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.