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Figure 1.Schematic representation of the wild-type RoGA and various mutant of RoGA. Full-length RoGA and its deletion mutants were cloned into the yeast expression plasmid pS1. Each construct was designed to have a natural signal sequence (SS) for secretion. The starch-binding and catalytic domains and the sequence of the linker region (amino acids 132–167) of RoGA are indicated. The residues in the linker which were subjected to mutagenesis are underlined. Closed circles indicate the potential N-linked glycosylation sites.
Figure 2.Expression of recombinant RoGAs in Saccharomyces. (A) Starch plate assay for GA expression in 10 μL (A600 = 2) S. cerevisiae cells carrying pS1 (lane 1), pS1/full-length GA (lane 2) and pS1/GAΔ132–167 (lane 3) plasmids growth on a SD plate containing 0.5% soluble starch at 30°C and stained with 0.01% iodine solution. (B) Yeast strain was transformed with expression plasmids containing the full-length or mutant GA insert. After incubation at 30°C for 3 days, the supernatants were collected and concentrated. Culture supernatant (10 μL) from each clone was examined by Western blotting using anti-GA antibody. Upper panel, lane 1, vector only; lane 2, full-length GA; lane 3, GAΔ168–604 and lane 4, GAΔ132–604; lower panel, lane 1, vector only; lane 2, full-length GA; lane 3, GAΔ132–167; lane 4, GAΔ26–131; lane 5, GAΔ26–145; lane 6, GAΔ26–160 and lane 7, GAΔ26–167.
Figure 3.Purification and deglycosylation of recombinant RoGAs. (A) Purified proteins (1 μg) obtained by cation-exchange chromatography were subjected to electrophoresis on a 15% SDS-PAGE and stained with Coomassie Brilliant Blue R-250. Lane 1, full-length GA; lane 2, GAΔ168–604; lane 3, GAΔ132–604; lane 4, GAΔ26–131; lane 5, GAΔ26–145 and lane 6, GAΔ26–160. (B) Protein samples were treated with PNGase F (250 U) and resolved by 10% SDS-PAGE. Symbols (+) and (-) indicate treatment with and without PNGase F, respectively. (C) Recombinant SBD was treated with Jack bean α-mannosidase and examined by 15% SDS-PAGE. Symbols (+) and (-) indicate treatment with and without Jack bean α-mannosidase, respectively.
Figure 4.Characterization of enzymatic function of recombinant RoGAs. (A) The extracellular activity of various recombinant GAs was tested. Lane 1, pS1 (empty vector); lane 2, full-length GA; lane 3, GAΔ26–167; lane 4, GAΔ132–167; lane 5, T165A and lane 6, N167D. (B) Western blot analysis of wild-type and mutant GAs grown at 30°C (upper panel) and 20°C (lower panel) for 3 days. Lane 1, pS1 (empty vector); lane 2, full-length GA; lane 3, GAΔ26–167; lane 4, GAΔ132–167; lane 5, T165A and lane 6, N167D. (C) Starch plate assays of mutant GAs. No. 1, S. cerevisiae MNN10 cells transformed with pS1 (empty vector); No. 2, pS1-full-length GA; No. 3, pS1-GAΔ26–167; No. 4, pS1-GAΔ132–167; No. 5, pS1-T165A and No. 6, pS1-N167D. (D) The electrophoretic mobilities of wild-type GA (lane 1) and N167D (lane 2) on an 8% SDS-PAGE.
Figure 5.Binding of purified SBD to corn-starch. SBD, at different concentrations, was allowed to adsorb to insoluble corn-starch (1 mg) to the point of equilibrium. Protein concentrations were determined by direct measurement of unbound protein in solution. The data shown are representative of three independent experiments. Symbols: GAΔ132–604 (open circle) and GAΔ168–604 (open triangle).
Figure 6.Effects of temperature and pH on the enzymatic activity of RoGA. The effect of temperature on enzymatic activity (solid curves) was determined by pre-incubating the enzyme at pH 4.5 for 30 min at different temperatures and then assayed for residual activity. The effect of pH on GA activity (dotted curves) was performed in 0.1 M glycine/acetate/phosphate/tris buffers at the indicated pH values at 25°C. The activities measured without any pretreatment were defined as 100%. Each data point represents the average of three measurements, and error bars represent the S.D. Symbols: full-length GA (open square), GAΔ26–131 (closed circle), GAΔ26–145 (closed triangle), and GAΔ26–160 (closed square).
Figure 7. Structural determination of wild-type and mutant RoGAs. (A) Each circular dichroism spectrum represents an average of three scans. The data were recorded at 25°C and corrected for the baseline contribution of the buffer. Spectra were truncated below 200 nm because of excessive noise at those wavelengths. Symbols: full-length GA (open square), GAΔ132–604 (open circle), GAΔ168–604 (open triangle), GAΔ26–131 (closed circular), GAΔ26–145 (closed triangle) and GAΔ26–160 (closed square). (B) Thermal denaturation curves of GAΔ132–604 (open circle) and GAΔ168–604 (open triangle) were obtained by monitoring changes in ellipticity at 215 nm.
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