How recombinant swollenin from Kluyveromyces lactisaffects cellulosicsubstrates and accelerates their hydrolysis

Abstract

BackgroundIn 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.ResultsAfter 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.ConclusionsRecombinant 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.

Cerato-platanin shows expansin-like activity on cellulosic materials

“…Consequently, almost all the expansin-like proteins from fungi and bacteria are nowadays tested in vitro on cellulosic materials, for obvious biotechnologically relevant purposes, i.e. to increase the efficiency of bioconversion of lignocellulose to ethanol (Arantes and Saddler 2010;Jäger et al 2011). These objectives are supported by a number of experimental data concerning the synergism with cellulases (Chen et al 2010;Kim et al 2009;Lee et al 2010;Quiroz-Castañeda et al 2011;Wang et al 2010Wang et al , 2011Zhou et al 2011).…”

The Use of Carbohydrate Binding Modules (CBMs) to Monitor Changes in Fragmentation and Cellulose Fiber Surface Morphology during Cellulase- and Swollenin-induced Deconstruction of Lignocellulosic Substrates

“…As previous work had suggested that Swollenin can weaken hydrogen bonding within amorphous regions of cellulose and hemicellulose (8,9,12), it is possible that Swollenin promotes fiber fragmentation by weakening hydrogen bonds and enhancing slippage between adjacent chains within fiber dislocations. This weakening of hydrogen bonding and chain slippage might be sufficient to cause fragmentation within certain severe dislocations, but not within the less severe dislocations.…”

“…These disruptive proteins include examples from plants (Expansins) (13), bacteria (Expansin-like proteins (14,15) and some carbohydrate binding modules (CBMs) 2 (16)), and fungi * This work was supported by the Natural Sciences and Engineering Council (Swollenin (8,9,12), Loosenin (17), and CBMs (18)). These proteins have also been shown to promote a variety of disruptive effects on cellulosic biomass, including filter paper dispersion, crystallinity reduction, particle size reduction, swelling of cotton fibers, and roughening of cotton fiber surface (7).…”

“…The non-hydrolytic non-oxidative proteins (also known as "disruptive" or "amorphogenesis-inducing" proteins) are of interest (7,9,12) due to their apparent ability to disrupt the structural matrix of biomass, consequently facilitating the subsequent depolymerization of the carbohydrate polymers by hydrolytic and oxidative enzymes (7). These disruptive proteins include examples from plants (Expansins) (13), bacteria (Expansin-like proteins (14,15) and some carbohydrate binding modules (CBMs) 2 (16)), and fungi * This work was supported by the Natural Sciences and Engineering Council (Swollenin (8,9,12), Loosenin (17), and CBMs (18)).…”

“…Swollenin, in particular, has been shown to effectively swell cotton fibers (9), disperse filter paper squares (12), roughen filter paper fibers (12), reduce the particle size of a number of model cellulosic substrates (12), enhance the accessibility of cotton fibers (19), and enhance hydrolysis (8,20). Recent work has shown that Swollenin targets the less-ordered regions of the cellulose, rather than directly disrupting the more crystalline regions of the substrate (8,19), and enhances the solubilization of the hemicellulosic fraction of steam-pretreated corn stover when it synergistically interacted with family 10 and 11 xylanases, promoting a 3-fold increase in xylose released by these hydrolases (8).…”