The following types of starch were used for carboxymethylation: native starch, oxidized starch (Starch Industry, Zrenjanin, Serbia, starch oxidized by hypochlorite solution, content of carboxyl groups between 0.40-0.60 mass%) and starch enriched with amylose (BAGKF, Germany, 70% amylose).All the other chemicals used in this investigation (NaOH, Carboxymethyl starch (CMS) was obtained under heterogeneous conditions as a product of the reaction of starch and monochloroacetic acid (MCA) in the presence of sodium hydroxide. The influence of the composition of the reaction mixture, the reaction time and temperature and the type of starch being carboxymethylated on the degree of substitution, DS, and limiting viscosity number, [η], was studied. The reaction temperature influenced the DS values, the highest values of the DS being obtained when the carboxymethylation was performed at 58°C. Increase of the molar ratio of NaOH to anhydroglycose units leads to an increase in DS, but only to certain extent. The values of the DS and [η] of CMS increased almost linearly with increasing MCA amount in the reaction mixture. The reaction mixture changes caused by adding water to ethanol influenced the DS values of the obtained CMS samples, the maximum DS value being obtained for CMS sample synthesized in ethanol with 25% (v/v) water.

The degree of substitution (DS) markedly affects the properties of sodium carboxymethyl starch (Na-CMS). In this work Na-CMS samples with different DS starting from both potato and corn starch were synthesized in an ethanol/water mixture and their DS was determined using three methods: direct titration of the acid form of the carboxymethyl starch (H-CMS), Cu salt precipitation and back titration. It was found that direct titration gave smaller DS values, while the Cu salt precipitation method gave higher DS values than the back titration method, which was found to be the most accurate. The values of the DS obtained by these methods were similar for lower DS while for higher values the discrepancy was more pronounced. Under the same experimental conditions the CMS obtained from corn starch had a higher DS than the one obtained from potato starch and on average, the discrepancies between the DS values obtained by the employed analytical methods were smaller for the CMS samples synthesized from corn starch.

Three samples of corn starch and three samples of potato starch were transformed to carboxymethyl starch (CMS) using a two‐step etherification with sodium monochloroacetate in ethanol as the reaction medium. The degree of substitution (DS) of the synthesized samples was between 0.40 and 1.10. The structure parameters – solution properties relationships of the CMS samples were investigated by static light scattering and dilute solution viscometry in water and sodium‐chloride aqueous solutions of different concentrations at 25°C. The values of the mass average molar mass, MW, radius of gyration, Rg, and second virial coefficient, A2, were determined for all samples together with the limiting viscosity number. The results indicated that the Mw ‐ Rg correlation was independent of the origin of the starch, as well as the exponent aη and constant Kw from the Kuhn–Mark–Houwink equation. Values of the exponent ν for CMS in all sodium‐chloride aqueous solutions were between 0.450 and 0.472, while for the CMS samples in aqueous solution the value of ν was 0.580. The values of the exponent aη were around 0.3, which is typical for branched macromolecules.

Soybean hull peroxidase (SHP, E.C. 1.11.1.7) was immobilized by a glutaraldehyde and periodate method onto series of macroporous copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA), poly(GMA-co-EGDMA) with various surface characteristics and pore size diameters ranging from 44 to 200 nm. Glutaraldehyde immobilization method and poly(GMA-co-EGDMA) named SGE 20/12 with pore sizes of 120 nm gave immobilized enzyme with highest specific activity of 25 U/g. Deactivation studies showed that immobilization increased stability of SHP and that surface characteristics of the used copolymer had a major influence on a stability of immobilized enzyme at high temperatures and in an organic solvent. The highest thermostability was obtained using the copolymer SGE 20/12 with pore size of 120 nm, while the highest stability in dioxane had SHP immobilized onto copolymer SGE 10/4 with pore size of 44 nm. Immobilized SHP showed a wider pH optimum as compared to the native enzyme especially at alkaline pH values and 3.2 times increased K m value for pyrogallol. After 6 cycles of repeated use in batch reactor, immobilized SHP retained 25 % of its original activity. Macroporous copolymers with different surface characteristics can be used for fine tuning of activity and stability of immobilized SHP to obtain a biocatalyst suitable for phenol oxidation or polymer synthesis in organic solvents.

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g(-1) dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K (m)) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.