Dataset Information

Hyperthermophilic endoglucanase for in planta lignocellulose conversion.

ABSTRACT: UNLABELLED: BACKGROUND:The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produced by heterologous expression in microbes, or even directly in the plant biomass. RESULTS:Here we show that a cellulase gene (sso1354) isolated from the hyperthermophilic archaeon Sulfolobus solfataricus can be expressed in plants, and that the recombinant enzyme is biologically active and exhibits the same properties as the wild type form. Since the enzyme is inactive under normal plant growth conditions, this potentially allows its expression in plants without negative effects on growth and development, and subsequent heat-inducible activation. Furthermore we demonstrate that the recombinant enzyme acts in high concentrations of ionic liquids and can therefore degrade ?-cellulose or even complex cell wall preparations under those pretreatment conditions. CONCLUSION:The hyperthermophilic endoglucanase SSO1354 with its unique features is an excellent tool for advanced biomass conversion. Here we demonstrate its expression in planta and the possibility for post harvest activation. Moreover the enzyme is suitable for combined pretreatment and hydrolysis applications.

SUBMITTER: Klose H

PROVIDER: S-EPMC3497586 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Hyperthermophilic endoglucanase for in planta lignocellulose conversion.

Klose Holger H Röder Juliane J Girfoglio Michele M Fischer Rainer R Commandeur Ulrich U

Biotechnology for biofuels 20120828 1

<h4>Unlabelled</h4><h4>Background</h4>The enzymatic conversion of lignocellulosic plant biomass into fermentable sugars is a crucial step in the sustainable and environmentally friendly production of biofuels. However, a major drawback of enzymes from mesophilic sources is their suboptimal activity under established pretreatment conditions, e.g. high temperatures, extreme pH values and high salt concentrations. Enzymes from extremophiles are better adapted to these conditions and could be produc ...[more]

PMID: 22928996

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Project description:BACKGROUND: Although many hyperthermophilic endoglucanases have been reported from archaea and bacteria, a complete survey and classification of all sequences in these species from disparate evolutionary groups, and the relationship between their molecular structures and functions are lacking. The completion of several high-quality gene or genome sequencing projects provided us with the unique opportunity to make a complete assessment and thorough comparative analysis of the hyperthermophilic endoglucanases encoded in archaea and bacteria. RESULTS: Structure alignment of the 19 hyperthermophilic endoglucanases from archaea and bacteria which grow above 80°C revealed that Gly30, Pro63, Pro83, Trp115, Glu131, Met133, Trp135, Trp175, Gly227 and Glu229 are conserved amino acid residues. In addition, the average percentage composition of residues cysteine and histidine of 19 endoglucanases is only 0.28 and 0.74 while it is high in thermophilic or mesophilic one. It can be inferred from the nodes that there is a close relationship among the 19 protein from hyperthermophilic bacteria and archaea based on phylogenetic analysis. Among these conserved amino acid residues, as far as Cel12B concerned, two Glu residues might be the catalytic nucleophile and proton donor, Gly30, Pro63, Pro83 and Gly227 residues might be necessary to the thermostability of protein, and Trp115, Met133, Trp135, Trp175 residues is related to the binding of substrate. Site-directed mutagenesis results reveal that Pro63 and Pro83 contribute to the thermostability of Cel12B and Met133 is confirmed to have role in enhancing the binding of substrate. CONCLUSIONS: The conserved acids have been shown great importance to maintain the structure, thermostability, as well as the similarity of the enzymatic properties of those proteins. We have made clear the function of these conserved amino acid residues in Cel12B protein, which is helpful in analyzing other undetailed molecular structure and transforming them with site directed mutagenesis, as well as providing the theoretical basis for degrading cellulose from woody and herbaceous plants.

Project description:Background:Expression of glycosyl hydrolases in lignocellulosic biomass has been proposed as an alternative to improve efficiency of cellulosic ethanol production. In planta production of hyperthermophilic hydrolytic enzymes could prevent the detrimental effects often seen resulting from the expression of recombinant mesophilic enzymes to plant hosts. Utilizing lignocellulosic feedstocks to produce hyperthermophilic hydrolases provides additional benefits for ethanol production in the way of transgenic feedstocks serving as both enzyme providers and cellulosic substrates. Results:In this study, transgenic hybrid poplar (Populus alba?×?grandidentata) was generated to express a hyperthermophilic endoglucanase from Thermotoga neapolitana with an optimal temperature over 100 °C. Functional hyperthermoactive endoglucanase was successfully produced in the transgenic events, and altered phenotypic growth was observed in transgenic lines. Moreover, the line with the highest TnCelB expression in both leaf and developing xylem had reduced lignin content and cellulose crystallinity, resulting in a more digestible cell wall. The activation of TnCelB by a post-harvest heat treatment resulted in enhanced saccharification efficiencies of transgenic poplar lines with moderate TnCelB expression and without alteration of cellulose and lignin when not heat-treated. In planta high-level overexpression of a hyperthermophilic endoglucanase paired with heat treatment following harvest, resulted in biomass that was comparable with wild-type lines that underwent a traditional pretreatment for saccharification. Conclusions:Overexpression of hyperthermophilic endoglucanase in feedstock had impacts on plant growth and cell wall composition, especially when the enzyme was highly expressed. Improved glucan saccharification efficiencies from transgenic lines before and after heat treatment could reduce both the economic and environmental costs associated with ethanol production from lignocellulosic biomass.

Dataset Information

Hyperthermophilic endoglucanase for in planta lignocellulose conversion.

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Hyperthermophilic endoglucanase for in planta lignocellulose conversion.

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