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Analysis of exposed cellulose surfaces in pretreated wood biomass using carbohydrate‐binding module (CBM)–cyan fluorescent protein (CFP)
Author(s) -
Kawakubo Takeshi,
Karita Shuichi,
Araki Yuko,
Watanabe Shota,
Oyadomari Masafumi,
Takada Rie,
Tanaka Fumio,
Abe Kentaro,
Watanabe Takahito,
Honda Yoichi,
Watanabe Takashi
Publication year - 2009
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.22550
Subject(s) - cellulose , chemistry , hydrolysis , kraft process , organosolv , cellulase , enzymatic hydrolysis , adsorption , pulp (tooth) , lignin , chromatography , kraft paper , nuclear chemistry , biochemistry , organic chemistry , materials science , composite material , medicine , pathology
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 ( Cj CBM3) and non‐crystalline cellulose ( Cj CBM28) 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 Cj CBM–cyan fluorescent proteins (CFPs) was below 5%. Adsorption of Cj CBM–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 Cj CBM3–CFP and Cj CBM28–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. Biotechnol. Bioeng. 2010; 105: 499–508. © 2009 Wiley Periodicals, Inc.