Characterization of an Endoglucanase Belonging to a New Subfamily of Glycoside Hydrolase Family 45 of the Basidiomycete<i>Phanerochaete chrysosporium</i>

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 celluloseaffinity 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.Cellulose is the primary polysaccharide of plant cell wall and the most abundant renewable biomass resource. Biological degradation of cellulose to soluble sugars has long been considered an alternative to the use of starch feedstocks for bioethanol production. Natural cellulose is an ordered, linear polymer of thousands of D-glucose residues linked by ␤-1,4-glucosidic bonds. Spontaneous crystallization of cellulose molecules due to chemical uniformity of glucose units and the high degree of hydrogen bonding in cellulose can often result in the formation of tightly packed microfibrils (8), which remain inaccessible to cellulolytic enzymes. No single enzyme is able to hydrolyze crystalline cellulose microfibrils completely. Synergistic effects of cellulase mixtures on crystalline cellulose degradation are well known (1,7,21). Nevertheless, cost-competitive technology for overcoming the recalcitrance of cellulosic biomass to enhance enzymatic saccharification is still a major impediment to the utilization of cellulosic materials in bioenergy generation.Expansins are plant cell wall proteins that cause cell wall enlargement by a unique loosening effect in an acid-induced manner (15,20). They are also involved in many physiological processes where cell wall extension occurs, such as pollination, fruit ripening, organ abscission, and seed germination (13,14). It has been proposed that plant expansins disrupt hydrogen bonding between cellulose microfibrils and other cell wall polysaccharides without hydrolytic activity, causing sliding of cellulose fibers or expansion of the cell wall (18,19,27). Swollenin, an expansin-like protein, was isolated and characterized from the cellulolytic filamentous fungus Trichoderma reesei. It has a bimodular structure consisting of a carbohydrate-binding module fami...

Section: Nidulans a Niger A Oryzae A Flavus A Clavatus A Tementioning

confidence: 90%

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Chen

Ishida

Todaka

et al. 2010

“…Shodex KS-801 and KS-802 (Showa Denko K.K., Kanagawa, Japan) columns operated at 80°C with a flow rate of 0.6 ml/min. Hydrolysis products were also viewed on thin-layer chromatography (TLC) by using silica gel 60 TLC sheets (Fisher), following protocols described previously (27). EtOAc-CH 3 COOH-H 2 O (3:2:1 by volume) was used to develop the TLC plates; reducing sugars were detected by the orcinol reagent (1% orcinol in 10% H 2 SO 4 dissolved in ethanol).…”

Section: Methodsmentioning

confidence: 99%

S-Layer Homology Domain Proteins Csac_0678 and Csac_2722 Are Implicated in Plant Polysaccharide Deconstruction by the Extremely Thermophilic Bacterium Caldicellulosiruptor saccharolyticus

Ozdemir

Blumer-Schuette

Kelly

2012

The genus Caldicellulosiruptor contains extremely thermophilic bacteria that grow on plant polysaccharides. The genomes of Caldicellulosiruptor species reveal certain surface layer homology (SLH) domain proteins that have distinguishing features, pointing to a role in lignocellulose deconstruction. Two of these proteins in Caldicellulosiruptor saccharolyticus (Csac_0678 and Csac_2722) were examined from this perspective. In addition to three contiguous SLH domains, the Csac_0678 gene encodes a glycoside hydrolase family 5 (GH5) catalytic domain and a family 28 carbohydrate-binding module (CBM); orthologs to Csac_0678 could be identified in all genome-sequenced Caldicellulosiruptor species. Recombinant Csac_0678 was optimally active at 75°C and pH 5.0, exhibiting both endoglucanase and xylanase activities. SLH domain removal did not impact Csac_0678 GH activity, but deletion of the CBM28 domain eliminated binding to crystalline cellulose and rendered the enzyme inactive on this substrate. Csac_2722 is the largest open reading frame (ORF) in the C. saccharolyticus genome (predicted molecular mass of 286,516 kDa) and contains two putative sugar-binding domains, two Big4 domains (bacterial domains with an immunoglobulin [Ig]-like fold), and a cadherin-like (Cd) domain. Recombinant Csac_2722, lacking the SLH and Cd domains, bound to cellulose and had detectable carboxymethylcellulose (CMC) hydrolytic activity. Antibodies directed against Csac_0678 and Csac_2722 confirmed that these proteins bound to the C. saccharolyticus S-layer. Their cellular localization and functional biochemical properties indicate roles for Csac_0678 and Csac_2722 in recruitment and hydrolysis of complex polysaccharides and the deconstruction of lignocellulosic biomass. Furthermore, these results suggest that related SLH domain proteins in other Caldicellulosiruptor genomes may also be important contributors to plant biomass utilization.

“…The culture supernatant was boiled for 5 min to inactivate enzymes secreted by the fungus. The concentrations of glucose and cellooligosaccharides in the supernatant were measured by high-performance liquid chromatography (HPLC; LC-2000 series; Jasco, Tokyo, Japan), using a corona-charged aerosol detector (ESA Biosciences, Chelmsford, MA) based on our previous report (21). The supernatants were filtered using a MultiScreen HTS 96-well filtration system (Millipore Corporation, Billerica, MA) and then separated on a Shodex Asahipak NH2P-50 4E (Showa Denko K.K., Kanagawa, Japan) with isocratic elution (65% acetonitrile, 35% H 2 O [vol/vol]).…”

Section: Methodsmentioning

confidence: 99%

Cellotriose and Cellotetraose as Inducers of the Genes Encoding Cellobiohydrolases in the Basidiomycete Phanerochaete chrysosporium

Suzuki

Igarashi

Samejima

2010

Self Cite

The wood decay basidiomycete Phanerochaete chrysosporium produces a variety of cellobiohydrolases belonging to glycoside hydrolase (GH) families 6 and 7 in the presence of cellulose. However, no inducer of the production of these enzymes has yet been identified. Here, we quantitatively compared the transcript levels of the genes encoding GH family 6 cellobiohydrolase (cel6A) and GH family 7 cellobiohydrolase isozymes (cel7A to cel7F/G) in cultures containing glucose, cellulose, and cellooligosaccharides by real-time quantitative PCR, in order to evaluate the transcription-inducing effect of soluble sugars. Upregulation of transcript levels in the presence of cellulose compared to glucose was observed for cel7B, cel7C, cel7D, cel7F/G, and cel6A at all time points during cultivation. In particular, the transcription of cel7C and cel7D was strongly induced by cellotriose or cellotetraose. The highest level of cel7C transcripts was observed in the presence of cellotetraose, whereas the highest level of cel7D transcripts was found in the presence of cellotriose, amounting to 2.7 ؋ 10 6 and 1.7 ؋ 10 6 copies per 10 5 actin gene transcripts, respectively. These numbers of cel7C and cel7D transcripts were higher than those in the presence of cellulose. In contrast, cellobiose had a weaker transcription-inducing effect than either cellotriose or cellotetraose for cel7C and had little effect in the case of cel7D. These results indicate that cellotriose and cellotetraose, but not cellobiose, are possible natural cellobiohydrolase gene transcription inducers derived from cellulose.