Cross-Linked Crystals

One enzyme form that has received considerable attention is based on enzyme crystals. Production of protein microcrystals from aqueous solution is often quite easy, and is increasingly used as a step in the manufacture of enzymes on an industrial scale. (Many people have the impression that protein crystallization is very difficult, but this stems from the problems in growing large near-perfect crystals for 

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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],... 

Costes, D., Wehtje, E. and Adlercreutz, P. (2001) Cross-linked crystals of hydroxynitrile lyase as catalyst for the synthesis of optically active cyanohydrins. Journal of Molecular Catalysis B-Enzymatic, 11, 607-612. 

Wang, Y.F., Yakovlevsky, K., Zhang, B.L. and Margolin, A.L. (1997) Cross-linked crystals of subtilisin— versatile catalyst for organic synthesis. Journal of Organic Chemistry, 62(11), 3488-3495. 

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... 

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. 

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],... 

Reduction of achiral precursors is often used to produce chiral products. The advantage of this approach is that the theoretical yield of product is 100% compared to the 50% theoretical maximum for the resolution of racemates. Cross-linked crystals of lactate dehydrogenase have been used to prepare L-lactic acid from pyruvic acid in an electrolytic cell. The LDH CLCs maintained constant... 

In the very important therapeutic class of antibiotics, both 6-AP A (6-amino-penicillinic acid) and 7-ADC A (7-aminodeacetoxycephalosporanic acid) are being produced today on multi-ton scale using the cross-linked crystal form of peni-cillin-G amidase (SynthaCLEC -PA) (P. Lanciano, President, Altus Biologies,... 

JJ Lalonde, C Govardhan, N Khalaf, AG Martinez, K Visuri, AL Margolin. Cross-linked crystals of Candida rugosa lipase highly efficient catalysts for the resolution of chiral esters. J Am Chem Soc 117 6845-6852, 1995. 

Y-F Wang, K Yakovlevsky, B Zhang, AL Margolin. Cross-linked crystals of subti-lisin versatile catalyst for organic synthesis. J Org Chem 62 3488-3495, 1997. 

ChiroCLEC -EC is cross-linked crystals of penicillin-G acylase and is available from Altus Biologies, Inc., Cambridge, MA. 

The sp modification carbyne briefly described below is characterized by a different intrinsic structure and can be obtained only in the form of very small particles. Another analogous example is the sp molecular crystals which are composed of short sp -carbon chains with organic caps on the ends of the chains, which prevents them from collapsing into the amorphous state [28]. These crystals are not composed of pure sp -hybridized carbon and are stacked into a cross-linked crystal structure, which is easily dissolved in many organic solvents and is completely different from Tetracarbon . 

Immobilized enzymes are currently the object of considerable interest. This is due to the expected benefits over soluble enzymes or alternative technologies. The number of applications of immobilized enzymes is increasing steadily [5]. Occasionally, however, experimental investigations have produced unexpected results such as a significant reduction or even an increase in activity compared with soluble enzymes. Thus, cross-linked crystals of subtihsin showed 27 times less activity in the aqueous hydrolysis of an amino acid ester compared to equal amounts of soluble enzyme [6]. On the other hand, in the application of hpo-protein lipase in the solvent-mediated synthesis of esters there was a 40-fold increase in activity using immobihzed or otherwise modified enzyme preparations as compared to enzyme powder [7]. 

Cross-linked crystals from subtilisin exhibited 27 times less activity than soluble... 

Cross-linked crystals of lipase from Candida rugosa (CRL) were applied in the resolution of racemic ketoprofen chloroethyl ester. In batch-wise operation, the half-life of the catalyst was reached after about 18 cycles or, in terms of enzyme consumption, about 5.6 g of enzyme protein were consumed to prepare 1 kg of (S)-ketoprofen. CRL suffers from a low specific activity towards this poorly water-soluble substrate which may explain the high enzyme input [117]. 

When cross-linked crystals of thermolysin were applied in peptide synthesis in ethyl acetate, they were stable for several hundred hours at amazingly low enzyme consiunption, whereas a soluble enzyme preparation became inactive within a short period of time. Again it is worthwhile to consider the quality of the soluble enzyme preparation. When soluble thermolysin was stored in mixed aqueous-organic solutions, it lost about 50% of its activity within the first day of incubation only to be then quite stable for the next 15 days. It is possible that the initial inactivation was caused by an unstable fraction of thermolysin and that crystals of thermolysin no longer contained this unstable fraction [118]. Productivity comparable to that of crystals was achieved with thermolysin adsorbed on Amberlite XAD-7 resin which was employed in continuous plug flow reactors with tert-amyl alcohol as solvent [119]. 

Altus Biologies Cambridge, MA www.altus.com Enzyme cross-linked crystals... 

Penicillin G amidase was immobilized on pre-fabricated carriers or insolubilized as cross-linked crystals. Eupergit-related value for R (mean particle radius of swelled carrier) was 80 pm [87]. Vm (assuming maximum intrinsic activity per accessible catalyst volume, based on active enzyme molecules 1 unit=l pmol min-1 at 28°C) was 90 and 170 U cnr3 for Eupergit C and 250L, respectively [87], Deff (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. 

Cross-linked crystals from subtilisin exhibited 27 times less activity than soluble subtilisin in the hydrolysis of benzoyl-L-phenylalanine ethyl ester. Denaturation of the enzyme and restrictions from substrate-dependent internal diffusion were ruled out. A shift in the pH-dependence of the maximum activity to higher pH-values was observed which was explained by inter molecular electrostatic... 

X-ray diffraction.) The protein molecules in the microcrystals are then covalently cross-linked by treatment with an appropriate multi-functional reagent, usually glutaraldehyde. This renders the crystals insoluble on transfer to different aqueous media. The cross-linked crystals are effectively another form of immobilized enzyme, and can be dried for transfer to low-water media by the same methods (again see further details in the discussion of water effects below). Cross-linked crystals are available commercially for a number of enzymes. Figure 8-1 shows a diagrammatic representation of the organization of the protein molecules in lyophilized powders, immobilized enzymes and cross-linked crystals. 

A decision must be made about the sequence and timing in which components are combined to make the final reaction mixture. The choices made can have large effects on the final hydration conditions and biocatalyst behavior. It is usually best initially to prepare as separate phases (i) a non-aqueous solution or mixture of the reactants and (ii) the solid biocatalyst preparation (lyophilized powder, immobilised enzyme, cross-linked crystal etc). The best treatment to apply then depends on the objective of the experiment. 

In MIMIC, a PDMS mold is brought into contact with a rigid support, forming microchannels between the substrate and the recessed regions of the PDMS mold as shown in Figure 5.5.28. The fluid, placed at the end of mold, hlls the channels of the mold by capillary action. The material is then cross-linked, crystallized, or... 

Figure 8.4 Still diffraction patterns recorded from native and modified crystals of gn crystallin. (a) Native crystal, (b) cross-linked crystal (2.5% gluteraldehyde). Resolution limit approximately 2.25 A with noticeable diffuse scattering.

Figure 8.4 (cont.) (c) Cross-linked crystal soaked in 10 nM thiomersal. 

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. 

Cross-linked crystals of Candida rugosa lipase are highly efficient catalysts for resolving racemic esters, whereas PPL immobilized in microemulsion-based gel has been used to hydrolyze one of the polyphenolic perpropanoates. Actually, symmetrical diacetates are readily cleaved to provide the monoesters by PPL. ... 

Persichetti R, St Clair N, Griffith J et al. (1995) Cross-linked crystals (CLECs) of thermolysin in the synthesis of peptides. J Am Chem Soc 117 2732-2737 Pierre AC (2004) The sol-gel encapsulation of enzymes. Biocatal Biotransform 22(3) 145-170... 

One interesting technology uses lipases in the form of cross-linked enzyme crystals (CLECs) (Margolin 1996). This immobilization method does not use any solid support and the lipase specific activity (units of activity/g of immobilized catalyst) of the immobilized lipase derivative can be enhanced by 10-fold because there is no inert support, that usually represent more than 90% of the catalyst weight in the case of carrier-bound enzymes. These cross-linked crystals have been used for the chiral resolution of commercially important organic compounds, such as ibuprofen. 

J. J. Lalonde and C. Govardhan, Cross-Linked Crystals of Candida Rugosa Lipase Highly Efficient Catalyst for the Resolution of Chiral Esters, /. Am. Chem. Soc., 117(1995)6845. 

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