Characterization of cellulolytic enzyme system of <i>Schizophyllum commune</i> mutant and evaluation of its efficiency on biomass hydrolysis

<p><i>Schizophyllum commune</i> is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from <i>S. commune</i> mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and β-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external β-xylosidase.</p> <p>Enzymatic hydrolysis of alkaline-pretreated biomass by Sc-Cel and yield enhancement by external β-xylosidase (BX).</p

Validation of proteins differentially expressed in response to CHIKV infection in CHME-5 cells.

<p>(A) CHME-5 cells were either mock infected or infected with CHIKV at MOI 0.1 before extraction of proteins and analysis by Western blot analysis on 1 and 2 d.p.i. hnRNP: heterogeneous nuclear ribonucleoprotein; NCL: nucleolin; JAK2: tyrosine-protein kinase JAK2; Hsp70: heat shock protein 70; Hsp90: heat shock protein 90. (B). CHME-5 cells were either mock infected or infected with CHIKV at MOI 0.1 before extraction of total RNA and analysis by RT-PCR on 1, 2 and 3 d.p.i. BRE1B: E3 ubiquitin-protein ligase; CUL9: Cullin-9; CHD2: chromodomain-helicase-DNA binding protein 2; MTERF: mitochondrial precursor transcription termination factor; ROD1: regulator of differentiation 1 isoform; PIK3CD: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta; GCDH: mitochondrial glutaryl-CoA dehydrogenase isoform precursor; HSDL2: hydroxysteroid dehydrogenase-like protein 2; PLCH2: 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase eta-2; ALOX12: 12-lipoxygenase; DRPLA: Dentatorubral pallidoluysian atrophy protein; DENND3: DENN domain-containing protein 3; HIS1H2B: Histone 2B.</p

Sub-cellular distribution and functional annotation of proteins significantly differentially expressed in response to CHIKV infection of CHME-5 cells.

<p>The sub-cellular (A) and functional (B) categorization of the proteins was performed using the GoCat software.</p

Values of fold change in response to CHIKV infection, significance and half life of selected proteins.

<p>Values of fold change in response to CHIKV infection, significance and half life of selected proteins.</p

Infection and apoptosis in CHIKV infected CHME-5 cells.

<p>(A and B) CHME-5 cells either mock infected or infected with CHIKV at MOI 2.5 or 5 were collected at day 2 p.i. and (A) cells were stained with an anti-alphavirus antibody and the percentage of infected cells was determined by flow cytometry or (B) cells were stained with Annexin V-FITC and PI and the percentage of induced apoptosis determined by flow cytometery. Bar graphs represent the means ± SD of 3 replications per group. (C and D) CHME-5 cells either mock infected or infected with CHIKV at MOI 2.5 or 5 were collected on days 2 and 4 p.i. and analyzed by flow cytometry after double staining with antibodies directed against active caspase 3 and alphavirus. Experiment was undertaken in three independent replicates. Representative flow cytometry dot plot is shown in (C) and data is shown graphically in (D). Bar graphs represent the means ± SD of 3 replications per group.</p

Analysis of CHIKV infected CHME-5 cells.

<p>CHME-5 cells either mock infected or infected with CHIKV at MOI 0.1 were collected at day 2 p.i. (A, B, D) or on days 1 to 3 p.i. (C) and subsequently (A) stained with an anti-alphavirus antibody and the percentage of infected cells analyzed by flow cytometry or (B) stained with Annexin V-FITC and PI and the percentage of apoptotic cells analyzed by flow cytometery or (C, D) used for total protein extraction and (C) analyzed by western blotting with an anti-alphavirus monoclonal antibody and an anti-actin polyclonal antibody or (D) the differential proteome determined by 2D-PAGE. Representative gels from 6 biological replicates are shown. (A and B) Bar graphs represent the means ± SD of 6 replications.</p

GeLC-MS/MS analysis of the proteome of CHIKV infected CHME-5 cells.

<p>CHME-5 cells either mock infected or infected with CHIKV at MOI 2.5 were collected at day 2 p.i. and proteins extracted and the proteomes determined by GeLC-MS/MS. Each line of the graph represents a single protein and the intensity of individual proteins is shown. The upper panel of the graph shows differentially expressed proteins significant at <i>p</i><0.01. The lower panel shows proteins which are not significant at <i>p</i><0.01. Samples were analyzed as three independent replicates.</p