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A novel expansin protein from the white-rot fungus Schizophyllum commune - PubMed

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A novel expansin protein from the white-rot fungus Schizophyllum commune

Omar Eduardo Tovar-Herrera et al. PLoS One. 2015.

Abstract

A novel expansin protein (ScExlx1) was found, cloned and expressed from the Basidiomycete fungus Schizophylum commune. This protein showed the canonical features of plant expansins. ScExlx1 showed the ability to form "bubbles" in cotton fibers, reduce the size of avicel particles and enhance reducing sugar liberation from cotton fibers pretreated with the protein and then treated with cellulases. ScExlx1 was able to bind cellulose, birchwood xylan and chitin and this property was not affected by different sodium chloride concentrations. A novel property of ScExlx1 is its capacity to enhance reducing sugars (N-acetyl glucosamine) liberation from pretreated chitin and further added with chitinase, which has not been reported for any expansin or expansin-like protein. To the best of our knowledge, this is the first report of a bona fide fungal expansin found in a basidiomycete and we could express the bioactive protein in Pichia pastoris.

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Conflict of interest statement

Competing Interests: Jorge Luis Folch-Mallol is currently an academic editor for PLOS ONE but this does not alter the authors’ adherence to PLOS ONE Editorial policies and criteria.

Figures

Fig 1
Fig 1. Protein alignment of BsExlx1, PcExl1, EXPB1 and ScExlx1.

Darker background indicates high amino acid conservation among the sequences. M1, M2 and M3 indicate the three classic motifs of plant expansins. The boxed amino acids are important residues involved in binding and creep activity in BsExlx1. White square indicates a putative N-glycosylation site in ScExlx1 and white circles indicate the predicted residues to form disulfide bonds.

Fig 2
Fig 2. Phylogeny of ScExlx1.

A) Phylogenetic relation of ScExlx1 with expansin and expansin-like proteins from plants, bacteria and fungi. Branch lengths represent the amount of genetic change between a node and its descendant. B) Phylogenetic divergence between expansin subfamilies. Ac. Acidovorax citrulli (gi|120612050); Mx. Myxococcus xanthus (gi|108762346); Rsp. Roseiflexus sp. (gi|148655687); Cm. Clavibacter michiganensis (gi|148272660). Hc. Hahella chejuensis (Uniprot: K2SG15) Pc. Pectobacterium carotovorum (gi|577859988); Bs. Bacillus subtilis (PDB 3D30). Asper-EXP. Aspergillus niger (gi|350632840).

Fig 3
Fig 3. Structure of ScExlx1 and comparison with previously crystallized expansins.

A) Three-dimensional model proposed for ScExlx1. Most conserved amino acids between plant and microbial expansins in D1 are depicted in magenta (Thr-49 and Asp-116). Sugar-binding residues in D2 are showed in cyan (Tyr-160, Tyr-161 and Tyr-195). B) Three-dimensional models showing the positive charged amino acids (Arg+Lys) between different EXLX proteins reported previously and ScExlx1 (Lysine is depicted in cyan and Arginine is depicted in magenta). Top-left, PDB: 3D30. Top-right, PDB: 2HCZ. Bottom-left, PDB: 4JCW. Bottom-right, ScExlx1. C) BsExlx1 (cyan model) and ScExlx1 (yellow model) superimposed, showing an N-terminal extension in ScExlx1 that it is absent in BsExlx1. Amino acids depicted in silver, C-terminal in both proteins. Amino acid in green, Met-1 of BsExlx1. Amino acids in purple, Arg-20 and Asp-38 depicting the N-terminal extension in ScExlx1).

Fig 4
Fig 4. Purification and western blot analysis of recombinant ScExlx1.

A) SDS-PAGE of control and recombinant strains. Supernatant from wild type P. pastoris X-33 (WT). Supernatant from P. pastoris X-33 transformed with pPICZαA empty vector (EV). Supernatant from P. pastoris transformed with pPICZαA-ScExlx1 (CSN). Purified ScExlx1. B) Western blot analysis of recombinant ScExlx1.

Fig 5
Fig 5. Disrupting activity of ScExlx1 on cotton fibers and avicel.

Light microscopy (10X) of cotton fibers and avicel incubated with ScExlx1 or proteins from mock supernatant for 72 h at 25°C. A) Proteins from mock supernatant acting on cotton fibers. B) “Bubble” effect on cotton fibers generated by ScExlx1. C) Avicel incubated with proteins from mock supernatant. D) Reduction in avicel size particle mediated by ScExlx1.

Fig 6
Fig 6. Neutral salt addition effect on ScExlx1 binding to plant cell wall polysaccharides and chitin.

Nacl effect on ScExlx1 binding to: A) avicel; B) chitin; C) xylan.

Fig 7
Fig 7. Effect of ScExlx1 on the enzymatic hydrolysis of cellulose.

Mercerized cotton fibers (1 mg) were incubated with 20 μg of ScExlx1, 20 μg of BSA or sodium acetate buffer (pH 5) for 72 h at 25°C. After incubation, temperature was raised to 50°C and cellulase cocktail from T. reesei was added (0.25 U) in a 3 h experiment. Reducing sugars were quantified by DNS method and compared with a glucose standard curve. Experiments were performed in triplicate, and the data points and error bars indicate means ± standard deviations. *Statistical differences between treatments (p<0.05) at each point.

Fig 8
Fig 8. ScExlx1 is a chitin active protein that enhances chitin hydrolysis.

Chitin from shrimp shells (5 mg) was incubated with 50 and 100 μg of ScExlx1, 50 μg of BSA, or sodium phosphate buffer 100 mM pH 7 for 24 h at 25°C. After incubation, temperature was increased to 37°C and chitinase from S. griseus (0.25 U) was added. After 2 hours of incubation, released N-acetylglucosamine was measured by DNS method and compared with a standard curve. Experiments were performed in triplicate, and the data points and error bars indicate means ± standard deviations. Letters indicate statistical differences in each treatment (p<0.05).

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Grants and funding

This work was funded by grant CB-153789-Q and PAICYT-UANL CN-1008-11 from the National Council for Science and Technology (CONACyT) from the Mexican government: http://www.conacyt.gob.mx. RAB-G and OET-H received a scholarship from CONACyT. The in silico analyses were performed during RAB-G's research stay funded by the Europe Molecular Biology Organization (EMBO: ASTF503-2013) in the Environmental Research Institute, University College Cork, Cork (Ireland). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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