Polyvinylsaccharides are synthesized by reductive amination of sugars without protecting groups, by reaction with methacrylic acid anhydride to obtain monomers and finally by free radical homopolymerization. The characterization of the polymers is carried out by viscosity, light scattering and elemental analysis with respect to molecular seize and composition.[1]

Ionic polymers are produced by polymeranalogous reaction. Cationic polymers are obtained by reaction of neutral polyvinylsaccharides with 3-chloro-2-hydroxypropyltrimethylammonium-chloride and sodiumhydroxide.

Anionic polymers are modified by sulfatation using sulfur trioxid pyridine complex in pyridine or carbxymethylation using sodiumchloroacetate, sodiumhydroxide and formaldehyd . The polymers are purified by dialysis and characterized by viscosity, light scattering and elemental analysis with respect to molecular seize and composition. The degree of substitution is additionally examined by polyelectrolyte titration.

Examinations of the functional stabilization of horseradish peroxidase as model enzyme are carried out at elevated temperature (55°C) with the enzyme without additions as reference. The experiments are normally realized with a concentration of 2% of additive in a phosphate buffer (pH 5,0). The residual activities with additions of polymers are measured within certain timeintervals. [2, 3, 4] Neither the corresponding sugar nor the monomer show significant effect on the stability of the enzym whereas some neutral polymers indicate stabilization potential.[5] This ability for stabilization depends strongly on the structure of the carbohydrate side group of the polymers.

Cationic and anionic polymers have a greater potential to stabilize the enzyme than neutral polymers. Under certain conditions, these polymers are able to significantly enhance the functional stability of the enzyme. In this respect the type of ionic group is of importance. While sulfated polymers show excellent results, carboxymethylated polymers destroy the activity of POD, presumable by binding the calcium of POD and so affecting the structural stability of the enzyme.

The concentration of the additives has an important influence on the stabilization, to obtain satisfactory results, the concentration of polymer in the solution should exceed 0.5%.

Even the molecular weight plays an important role. The best neutral polymers concerning their stabilization potential exhibit a molecular weight of about 2 million dalton, ionic polymers show similar tendencies.

The degree of substitution (DS) has also to be considered. Polymers with low degrees of substitution containing less than one ionic group per sugar unit show better results than polymers with high DS.

The carbohydrate based acryl polymers show a significant higher potential to stabilize POD than normally used polymers like PEG and Dextran. Some microcalorimetric results leed to the conclusion that stabilizational effects primarily depend on environmental effects and not on specific protein polymer interactions. Comparable investigations related to the stabilization of other enzymes will be discussed.

[1] J.Klein, A.Haji Begli, Makromol.Chem., 190, 2527-2534, 1989

[2] T.D.Gibson, I.J.Higgins, J.R. Woodward, Analyst., 117, 1293-1297, 1992

[3] T.D.Gibson, J.N.Hulbert, B.Pierce, J.I. Webster, Stability and Stabilization of Enzymes, Proc. of an Intern. Symp. held in Maastricht, 22-25.11.1992, Elsevier Science Publisher, 1993

[4] T.D.Gibson, J.N.Hulbert, J.R. Woodward, Anal.Chim.Acta., 279, 185-192, 1993

[5] DE 4011084 A1 (1990), invs.: W.Tischer, J.Klein, R.-J.Müller, S.Engelke