How recombinant swollenin from Kluyveromyces lactisaffects cellulosicsubstrates and accelerates their hydrolysis

Abstract

Abstract Background In order to generate biofuels, insoluble cellulosic substrates are pretreated andsubsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safeand environmentally friendly manner is to apply, under mild conditions,non-hydrolyzing proteins such as swollenin - naturally produced in low yields bythe fungus Trichoderma reesei. To yield sufficient swollenin forindustrial applications, the first aim of this study is to present a new way ofproducing recombinant swollenin. The main objective is to show how swolleninquantitatively affects relevant physical properties of cellulosic substrates andhow it affects subsequent hydrolysis. Results After expression in the yeast Kluyveromyces lactis, the resultingswollenin was purified. The adsorption parameters of the recombinant swolleninonto cellulose were quantified for the first time and were comparable to those ofindividual cellulases from T. reesei. Four different insoluble cellulosicsubstrates were then pretreated with swollenin. At first, it could bequalitatively shown by macroscopic evaluation and microscopy that swollenin causeddeagglomeration of bigger cellulose agglomerates as well as dispersion ofcellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin oncellulose particle size, maximum cellulase adsorption and cellulose crystallinitywere quantified. The pretreatment with swollenin resulted in a significantdecrease in particle size of the cellulosic substrates as well as in theircrystallinity, thereby substantially increasing maximum cellulase adsorption ontothese substrates. Subsequently, the pretreated cellulosic substrates werehydrolyzed with cellulases. Here, pretreatment of cellulosic substrates withswollenin, even in non-saturating concentrations, significantly accelerated thehydrolysis. By correlating particle size and crystallinity of the cellulosicsubstrates with initial hydrolysis rates, it could be shown that theswollenin-induced reduction in particle size and crystallinity resulted in highcellulose hydrolysis rates. Conclusions Recombinant swollenin can be easily produced with the robust yeast K.lactis. Moreover, swollenin induces deagglomeration of celluloseagglomerates as well as amorphogenesis (decrystallization). For the first time,this study quantifies and elucidates in detail how swollenin affects differentcellulosic substrates and their hydrolysis.