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

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.

Loosening xyloglucan accelerates the enzymatic degradation of cellulose in wood.

In order to create trees in which cellulose, the most abundant component in biomass, can be enzymatically hydrolyzed highly for the production of bioethanol, we examined the saccharification of xylem from several transgenic poplars, each overexpressing either xyloglucanase, cellulase, xylanase, or galactanase. The level of cellulose degradation achieved by a cellulase preparation was markedly greater in the xylem overexpressing xyloglucanase and much greater in the xylems overexpressing xylanase and cellulase than in the xylem of the wild-type plant. Although a high degree of degradation occurred in all xylems at all loci, the crystalline region of the cellulose microfibrils was highly degraded in the xylem overexpressing xyloglucanase. Since the complex between microfibrils and xyloglucans could be one region that is particularly resistant to cellulose degradation, loosening xyloglucan could facilitate the enzymatic hydrolysis of cellulose in wood.

Degradation of water-insoluble dyes by microperoxidase-11, an effective and stable peroxidative catalyst in hydrophilic organic media.

Microperoxidase-11 (MP-11), a heme-containing undecapeptide, derived from horse heart cytochrome c was utilized as a peroxidative catalyst. Catalytic characteristics of MP-11 in hydrophilic organic media were studied using 2,6-dimethoxyphenol as a reducing substrate in a series of organic solvents at various concentrations, indicating that MP-11 was active in water-miscible organic solvents but at least 5% water was compulsory for the catalytic action. Thus, MP-11 was not active in hydrophobic solvents. The pH of the water portion in the media affected the reaction rate. The optimal pH was found to be 9, where a release of protons from either an oxidizing or reducing substrate to the media was facilitated. The decolorization of water-insoluble synthetic dyes by MP-11 in 90% methanol was attempted. MP-11 showed effective decolorization activities against either azo or anthraquinone dyes. The degradation pathway for Solvent Orange 7 was investigated in detail, showing that MP-11 catalyzed the oxidative cleavage of the azo linkage to generate 1,2-naphthoquinone and 2,4-dimethylphenol as key intermediates.