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Promotion of efficient Saccharification of crystalline cellulose by Aspergillus fumigatus Swo1 - PubMed

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Promotion of efficient Saccharification of crystalline cellulose by Aspergillus fumigatus Swo1

Xin-ai Chen et al. Appl Environ Microbiol. 2010 Apr.

Abstract

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.

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Figures

FIG. 1.
FIG. 1.

SDS-PAGE analysis of the AfSwo1 produced by A. oryzae. The culture supernatant (16 μl) of each transformant was analyzed by SDS-PAGE. Four transformants (strains TAFSN1 to TAFSN) were used for AfSwo1 production. The arrow points to the bands specifically seen in the TAFSN strains. C, marker-transformed control strain (S-tApE).

FIG. 2.
FIG. 2.

The purified AfSwo1 and its deglycosylation analysis. The AfSwo1 produced by A. oryzae was purified (− lane) and treated with glycopeptidase F at 37°C for 20 h (+ lane).

FIG. 3.
FIG. 3.

Binding assay of AfSwo1 to polysaccharides. AfSwo1 was subjected to a binding assay with a polysaccharide matrix. The bound (+) and unbound (−) fractions were analyzed by SDS-PAGE analysis. AV, Avicel; CH, chitin; XB, xylan from birch wood; XO, xylan from oat spelt.

FIG. 4.
FIG. 4.

Endoglucanase activity of AfSwo1 toward CMC. (A) Halo assay. Three microliters of the eluted fractions including AfSwo1 (see Materials and Methods) were dropped onto the CMC-containing plate. Aliquots of equivalent fractions of the wild-type parent strain (NS-tApE) were also dropped on the plate. The plate was incubated with Congo red solution and washed. Water was used as a control. (B) Optimal temperature and pH. Endoglucanase activity of AfSwo1 was measured under the indicated conditions, and results are represented as relative values (%).

FIG. 5.
FIG. 5.

Disruption activity of AfSwo1 toward crystalline celluloses. (A) Disruption activity of AfSwo1 toward filter paper. Filter paper (10 mg) was incubated with the purified AfSwo1 (0, 800, and 8,000 μg/g of substrate) at 40°C for 48 h by shaking. (B) Disruption activity of AfSwo1 toward Avicel crystalline cellulose. Light microscopy data that show the physical structure of the crystalline cellulose Avicel after incubation with 0 and 800 μg/g of substrate of AfSwo1 at 40°C for 72 h by shaking. (C) Size of the Avicel particles after the AfSwo1 treatment. Avicel was incubated in the presence (+) or absence (−) of AfSwo1 (800 μg/g of substrate) for 72 h at indicated temperatures and pH. Sizes of the Avicel particles in at least four independent experiments were averaged and are represented as arbitrary values with standard deviations.

FIG. 6.
FIG. 6.

Promoting effect of AfSwo1 on saccharification of crystalline cellulose by cellulase mixture. Avicel in acetate buffer was treated with the purified AfSwo1 (0, 100, 1,000, and 2,000 μg/g of substrate) and a cellulase mixture from T. reesei and A. niger at 40°C. The amount of released glucose was determined after the indicated times. Averages and standard deviations of glucose conversion (percent) for three independent experiments are represented.

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