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Supplemental document
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Measurement of each cellulase activity
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The activities of four cellulases were measured as follows; The activity assay of EG
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was performed at 30°C by using a 500-μL mixture containing 50 mM of acetate buffer
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(pH 5.0) and 25 μL of the culture supernatant. The reaction was initiated by the
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addition of 0.5% (w/v) carboxymethyl cellulose (CMC). The reducing sugar liberated
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during the reaction was colorimetrically estimated by the Somogyi–Nelson method
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with glucose as the standard.1,2) The EG activity was calculated by determining the
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amount of reducing sugar liberated from CMC. The incubation time was adjusted such
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that the EG assay was linear with time. One unit of EG activity was defined as the
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amount of enzyme that liberated 1 μmol of reducing sugar/min.
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The CBHI activity assay was performed at 30°C by using a mixture (500 μL) of 50
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mM acetate buffer (pH 5.0) and 100 μL of culture supernatant. The reaction was
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initiated by the addition of 4 mM p-nitrophenyl-β-lactopyranoside and terminated by
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the addition of 0.5 M sodium carbonate solution. p-Nitrophenol liberated during the
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reaction was colorimetrically estimated at 405 nm. The CBHI activity was calculated
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by determining the amount of p-nitrophenol liberated from
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p-nitrophenyl-β-lactopyranoside. The incubation time was adjusted such that the CBHI
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assay was linear with time. One unit of CBHI activity was defined as the amount of
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enzyme that liberated 1 μmol of p-nitrophenol/min.
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The CBHII activity was measured by the same method as that used to measure EG
activity, using phosphoric acid swollen cellulose (PASC) instead of CMC as the
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substrate. PASC was prepared from Avicel PH-101 (Fluka Chemie GmbH, Buchs,
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Switzerland) as amorphous cellulose.3) One unit of CBHII activity was defined as the
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amount of enzyme that liberated 1 μmol of reducing sugar/min.
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The BGL activity was measured by the same method as that used to measure CBHI
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activity, using p-nitrophenyl-β-glucopyranoside instead of
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p-nitrophenyl-β-lactopyranoside as the substrate. One unit of BGL activity was defined
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as the amount of enzyme that liberated 1 μmol of p-nitrophenol/min.
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References
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1) Somogyi M. Micromethods for the estimation of diastase. J. Biol. Chem.
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1938;125:399–414.
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2) Nealson N. A photometric adaptation of the Somogyi method for the determination
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of Glucose. J. Biol. Chem. 1944;153:375–380.
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3) Den Haan R, Rose SH, Lynd LR, van Zyl WH. Hydrolysis and fermentation of
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amorphous cellulose by recombinant Saccharomyces cerevisiae. Metab. Eng.
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2007;9:87–94.
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Supplemental figure
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Supplemental Fig. SF1. Cellulase production and secretion by the recombinant A.
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oryzae
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The time course of secreted protein concentration (A). Circle, EG; triangles, BGL;
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square, CBHI; and diamonds, CBHII. Data are the averages from three independent
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experiments. Error bars represent the standard deviation. SDS-PAGE analysis of the
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supernatant of the culture (B). Ten microliters of the culture supernatant was loaded in
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each lane: Lane W, IF4; Lane E, IF4/pIS1-EG; Lane B, IF4/pIS1-BGL; Lane CI,
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IF4/pIS1-CBHI; Lane CII, IF4/pIS1-CBHII; and Lane M, Precision Protein Standard
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marker.
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Supplemental Fig. SF2. Simultaneous saccharification and fermentation of ethanol
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from kraft pulp
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Filled circles, with modified cellulase mixture; open circles, without cellulase. Data are
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the averages from three independent experiments. Error bars represent the standard
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deviation.
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