Cross-linked protein crystals

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. 

St Clair, N. Shenoy, B. Jacob, L.D. Margolin, A.L. Cross-linked protein crystals for vaccine delivery. Proc. Natl. Acad. Sc. USA 1999, 96, 9469-9474. 

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. 

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... 

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). 

The determination of binding and conformational changes leaves the question of the detailed structure of complexes unanswered. At present there is no absolute method for structure determination of protein-surfactant complexes apart from x-ray diffraction, which has only been applied to lysozyme with three bound SDS molecules [49]. X-ray diffraction requires a crystal, so in the case of lysozyme cross-linked triclinic crystals of the protein were soaked in 1.1 M SDS and then transferred to water or a lower concentration (0.35 M) of SDS to allow the protein to refold. It was necessary to use cross-linked crystals to prevent them dissolving when exposed to a high SDS concentration. The resulting denatured-renatured crystals were found to have three SDS molecules within a structure that was similar but not identical to that of native lysosyme. Neutron scattering has been applied in a few cases (see Sec. IX), but this is a model-dependent technique. 

Enzyme Crystals and Precipitates. Solid enzyme preparations can be cross-linked with a bifunctional reagent to yield cross-linked enzyme crystals (CLEG) (21), or cross-linked enzyme aggregates (CLEA) (22) that are insoluble in aqueous as well as nonaqueous media. This maintains their catalytic activity and increases their thermostability and resistance to proteases. The activity of the preparation remains high because of the high purity of the protein. CLECs are not commercially available anymore. CLEAs can be dried and suspended in any type of solvent (23). 

As an alternative to native protein crystals, protein crystals cross-linked by glutaraldehyde provide attractive solid reaction vessels for preparing novel nanomaterial-in-crystal hybrids with potential application in catalysis. Au nanoparticles were S5mthesized within the solvent channels of cross-linked lysoz5mie crystals in situ without the introduction of extra chemical reagents or physical treatments (Liang et al., 2013). 

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],... 

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