title: Functional characterization of the native swollenin from Trichoderma reesei: study of its possible role as C1 factor of enzymatic lignocellulose conversion.
abstract: BACKGROUND
  Through binding to cellulose, expansin-like proteins are thought to loosen the structural order of crystalline surface material, thus making it more accessible for degradation by hydrolytic enzymes. Swollenin SWO1 is the major expansin-like protein from the fungus Trichoderma reesei. Here, we have performed a detailed characterization of a recombinant native form of SWO1 with respect to its possible auxiliary role in the enzymatic saccharification of lignocellulosic substrates.
  
  
  RESULTS
  The swo1 gene was overexpressed in T. reesei QM9414 Δxyr1 mutant, featuring downregulated cellulase production, and the protein was purified from culture supernatant. SWO1 was N-glycosylated and its circular dichroism spectrum suggested a folded protein. Adsorption isotherms (25 °C, pH 5.0, 1.0 mg substrate/mL) revealed SWO1 to be 120- and 20-fold more specific for binding to birchwood xylan and kraft lignin, respectively, than for binding to Avicel PH-101. The SWO1 binding capacity on lignin (25 µmol/g) exceeded 12-fold that on Avicel PH-101 (2.1 µmol/g). On xylan, not only the binding capacity (22 µmol/g) but also the affinity of SWO1 (K d = 0.08 µM) was enhanced compared to Avicel PH-101 (K d = 0.89 µM). SWO1 caused rapid release of a tiny amount of reducing sugars (<1 % of total) from different substrates (Avicel PH-101, nanocrystalline cellulose, steam-pretreated wheat straw, barley β-glucan, cellotetraose) but did not promote continued saccharification. Atomic force microscopy revealed that amorphous cellulose films were not affected by SWO1. Also with AFM, binding of SWO1 to cellulose nanocrystallites was demonstrated at the single-molecule level, but adsorption did not affect this cellulose. SWO1 exhibited no synergy with T. reesei cellulases in the hydrolysis of the different celluloses. However, SWO1 boosted slightly (1.5-fold) the reducing sugar release from a native grass substrate.
  
  
  CONCLUSIONS
  SWO1 is a strongly glycosylated protein, which has implications for producing it in heterologous hosts. Although SWO1 binds to crystalline cellulose, its adsorption to xylan is much stronger. SWO1 is not an auxiliary factor of the enzymatic degradation of a variety of cellulosic substrates. Effect of SWO1 on sugar release from intact plant cell walls might be exploitable with certain (e.g., mildly pretreated) lignocellulosic feedstocks.
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, 8010 Graz, Austria.
authors: Manuel
authors: M
authors: Eibinger
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, 8010 Graz, Austria.
authors: Karin
authors: K
authors: Sigl
authors: Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Steyrergasse 17, 8010 Graz, Austria.
authors: Jürgen
authors: J
authors: Sattelkow
authors: Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Steyrergasse 17, 8010 Graz, Austria.
authors: Thomas
authors: T
authors: Ganner
authors: Research Division Biochemical Technology, Institute of Chemical Engineering, TU Wien, Gumpendorferstrasse 1A/166, 1060 Vienna, Austria.
authors: Jonas
authors: J
authors: Ramoni
authors: Research Division Biochemical Technology, Institute of Chemical Engineering, TU Wien, Gumpendorferstrasse 1A/166, 1060 Vienna, Austria.
authors: Bernhard
authors: B
authors: Seiboth
authors: Institute for Electron Microscopy and Nanoanalysis, Graz University of Technology, Steyrergasse 17, 8010 Graz, Austria.|Graz Centre for Electron Microscopy, Steyrergasse 17, 8010 Graz, Austria.
authors: Harald
authors: H
authors: Plank
authors: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, 8010 Graz, Austria.|Austrian Centre of Industrial Biotechnology, Petersgasse 14, 8010 Graz, Austria.
authors: Bernd
authors: B
authors: Nidetzky
keywords: Amorphogenesis; Atomic force microscopy; Cellulose degradation; Expansin; Glycoprotein; SWO1; Swollenin; Synergism; Trichoderma reesei