Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)

Abstract

In enzymatic saccharification of lignocellulosics, the access of the enzymes to exposed cellulose surfaces is a key initial step in triggering hydrolysis. However, knowledge of the structure-hydrolyzability relationship of the pretreated biomass is still limited. Here we used fluorescent-labeled recombinant carbohydrate-binding modules (CBMs) from Clostridium josui as specific markers for crystalline cellulose (CjCBM3) and non-crystalline cellulose (CjCBM28) to analyze the complex surfaces of wood tissues pretreated with NaOH, NaOH-Na(2)S (kraft pulping), hydrothermolysis, ball-milling, and organosolvolysis. Japanese cedar wood, one of the most recalcitrant softwood species was selected for the analysis. The binding analysis clarified the linear dependency of the exposure of crystalline and non-crystalline cellulose surfaces for enzymatic saccharification yield by the organosolv and kraft delignification processes. Ball-milling for 5-30 min increased saccharification yield up to 77%, but adsorption by the CjCBM-cyan fluorescent proteins (CFPs) was below 5%. Adsorption of CjCBM-CFPs on the hydrothermolysis pulp were less than half of those for organosolvolysis pulp, in coincidence with low saccharification yields. For all the pretreated wood, crystallinity index was not directly correlated with the overall saccharification yield. Fluorescent microscopy revealed that CjCBM3-CFP and CjCBM28-CFP were site-specifically adsorbed on external fibrous structures and ruptured or distorted fiber surfaces. The assay system with CBM-CFPs is a powerful measure to estimate the initiation sites of hydrolysis and saccharification yields from chemically delignified wood pulps.

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

“…In earlier work, Type B CBMs, including members of families CBM4, CBM6, and CBM28, were shown to preferentially bind to fiber dislocations (32,33), agreeing with the preferential adsorption of CBM44 to the fiber dislocations observed here, whereas related work using bacterial Type A CBMs (such as CBM2a) showed that these CBMs do not preferentially bind to these types of dislocations. For example, two Type A family 3 CBMs from the cellulosome complexes of Clostridium josui and C. thermocellum did not preferentially bind to dislocations (28).…”

“…The CBM adsorption technique used in the work described here compares the adsorption of a Type A and a Type B CBM with cellulose where the Type A CBM has been shown to preferentially adsorb to the more crystalline regions of the cellulose surface (29), whereas the Type B CBM preferentially binds to the more amorphous regions (30). Several previous studies have used CBMs with different specificities for crystalline and amorphous regions of the cellulose to try to reveal the differences in surface morphology between the surrounding fiber and the cellulose within fiber dislocations (21,27,(31)(32)(33). However, no clear consensus was reached, with some Type A CBMs binding to dislocations (21,31), whereas other Type A CBMs did not localize to these dislocations (32,33).…”

“…Several previous studies have used CBMs with different specificities for crystalline and amorphous regions of the cellulose to try to reveal the differences in surface morphology between the surrounding fiber and the cellulose within fiber dislocations (21,27,(31)(32)(33). However, no clear consensus was reached, with some Type A CBMs binding to dislocations (21,31), whereas other Type A CBMs did not localize to these dislocations (32,33). However, all of the Type B CBMs tested to date do appear to localize to fiber dislocations (31-33).…”

Recognition of cellooligosaccharides by a family 28 carbohydrate‐binding module

“…In summary, the cellooligosaccharides bound to the two CBMs adopt conformations basically similar to those found in crystals and solution, but they are clearly different from the cellulose structure. This result is consistent with the analysis using fluorescently-labeled CjCBM28 that revealed specific binding to exposed surfaces near the bent or distorted parts of the pulp fibers [9].…”

Nanostructure of cellulose microfibrils in spruce wood

“…However Family 1 cellulose binding domains bind to cellulose in coniferous wood (68) and Arabidopsis secondary cell walls (69) through a planar hydrophobic binding region that is specific for the (200) face when these proteins bind to crystalline celluloses (70) [again we use the Iβ indexing: the same face is indexed as (110) on the Iα lattice]. These findings suggest that cellulose microfibrils in higher plants may have the (200) faces exposed as in the rectangular model, contrary to what has often been assumed.…”