Organic Polymers in Enzyme Immobilization

Abstract

Structural stability is a critical factor in the practical application of enzymes as catalysts. Evidence based on structural analysis has shown that thermophilic enzymes usually differ from their mesophilic counterparts by only insignificant alteration of their primary structure, and the three dimensional structure of such enzymes are largely similar. The observed difference in operational temperature corresponds to an increase in stability by 5–7 kcal/mol. A free energy change of this order could be derived from one additional salt bridge or several additional hydrogen bonds inside the protein molecule. Perception of these differences and their structural determinants would allow manipulation of proteins to produce stability enhancement. Many methods of enzyme stabilization have been developed but none of them is generally applicable. These include covalent bonding to activated supports, physical adsorption to support matrixes, electrostatic entrapment, and suspension in semipermeable membranes. Another method used to stabilize enzymes is to introduce additional internal binding forces via site-specific mutagenesis. Procedures are described here for the preparation of water soluble immobilized enzymes of interest as catalysts in organic synthesis. Multipoint attachment between the enzyme and the water-soluble polymer poly(acrylamide-co-N-acrylamide- co-N-acryloxysuccinimide), PAN,was used to stabilize the enzyme in solution. This approach takes advantage of the flexibility of the polymer to react in a manner which keeps disruption of the preferred enzyme conformation to a minimum. Application of this technique was demonstrated with a-chymotrypsin, trypsin, FDP-aldolase, pig liver esterase, horse liver alcohol dehydrogenase, and lactate dehydrogenase. Enhancements in enzyme stability of greater than 100 times were observed in some cases. Model studies indicated that the PAN polymer reacts selectively with primary amines. These results can be related to the number and type of nucleophile containing amino acids on the surface of the enzyme and allow for a better determination of the optional reaction conditions for increased enzymatic stability. A new copolymer of styrene and acryloxysuccinimide was also prepared and used to immobilize several enzymes. Unlike the water-soluble PAN, this copolymer is insoluble in water but soluble in many organic solvents. The crosslinked enzymes were tested at 25°C in dioxane and water mixtures, and other organic solvents. In all cases, the immobilized enzyme was found to retain much greater stability than the corresponding non-treated enzyme.