Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 1/23/2006
Publication Date: 4/2/2006
Citation: Wong, D., Robertson, G.H., Lee, C.C., Wagschal, K.C. 2006. Synergistic action of barley alpha-amylase and lentinula edodes glucoamylase on raw starch hydrolysis. Approved 10/27/2005. American Society of Biochemistry & Molecular Biology, San Francisco, CA. April 1-4, 2006, Program 73.16, vol A27.
Technical Abstract: The genes encoding barley alpha-amylase (AMY1) and Lentinus edodes glucoamylase (GLA) were cloned into Saccharomyces cerevisiae, expressed and constitutively secreted in active forms. The two enzymes were purified by (NH4)2SO4 fractionation and affinity chromatography. AMY1 hydrolyzed starch granules at rates 2 times that of GLA under the same reaction conditions. The recombinant enzymes were mixed in various combinatorial ratios to determine the optimum conditions for hydrolysis of raw starch granules at 37 oC. AMY1 and GLA synergistically increased the rate by 2 and 4 times, respectively, compared to the use of AMY1 and GLA alone. The synergistic effect on the initial rate of hydrolysis did not change by varying the ratio of the two enzymes, using either raw corn or wheat starch as the substrate. The temperature stability of the enzyme mixtures was < 50 degree C, but the extent of hydrolysis continued to increase with higher temperatures. Ca++ increased the stability of the free enzymes at 50 degree C incubation, and EDTA caused a significant decrease in the subsequent activity. Inhibition was observed when 10 mM Fe++ or Cu++ was added to the reaction, with yield losses of 29 and 53%, respectively, while Mg++ and EDTA showed lesser effects. Under the same conditions, Mn++ enhanced hydrolysis, with a maximum yield increase of 44% at 1 mM and higher concentrations.