a-Amylases and glucoamylases free or immobilized in calcium alginate gel capsules for synergistic hydrolysis of crude starches

The hydrolysis of starch is usually carried out in two steps, consisting in a first stage of simultaneous gelatinization and liquefaction with a thermostable a-amylase and a second one of saccharification with a glucoamylase. However, when liquefaction and saccharification are separated processes, relatively long reaction times are needed. Reduction of hydrolysis time has been attempted by the use of a mixture of a-amylase and glucoamylase in a single step process. For this purpose a Bacillus subtilis thermostable a-amylase developed by our laboratory, as well as three commercial a-amylases and two commercial glucoamylases, in 8 combinations, were employed, free or immobilized in calcium alginate gel capsules, in the synergistic hydrolysis of crude starches. Enzyme immobilization in capsules prepared from 2% (w/v) sodium alginate and 5% (w/v) CaCl2 was attempted in order to assess the activity retained upon immobilization in comparison to the free enzymes. Although the immobilized enzymes appeared to hydrolyze crude starches satisfactorily, lower hydrolysis rates were achieved in comparison to free enzymes. The synergism of a-amylase and glucoamylase was observed by the higher reaction rates attained in the mixed-enzyme systems in comparison to the corresponding rates of sole glucoamylase and sole a-amylase systems. Generally the combinations containing Aspergillus niger glucoamylase were more efficient than the combinations employing Rhizopus mold glucoamylase. The enzyme system comprising B. subtilis thermostable a-amylase developed by our laboratory and commercial A. niger glucoamylase led to the highest degradation of all the crude starches tested. In an attempt to further improve the performance of the laboratory coenzyme system the effect of total enzyme concentration and the ratio of a-amylase=glucoamylase in the reaction mixture was studied. In the assayed range, higher reducing sugars yield was observed with increasing glucoamylase activity in the co-enzyme system. The a-amylase/glucoamylase ratio of 1:2 was the most effective for starch hydrolysis since more than 90 and 73% of substrate degradation was achieved in the case of free and immobilized co-enzyme system, respectively. Finally the immobilization of the co-enzyme system in calcium alginate gel capsules enabled its repeated use in 10 successive and efficient starch hydrolysis operations and the production of more than 3800 mg of reducing sugars during that period.