Cross enzyme crystals

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]. 

Purified MeHNL was crystallized by the sitting-drop vapor-diffusion method. The 10-20 mm bipyramidal crystals formed were cross-linked with glutaraldehyde and used as biocatalyst for the synthesis of optically active cyanohydrins. The cross-linked crystals were more stable than Celite-immobilized enzymes when incubated in organic solvents, especially in polar solvents. After six consecutive batch reactions in dibutyl ether, the remaining activity of the cross-linked crystals was more than 70 times higher than for the immobilized enzymes. Nevertheless, the specific activity of the cross-linked crystals per milligram protein was reduced compared with the activity of Celite-immobilized enzymes [53],... 

Enzyme crystallization, with or without cross-linking... 

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]. 

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... 

Another gap of almost 10 years occurred before work in the area of CLCs picked up again. In 1985, a group at the Louis Pasteur University in Strasbourg, France, prepared cross-linked crystals of horse liver alcohol dehydrogenase [4], The activity of the enzyme in CLC form was maintained and the coenzyme was found to be firmly bound to the crystals. The cross-linked crystals could be used as redox catalysts with no addition of coenzyme. The authors also reported the increased stability of CLCs toward organic solvents. 

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. 

Enzymes in the cross-linked crystal form are essentially impervious to degradation by exogenous proteases and from autolysis, in the case of CLCs of proteases themselves [5], This stability makes the enzyme-catalyzed preparation of peptides and peptide mimics truly practical [6], Examples will be discussed in more detail in Sec. IV. Further, one could conceive of using multiple enzymes in one-pot reaction systems mimicking natural biosynthetic cascades. Indeed, the application of this concept has been reported for a mixture of lipoamide dehydrogenase and lactate dehydrogenase [19],... 

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. 

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