Heterologous Expression and Characterization of an Endoglucanase from a Symbiotic Protist of the Lower Termite, Reticulitermes speratus

Todaka, Nemuri; Lopez, Crisanto M.; Inoue, Tsuneo; Saita, Kanako; Maruyama, Jun‐ichi; Arioka, Manabu; Kitamoto, Katsuhiko; Kudo, Toshiaki; Moriya, Shigeharu

doi:10.1007/s12010-009-8626-8

Cited by 31 publications

(32 citation statements)

References 35 publications

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“…These results indicated that AfSwo1 exhibited endoglucanase activity toward CMC. However, compared with other endoglucanases (26), the specific activity of endoglucanase in AfSwo1 was very low at 2.6 U/mg. Disruption activity of AfSwo1 toward crystalline cellulose.…”

Section: Downloaded Frommentioning

confidence: 73%

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Chen

Ishida

Todaka

et al. 2010

Appl Environ Microbiol

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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...

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Section: Downloaded Frommentioning

confidence: 73%

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Chen

Ishida

Todaka

et al. 2010

Appl Environ Microbiol

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“…With respect to microbial and termite cellulases, recent literature surveys of native and recombinant enzymes identified optimal pH and temperature ranges, respectively, of 3.6-9.0 and 45-1001C (Viikari et al, 2007;Todaka et al, 2010). Results for recombinant R. flavipes Cell-1 and engineered versions of a homologous endoglucanase from R. speratus expressed in E. coli (Ni et al, 2007) were very similar (pH$7, 40-501C) and were intermediate relative to the microbial activity ranges noted above.…”

Section: Stability Of Bev-expressed Cell-1mentioning

confidence: 86%

“…Alternatively, a homologous endoglucanase from C. formosanus showed highest CMC activity at pH 5 and reduced temperature stability above 371C (Zhang et al, 2009). Similarly, a recombinant symbiotic GHF 7 exoglucanase from R. speratus expressed in Aspergillus oryzae showed optimal CMC activity at pH 6.5 and 451C (Todaka et al, 2010). Thus, BEVS-expressed recombinant Cell-1 shows slightly improved temperature stability characteristics at neutral pH relative to a number of other recombinant termite endoglucanases.…”

Section: Stability Of Bev-expressed Cell-1mentioning

confidence: 95%

“…There have been several successful attempts to functionally express recombinant digestive enzymes from termites and their symbionts in both E. coli and Aspergillus oryzae. These include GHF9 endoglucanases from Coptotermes formosanus and C. acinaciformis in E. coli (Tokuda et al, 1999;Nakashima et al, 2002;Watanabe et al, 2002;Inoue et al, 2005;Ni et al, 2005Ni et al, , 2007Zhang et al, 2009); GHF5, GHF9 and GHF45 cellulases from Nasutitermes hindgut bacteria in E. coli (Warnecke et al, 2007); and R. speratus protist symbiont GHF7 exoglucanases in A. oryzae (Sasaguri et al, 2008;Todaka et al, 2010). With the current emphasis on biofuels and improved lignocellulose processing, there is also much interest in the development and characterization of recombinant termite and symbiont lignocellulases for biofuel production purposes (Scharf and Tartar, 2008;Matsui et al, 2009).…”

Section: Introductionmentioning

confidence: 99%

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Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

Zhou

Kovaleva²,

Wu-Scharf

et al. 2010

Arch Insect Biochem Physiol

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Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli-expressed enzyme. K(m) and V(max) values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056 micromol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5-7.5 and 50-60 degrees C, is inhibited by EDTA, and displays enhanced activity up to 70 degrees C in the presence of CaCl(2). These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others.

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“…Enzymes such as glycosyl hydrolases which are highly active in the hydrolysis of crystalline cellulose, have been revealed by metatranscriptomic analyses of symbiotic protozoal communities from four termites species and a wood-eating cockroach (Todaka et al 2009). Enzymatic activities such as amylases and proteases have also been detected in crude extracts of hindgut's protozoa (Kane 1997).…”

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confidence: 99%

Animal digestive strategies versus anaerobic digestion bioprocesses for biogas production from lignocellulosic biomass

Bayané

Guiot

2010

Rev Environ Sci Biotechnol

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Herbivorous mammals and wood-eating insects are fairly effective in the digestion of plant polymers, such as lignocellulosics. In order to improve methane production from the lignocellulosic biomass, several kinds of anaerobic digestion processes derived from animal models have been devised. However, the rates of biodegradation occurring in the anaerobic bioreactors still remain lower than in animal guts. The effectiveness of the digestive systems of those animals results from the concerted action of the various enzymes (e.g. cellulases, xylanases, esterases, ligninases) produced in their guts as well as their integration with mechanical and chemical actions. Powerful pretreatment (prefermentation) operations are integrated to and support efficiently the microbial fermentation system, e.g. the rumination (i.e. mechanical) in ruminants and the secretion of endogenous cellulases (i.e. enzymatic) or the alkaline treatment (chemical) at mid-way in xylophagous insects. The oxygen gradients along the gastrointestinal tract may also stimulate the hydrolytic activities of some microbial populations. In addition, the solid retention time, the digesta flow and the removal of the end-products are well ordered to enable animals to thrive on a complex polymer such as lignocellulose. At the same time, technologies were developed to degrade lignocellulosic biomass, such as the rumen derived anaerobic digestion (RUDAD) process and the rumen simulating technique (RUSITEC), more elaborated and using rumen microbial consortia. Overall, they showed that the fermentation taking place in the rumen fermentation and even in the hindgut are biological processes that go beyond the limited environmental conditions generally found in anaerobic digesters. Hence, knowledge on herbivores' digestion mechanisms might be better exploited in the design and operation of anaerobic digesters. This literature review is a cross-analysis of the relevant information about the digestive strategies of herbivorous and wood-eating animals and the bioengineering techniques in lignocelluloses degradation. The aim is to highlight strategies of animals' digestion simulation for more effective anaerobic digestion of lignocellulosic compounds and other solid residues.The main structural polysaccharides of plant tissues, cellulose and hemicelluloses, are linked with lignin and together called lignocellulose. Lignocellulosic biomass is more and more considered as a potential

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Heterologous Expression and Characterization of an Endoglucanase from a Symbiotic Protist of the Lower Termite, Reticulitermes speratus

Cited by 31 publications

References 35 publications

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Production and characterization of a recombinant beta‐1,4‐endoglucanase (glycohydrolase family 9) from the termite Reticulitermes flavipes

Animal digestive strategies versus anaerobic digestion bioprocesses for biogas production from lignocellulosic biomass

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