florante Quiocho and Frederic Richards at Yale university cross-linked crystals of carboxy-peptidase-A with glutaraldehyde (pentane-1,5-dial), hoping to get stable crystals for X-ray diffraction studies. They noted that these cross-linked enzyme crystals (now... 

Cross-Linked Enzyme Crystals Biocatalysts for the Organic Chemist... 

Throughout the bulk of this chapter, CLC will be used as an abbreviation for cross-linked enzyme crystal. Occasionally, the abbreviation CLEC will also be used to indicate cross-linked enzyme crystal. This acronym is a registered trademark of Altus Biologies, Inc. (Cambridge, MA) and will be used in discussing work done with various cross-linked enzyme crystals which are commercially available from Altus (Table 1). Finally, the notation CPC will be used to denote cross-linked protein crystal. 

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... 

Having briefly outlined the historical development of cross-linked enzyme crystals, a discussion of their properties as compared to soluble and immobilized enzymes is in order. 

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. 

The primary focus of this section will be the use of cross-linked enzyme crystals as catalysts of chemical reactions and particularly those reactions of relevance... 

The major application of cross-linked enzyme crystals in the pharmaceutical and fine chemicals industry is in biocatalysis or the use of CLCs to catalyze various... 

Peptide synthesis is an extremely important area of chemistry for the pharmaceutical industry, and like any specialized area of chemistry, has its own set of unique problems associated with it. Racemization and purification of final products are two of the most difficult problems in this area. The use of enzymes has been explored as a possible answer to these problems since 1938 [29]. However, proteases needed to catalyze peptide synthesis are subject to rapid autolysis under the conditions needed to affect peptide coupling, so this has generally not been a practical approach until cross-linked enzyme crystals of proteases became available. The synthetic utility of protease-CLCs was demonstrated by the thermolysin CLC (PeptiCLEC -TR)-catalyzed preparation of the aspartame precursor Z-... 

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. 

The first large-scale commercial application of cross-linked enzyme crystals was the use of glucose isomerase CLCs to produce high-fructose com syrup. While this is not a pharmaceutical or a biotechnological application, it is included here because it serves to demonstrate the economic viability of the technology in a very cost-sensitive business. In this application the CLCs were attached to the surface of a polystyrene-cellulose-titanium oxide composite carrier in a ratio of 9 1 carrier enzyme. The catalyst had a half-life of 150 days at 57°C, and 12-18 tons of dry sugar product could be produced per kilogram of enzyme [37],... 

A group at Industrial Research Limited in New Zealand recently reported the results of a study to determine if a cross-linked enzyme crystal-catalyzed resolution can compete with alternative chiral technologies in the pharmaceutical and chemical process industries. The group used the enantioselective hydrolysis of a-phenylethyl acetate catalyzed by ChiroCLEC -PC as a model system (Fig. 13). Based on their results with 270 kg of racemate, they evaluated the economics of mnning the process at the 600-kg batch scale and concluded that this process is economically feasible [38],... 

Like all new technologies, cross-linked enzyme crystals were not developed overnight. Almost 30 years separate the first report of cross-linked enzyme crystals from the introduction of truly useful, commercially available CLEC products. 

It is a certainty that many of the exciting new therapeutic agents which will reach the market in the coming years will be manufactured by processes which include the use of cross-linked enzyme crystals. 

NL St. Clair, M Navia. Cross-linked enzyme crystals as robust biocatalysts. J Am Chem Soc 114 7314-7316, 1992. 

D Flaring, P Schreier. Cross-linked enzyme crystals. Curr Opin Chem Biol 3 35-38, 1999. 

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