Submitted to: Protein and Peptide Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/3/2006
Publication Date: 4/1/2007
Citation: Wong, D., Robertson, G.H., Batt, S.B., Lee, C.C., Wagschal, K.C. 2007. Synergistic action of alpha-amylase and glucoamylase on the hydrolysis of native starch granules. Protein and Peptide Letters. 26:159-164.
Interpretive Summary: In the production of biofuel, raw starch granules are cooked at high temperatures, and the pre-cooked starch is converted completely to fermentable sugars by two enzymes, alpha-amylase and glucoamylase, acting sequentially. We have isolated, cloned, and evolved the genes of alpha-amylase from plants and glucoamylase from fungus. These individual enzymes are effective in degrading starch at low temperatures without prior cooking. In this study, we combined the two enzymes in pure forms and characterized their synergistic actions on corn and wheat raw starch. Significant enhancement of digestion rates was observed, because both enzymes showed starch-binding activities and functioned optimally under the same set of conditions. These results will facilitate further optimization of simultaneous liquefaction and saccharification of raw starch granules. Potential delivery systems for commercial use of these molecules include expression in fungal systems or in the fermentative yeast systems.
Technical Abstract: Barley alpha-amylase 1 mutant (AMY) and Lentinula edodes glucoamylase (GLA) were cloned and expressed in Saccharomyces cerevisiae. AMY hydrolyzed wheat and corn starch granules, respectively, at rates 2.5 and 1.7 times that of GLA under the same reaction conditions. AMY and GLA synergistically enhanced the rate of hydrolysis by ~3X for wheat and corn 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 ~40% conversion was obtained after 25 min 37 oC incubation (1 unit total enzyme, 15 mg raw starch granules, pH 5.3). The temperature stability of the enzyme mixtures was < 50 oC, but the initial rate of hydrolysis continued to increase with higher temperatures. Ca++ enhanced the stability of the free enzymes at 50 oC incubation. Inhibition was observed with addition of 10 mM Fe++ or Cu++, while Mg++ and EDTA had lesser effect. Mn++ enhanced the rate of hydrolysis with a maximum yield increase of 48% at 1 mM and higher concentrations.