Crosslinking of enzymes

Crosslinking of enzyme crystals with e.g. glutaraldehyde results in the so-called CLECs produced by Altus Biologies inc. 

Govardhan, C.P. (1999) Crosslinking of enzymes for improved stability and performance. Curr. Opin. Biotechnol.,... 

With Georgios D. Gianitsopoulos. Intermolecular Crosslinking of Enzymes . In Proceedings of the Third European Congress on Biotechnology (Munich, Sept. 10-14 1984), Vol. I, 299-305. Weinheim Verlag Chemie, 1984. 

The steady structure determined by the value of Kw (Fig. 1) for the entire class of carboxylic CP obtained by precipitation copolymerization is one of the most important factors determining the possibility of reversible bonding of proteins absorbed by carboxylic CP with a high sorption capacity [16,19]. Thus, for the MA-HHTT system (Fig. 2), a complete desorption of enzyme is carried out on crosslinked copolymers characterized by low Kw values. In crosslinked structures exhibiting looser structure (Kw P 1), owing to the mobility of chain fragments of CP especially in the process of desorption, the macromolecules of sorbed protein are irreversibly captured as a result of a marked polyfunctional interaction. 

Fig. 7. Quantity of enzymes (m) ]) terrilytin and 2) pancreatic ribonuclease, bonded by crosslinked MA-DMEG copolymers with different contents of the crosslinking agent...

The immobilization of enzymes with the formation of insoluble forms is usually intended for the development of specific catalysts for technical purposes. Here, we consider another medico-biological problem of the preparation of insoluble enzymatic systems based on crosslinked polyelectrolytes, used in the replacement therapy for oral administration. 

The reactivation of enzymes (after their partial inactivation in an acid medium) upon passing into a medium of pH 8 is also of great importance for oral use (Fig. 25). Enzymes immobilized in crosslinked polyelectrolytes are characterized by a structural memory even after considerable inactivation. Under changed conditions, this leads to a considerable or almost complete reactivation of the enzyme, whereas in the reactivation of a free enzyme in solution under similar conditions the enzymatic activity is restored on a lower level. 

Activation of Enzymes with NHS Ester-Maleimide Crosslinkers... 

The two-step nature of SPDP crosslinking provides control over the conjugation process. Complexes of defined composition can be constructed by adjusting the ratio of enzyme to secondary molecule in the reaction as well as the amount of SPDP used in the initial activation. The use of SPDP in conjugation applications is extensively cited in the literature, perhaps making it one of the more popular crosslinkers available. It is commonly used to form immunoto-xins, antibody-enzyme conjugates, and enzyme-labeled DNA probes. A standard activation and coupling procedure can be found in Chapter 5, Section 1.1. 

Several examples of the binding of enzymes to poly(vinyl alcohol) are in the literature. These could possibly be used to treat enzyme deficiency diseases. In a recent example, trypsin was immobilized on poly(vinyl alcohol) fibers using maleic dialdehyde or bromal. While the reaction was more complete with bromal, the reaction with maleic dialdehyde gave a better support which showed decreasing activity with increasing enzyme content. The activity of the bromal activated system was independant of the enzyme content (52 ). Trypsin and papain were attached to poly(vinyl alcohol) by the reaction sequence shown in Equation 13. In this case, the crosslinked poly(vinyl alcohol) is treated by the 1,3-dioxalone derivative and then converted to either the isothiocyanate or the diazonium salt for coupling with the enzyme. The bound enzymes showed significant, altho reduced, activity in each case (53). 

A recent addition to General Chapter Dissolution <711> allows for the addition of enzyme to the dissolution medium. This is justified when gelatin-coated tablets or gelatin-encapsulated products exhibit a slow-down in the dissolution rate attributed to the crosslinking of the gelatin. The addition of enzyme will break up the crosslinking and therefore remove the... 

Answer A. Homocysteine, the substrate for the enzyme, accumulates increasing the risk of deep vein thrombosis and disrupting the normal crosslinking of fibrillin. Deficiency of homocysteine methyl transferase would cause homocystinuria, but would also predispose to megaloblastic anemia. 

Crosslinked polymers with functional groups have recently been used even more frequently as reagents for the synthesis of low-molecular-weight organic compounds since they are easily separated after conversion and sometimes can easily be regenerated. The immobilization of enzymes by attaching them to crosslinked polymers should also be mentioned. This technique has already found industrial applications. 

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