Submitted to: Protein Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 7, 2005
Publication Date: May 9, 2005
Citation: Wong, D., Batt, S.B., Lee, C.C., Wagschal, K.C., Robertson, G.H. 2005. Characterization of active lentinula edodes glucoamylase expressed and secreted by saccharomyces cerevisiae. Protein Journal. vol 24, pp 455-463. Interpretive Summary: In starch processing, starch granules are cooked at high temperatures before enzymes can be added to break down the starch to simple sugars for fermentation to produce ethanol or for further enzyme treatment to produce high fructose corn syrup. The pre-cooked starch is converted completely to simple sugars by two enzymes, a-amylase and glucoamylase, acting sequentially and in synergy. The fungal glucoamylase in this study is effective in degrading starch at low temperatures, an important property that may eliminate the need for the energy intensive and costly cooking process. The glucoamylase gene was cloned into yeast, which secreted the enzyme in an active form. This recombinant enzyme was purified, and its physiochemical properties and kinetic behavior were characterized. The results obtained in this investigation facilitate the ability to genetically modify the enzyme, to improve its ability to digest starch, and to decrease the chemical and energy costs of starch processing.
Technical Abstract: The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and 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 efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4-6.4 stability range. 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), with an apparent kcat of 2.9 x 105 min-1. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10X the amount of GLA.