Author
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Wong, Dominic |
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Batt-Throne, Sarah |
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Lee, Charles |
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Wagschal, Kurt |
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Robertson, George |
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Submitted to: Meeting Abstract
Publication Type: Abstract Only Publication Acceptance Date: 3/4/2005 Publication Date: 4/5/2005 Citation: Wong, D., Batt, S.B., Lee, C.C., Wagschal, K.C., Robertson, G.H. 2005. Expression and characterization of active. lentinus edodes glucoamylase in saccharomyces cerevisiae. Presentation 04:05. Paper No 11277. Interpretive Summary: Technical Abstract: The gene coding for Lentinus edodes glucoamylase (GLA) was isolated by PCR of the cDNA obtained by transcribing the total mRNA collected from the mycelium. The GLA gene was cloned into Saccharomyces cerevisiae, expressed and constitutively secreted in an active form. The enzyme was purified to homogeneity by (NH4)2SO4 fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was correctly processed. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a stability range of pH 3.4-6.4. The temperature optimum was 50 oC with stability < 50 oC. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn++ activated the enzyme by 45%, while Li+, Zn++, Mg++, Cu+, Ca++, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Apparent Km and Vmax for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40 oC, pH 5.3). Soluble starch was hydrolyzed 150 and 290 times faster than wheat and corn raw starch, respectively. |
