Enzyme hydrolysis was performed in a 96-deep well format using the G LBRC En zyme Plat form (GENPLAT) as described earlier [4 (link),5 (link)]. Feedstocks were suspended and dispensed at 0.5% glucan, and final glucan loadings were 0.2%. Unless otherwise specified, enzyme loadings for all commercial benchmarks and for all mixture experiments were kept constant at 15 mg/g glucan, and reaction mixtures were incubated for 48 h at 50°C.
Design-Expert software (Stat-Ease Inc., Minneapolis, MN, USA) was used for experimental design and analysis. An augmented quadratic design was used throughout; thus, mixtures containing 6 and 16 components required 28 and 153 individual reactions, respectively. The lowest proportion of any enzyme in the core set (defined as CBH1, CBH2, EG1, BG, EX3, and BX) was set to 4%, because earlier studies indicated that for most of the core set, allowing them to go to 0% led to such poor Glc yields that reliable models could not be predicted [4 (link)]. The lowest proportion of all other enzymes ("accessory" proteins) was set to 0%. All assays were replicated once, sampled twice and assayed for Glc and Xyl twice, for a total of eight replicates of each mixture. Glc and Xyl were assayed colorimetrically [4 (link)]. Model predictions were tested experimentally as indicated in each table.
The monosaccharide composition of feedstocks was determined by the GLBRC Analytical Laboratory at Michigan State University. Briefly, samples were ground and washed sequentially with water, 70% ethanol, 1:1 chloroform:methanol, and acetone. The samples were then treated with amyloglucosidase + α-amylase, and the released Glc was quantitated as starch. The remaining material was then hydrolyzed with 2 N trifluoroacetic acid, and the released sugars were quantitated by GC of the alditol acetates. The insoluble residue from this step was treated with Updegraff's reagent, and the insoluble material was hydrolyzed with strong sulfuric acid and quantitated as cellulose using anthrone [16 (link),17 (link)].
The proteins in the commercial preparation Novozyme 188 and in β-mannanase (Megazyme catalog E-BMANN) were analyzed using standard mass spectrometry-based proteomics [3 (link)]. Scaffold version 01_07_00 (Proteome Software, Portland, OR, USA) was used to probabilistically validate protein identifications (DOE Joint Genome Institute) using the X!Tandem and ProteinProphet computer algorithms.
Design-Expert software (Stat-Ease Inc., Minneapolis, MN, USA) was used for experimental design and analysis. An augmented quadratic design was used throughout; thus, mixtures containing 6 and 16 components required 28 and 153 individual reactions, respectively. The lowest proportion of any enzyme in the core set (defined as CBH1, CBH2, EG1, BG, EX3, and BX) was set to 4%, because earlier studies indicated that for most of the core set, allowing them to go to 0% led to such poor Glc yields that reliable models could not be predicted [4 (link)]. The lowest proportion of all other enzymes ("accessory" proteins) was set to 0%. All assays were replicated once, sampled twice and assayed for Glc and Xyl twice, for a total of eight replicates of each mixture. Glc and Xyl were assayed colorimetrically [4 (link)]. Model predictions were tested experimentally as indicated in each table.
The monosaccharide composition of feedstocks was determined by the GLBRC Analytical Laboratory at Michigan State University. Briefly, samples were ground and washed sequentially with water, 70% ethanol, 1:1 chloroform:methanol, and acetone. The samples were then treated with amyloglucosidase + α-amylase, and the released Glc was quantitated as starch. The remaining material was then hydrolyzed with 2 N trifluoroacetic acid, and the released sugars were quantitated by GC of the alditol acetates. The insoluble residue from this step was treated with Updegraff's reagent, and the insoluble material was hydrolyzed with strong sulfuric acid and quantitated as cellulose using anthrone [16 (link),17 (link)].
The proteins in the commercial preparation Novozyme 188 and in β-mannanase (Megazyme catalog E-BMANN) were analyzed using standard mass spectrometry-based proteomics [3 (link)]. Scaffold version 01_07_00 (Proteome Software, Portland, OR, USA) was used to probabilistically validate protein identifications (DOE Joint Genome Institute) using the X!Tandem and ProteinProphet computer algorithms.
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