title: Characterization of active Lentinula edodes glucoamylase expressed and secreted by Saccharomyces cerevisiae.
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 degrees C with stability <or=50 degrees C. 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. 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. Apparent Km and Vmax for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40 degrees C, pH 5.3), with an apparent kcat of 2.9 x 10(5) min(-1).
authors.affiliation: Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, California 94710, USA. dwsw@pw.usda.gov
authors.forename: Dominic W S
authors.initials: DW
authors.lastname: Wong
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Batt
authors.forename: Charles C
authors.initials: CC
authors.lastname: Lee
authors.forename: Kurt
authors.initials: K
authors.lastname: Wagschal
authors.forename: George H
authors.initials: GH
authors.lastname: Robertson
mesh_terms: D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005947:Glucose / Q000096:biosynthesis; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D020942:Shiitake Mushrooms / Q000201:enzymology* / Q000235:genetics; D013213:Starch / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D009005:Monosaccharides; D011994:Recombinant Proteins; D013213:Starch; D005087:Glucan 1,4-alpha-Glucosidase; D005947:Glucose