title: How recombinant swollenin from Kluyveromyces lactis affects cellulosic substrates and accelerates their hydrolysis.
abstract: BACKGROUND
  In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.
  
  
  RESULTS
  After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.
  
  
  CONCLUSIONS
  Recombinant swollenin can be easily produced with the robust yeast K. lactis. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.
authors.affiliation: AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Gernot
authors.initials: G
authors.lastname: Jäger
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Michele
authors.initials: M
authors.lastname: Girfoglio
authors.affiliation: AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Dollo
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Roberto
authors.initials: R
authors.lastname: Rinaldi
authors.affiliation: Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, D-45470 Mülheim an der Ruhr, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Bongard
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Commandeur
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.|Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Forckenbeckstrasse 6, D-52074 Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: AVT-Aachener Verfahrenstechnik, Enzyme Process Technology, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Antje C
authors.initials: AC
authors.lastname: Spiess
authors.affiliation: AVT-Aachener Verfahrenstechnik, Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs