THIOLATION AND REVERSIBLE IMMOBILIZATION OF SWEET-POTATO BETA-AMYLASE ON THIOLSULFONATE-AGAROSE

Among the different methods for obtaining immobilized biocatalysts, those based on thiol-disulfide exchange reactions are unique because, simultaneously, they show a stable covalent bond and the possibility of eluting the protein by reduction when the enzymatic activity decays. The adsorbent can thus be reloaded. In this paper we report the use of the recently developed thiol-reactive adsorbent thiolsulfonate-agarose, for the immobilization of sweet potato beta-amylase. Since native beta-amylase thiol groups were not reactive towards the adsorbent, the enzyme was provided with 'de novo' thiol groups by reaction with the heterobifunctional reagent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). When the SPDP/beta-amylase molar ratio was changed between 3 and 100, up to sixteen exposed thiol groups per mol of enzyme were introduced. This was achieved without affecting the amylolytic activity. The immobilization yield for the intermediate thiolation level was 98%. However, only 19% of the applied enzyme activity was found in the gel suspension. Comparative studies were made on thiolsulfonate-agarose and on a commercial thiol-activated adsorbent (2-pyridyldisulfide-agarose). The immobilization of the thiolated enzyme through reversible disulfide bonds on both adsorbents showed similar results. A close analysis reveals that immobilization of proteins on thiolsulfonate-agarose is a very promising technique.