Enzymes cross-linking

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]. 

Halpin RA, GD Hegeman, GL Kenyon (1981) Carbon-13 nuclear magnetic resonance studies of mandelate metabolism in whole bacterial cells and in isolated, in vivo cross-linked enzyme complexes. Biochemistry 20 1525-1533. 

Cuticle Collagen/Cuticulin Cross-linking Enzymes... 

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

Immobilized enzymes, particularly LAC, have been employed in the degradation of triclosan. The immobilization of LAC from Coriolopsis polyzona through the formation of cross-linked enzyme aggregates (CLEAs) and their subsequent use in an FBR for the removal of endocrine disrupting compounds [42] produced the complete removal of triclosan, nonylphenol, and bisphenol A (5 mg L-1 each) at a HRT of 150 min. The application of CLEAs in a perfusion basket reactor [43]... 

Mateo, C., Palomo, J.M., van Lancen, L.M., van Rantwijk, E. and Sheldon, R.A., A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates. Biotech. Bioeng., 2004, 86, 273-276. 

The 3-pyridinecarboxyaldehyde 58 is highly water soluble, and so the spontaneous cyanide addition to give racemic cyanohydrin cannot be suppressed unless the aqueous pH is lowered below 3.5, which is not tolerated by the enzymes. The only available option is to operate in a 100% organic solvent system. This was recently made possible by the availability of the cross linked enzyme aggregate particles (CLEAs), which can tolerate organic solvents [64]. The individual precipitated protein molecules are chemically bonded to one another through the formation... 

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... 

Cao, L., van Rantwijk, F. and Sheldon, R.A. (2000) Cross-linked enzyme aggregates a simple and effective method for the immobilization of penicillin acylase. Org. Lett., 2, 1361. 

Sheldon, R.A., Schoevaart, R. and van Langen, L.M. (2006) Cross-linked enzyme aggregates. Meth. Biotechnol., 22, 31. 

Sheldon, R.A., Sorgedrager, M.J. and Janssen, M.H.A. (2007) Use of cross-linked enzyme aggregates (CLEAs) for performing biotransformations. Chim. Oggi — Chemistry Today, 25, 62. 

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. 

Table 1 Commercially Available Cross-Linked Enzyme Crystals11...

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. 

---