Synergistic action of recombinant α-amylase and glucoamylase on the hydrolysis of starch granules

Barley alpha-amylase 1 mutant (AMY) and Lentinula edodes glucoamylase (GLA) were cloned and expressed in Saccharomyces cerevisiae. The purified recombinant AMY hydrolyzed corn and wheat starch granules, respectively, at rates 1.7 and 2.5 times that of GLA under the same reaction conditions. AMY and GLA synergistically enhanced the rate of hydrolysis by similar to 3x for corn and wheat starch granules, compared to the sum of the individual activities. The exo-endo synergism did not change by varying the ratio of the two enzymes when the total concentration was kept constant. A yield of 4% conversion was obtained after 25 min 37 degrees C incubation (1 unit total enzyme, 15 mg raw starch granules, pH 5.3). The temperature stability of the enzyme mixtures was <= 50 degrees C, but the initial rate of hydrolysis continued to increase with higher temperatures. Ca++ enhanced the stability of the free enzymes at 50 degrees C incubation. Inhibition was observed with the addition of 10 mM Fe++ or Cu++, while Mg++ and EDTA had lesser effect.