Analysis of the structures and stabilities of mROL^WT and mROL^imp.

<p>(A) Circular dichroism spectra of mROL^WT and mROL^imp. (B) The thermal stabilities of mROL^WT and mROL^imp. The negative ellipticities at 222 nm were measured to determine the fraction folded. The values are presented as mean ± SEM based on three independent measurements. The <i>P</i>-value was determined using a two-factor ANOVA with mROLs and temperatures as independent factors.</p

Lipase activity assay with purified ROL.

<p>(A) Measurement of the lipase activities of mROL^WT and mROL^imp using <i>p</i>-nitrophenyl esters with various acyl chain lengths (C2–C16) as substrates. The resultant <i>p</i>-nitrophenol was quantified to estimate the lipase activities. (B) Relative lipase activities normalized with the values obtained for mROL^WT. The values are presented as mean ± standard error of the mean (SEM) based on at least three independent measurements. (C) Competitive lipase activity assay with purified mROL^WT and mROL^imp. Lipase activities were determined in the presence of the peptidase substrate, Suc-Ile-Ile-Trp-MCA, dissolved in dimethyl sulfoxide (DMSO). The <i>P</i>-values were determined using the Student’s <i>t</i>-test. * <i>P</i> < 0.05.</p

Activity assay using yeast cell surface engineering.

<p>(A) Peptidase activity assay of mROL^WT, mROL^imp, and mROL^imp2 displayed on the yeast cell surface. (B) Lipase activity assay of mROL^WT, mROL^imp, and mROL^imp2 displayed on the yeast cell surface. The resultant <i>p</i>-nitrophenol was quantified to estimate the lipase activities. The peptidase and lipase activities were corrected by the number of displayed enzymes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.s003" target="_blank">S3 Fig</a>). The values are presented as mean ± SEM based on three independent measurements. The <i>P</i>-values were determined using one-way analysis of variance followed by Tukey’s test for multiple comparisons. ** <i>P</i> < 0.01.</p

Sequence alignment of <i>Rhizopus oryzae</i> lipase (ROL) and ROL-related lipases.

<p>The full-length primary sequences of ROL, <i>R</i>. <i>niveus</i> lipase (RNL), <i>R</i>. <i>stolonifer</i> lipase (RSL), and <i>R</i>. <i>chinensis</i> lipase (RCL) are presented. Multiple-sequence alignments were generated using the ClustalW program (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). The underlined sequences in the propeptide of ROL (Ser20–Gly37 and Ser38–Glu57) indicate the regions that are essential for secretion and folding of mROL, respectively. The underlined sequences in the mature domain of ROL (Phe183–Asp189) indicate the lid domain. The shadowed region indicates residues that were replaced with hydrophilic amino acids (VDDDDK). In the original host, <i>R</i>. <i>oryzae</i>, the propeptide is also cleaved between the Ala97 and Ser98 residues [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.ref021" target="_blank">21</a>]; however in <i>P</i>. <i>pastoris</i> and <i>S</i>. <i>cerevisiae</i>, the secondary cleavage has not been observed [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.ref014" target="_blank">14</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0124545#pone.0124545.ref022" target="_blank">22</a>]. Therefore, in this study, we defined the propeptide domain as the region between residues 1 and 69 and the mature domain as the region between residues 70 and 366 of ROL.</p

Structure of mROL^WT, modeled using the SWISS-MODEL program.

<p>The structure of mROL^WT was modeled based on the open-lid structure of <i>Rhizomucor miehei</i> lipase (Protein Data Bank [PDB] ID: 4TGL), and visualized using PyMOL. The active site residues, S242, D301, and H354, are colored orange. The magenta-colored α-helix represents the lid domain. Green residues indicate hydrophilic amino acids and white residues indicate hydrophobic amino acids.</p

Additional file 2: Table S2. of Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis

All proteins identified in the qualitative proteome analysis. Supplemental qualitative proteome analysis was carried out to strengthen the quantitative proteome analysis. Three independent biological experiments were carried out, and proteins with MASCOT scores >10 were accepted as identified protein. Score: MASCOT score; PSMs: Peptides spectrum matches

Additional file 1: Table S1. of Elucidation of the recognition mechanisms for hemicellulose and pectin in Clostridium cellulovorans using intracellular quantitative proteome analysis

The 734 proteins identified in quantitative proteome analysis. Proteome analytes were injected to LC–MS/MS system. Obtained data were used for protein identification with Proteome Discoverer software 1.4. Three independent biological experiments were carried out, and proteins with no missing values were accepted as identified protein. As a result, 734 proteins were successfully identified

Direct bioethanol production from brown macroalgae by co-culture of two engineered <i>Saccharomyces cerevisiae</i> strains

<p>A co-culture platform for bioethanol production from brown macroalgae was developed, consisting of two types of engineered <i>Saccharomyces cerevisiae</i> strains; alginate- and mannitol-assimilating yeast (AM1), and cellulase-displaying yeast (CDY). When the 5% (w/v) brown macroalgae <i>Ecklonia kurome</i> was used as the sole carbon source for this system, 2.1 g/L of ethanol was produced, along with simultaneous consumption of alginate, mannitol, and glucans.</p

Protein sequence alignment between Sap1–3 and Sap7.

<p>Multiple-sequence alignments were performed with CLUSTAL W. The active site of Sap7 was predicted to be D244 and D464 by homology. The cleavage point of Sap7 was found in the Sap7-specific insertion sequence N422-N451, which did not exist in the sequences of Sap1–3.</p

Additional file 5: of Description of the interaction between Candida albicans and macrophages by mixed and quantitative proteome analysis without isolation

Proteins identified in this study for C. albicans monoculture (976 proteins) and macrophage monocultre (1,357 proteins)