A Thermostable Xylanase from Clostridium thermocellum Expressed at High Levels in the Apoplast of Transgenic Tobacco Has No Detrimental Effects and Is Easily Purified

Herbers, Karin; Wilke, Ingo; Sonnewald, Uwe

doi:10.1038/nbt0195-63

Cited by 107 publications

(53 citation statements)

References 19 publications

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“…The b-D-xylanase (EC 3.2.1.8) gene from Clostridium thermocellum (Herber et al, 1995; GenBank accession number: M22624) was previously synthesized using the tobacco codons for expression in plants. pET29b (Novagen, Madison, WI) was used for expression of the chimeric genes.…”

Section: Genes and Plasmidsmentioning

confidence: 99%

The construction and characterization of two xylan‐degrading chimeric enzymes

Fan

Wagschal

Lee

et al. 2008

Biotech & Bioengineering

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Degradation of xylan requires several enzymes. Two chimeric enzymes, xyln-ara and xyln-xylo, were constructed by linking the catalytic portion of a xylanase (xyln) to either an arabinofuranosidase (ara) or a xylosidase (xylo) with a flexible peptide linker. The recombinant parental enzymes and chimeras were produced in E. coli at high levels and purified for characterization of their enzymatic and kinetic properties as well as activities on natural substrates. The chimeras closely resemble the parental enzymes or their mixtures with regard to protein properties. They share similar temperature profiles and have similar catalytic efficiencies as the parental enzymes when assayed using substrates 4-nitrophenyl-alpha-L-arabinofuranoside or 2-nitrophenyl- beta-D-xylopyranoside. The chimeras also show unique enzymatic characteristics. In xylanase activity assays using Remazol Brilliant Blue-xylan, while the parental xylanase has a pH optimum of pH 8, the chimeras showed shifted pH optima as a consequence of significantly increased activity at pH 6 (the optimal pH for ara and xylo). Both chimeras exhibited additive effects of the parental enzymes when assayed at wide ranges of pH and temperatures. The xyln-xylo chimera had the same activities as the xyln/xylo mixture in hydrolyzing the natural substrates oat spelt xylan and wheat arabinoxylan. Compared to the xyln/ara mixture, the xyln-ara chimera released the same amounts of xylose from oat spelt xylan and approximately 30% more from wheat arabinoxylan at pH 6. Our results demonstrate the feasibility and advantages of generating bifunctional enzymes for the improvement of xylan bioconversion.

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Section: Genes and Plasmidsmentioning

confidence: 99%

The construction and characterization of two xylan‐degrading chimeric enzymes

Fan

Wagschal

Lee

et al. 2008

Biotech & Bioengineering

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“…Proteins with molecular weights as small as 0.6 kD (Turpen et al, 1995) and as large as 80 kD (Verwoerd et al, 1995) per subunit have been produced (Table I). Although reported accumulation levels are difficult to compare because different plant expression systems Herbers et al, 1995 and extraction procedures were used, production levels above 1% of soluble protein were reported in several instances (Herbers et al, 1995;Hiatt et al, 1989;Hood et al, 1997;Verwoerd et al, 1995). Now that the transformation of major crops is becoming more routine, the choice of a plant for heterologous protein production will be increasingly governed by the ease of production of the plant, proper posttranslational processing, the plant's capabilities for protein accumulation, and the availability of methods for extraction and purification of the recombinant protein (Krebbers et al, 1992;Whitelam et al, 1993).…”

Section: Historical Perspectivementioning

confidence: 99%

“…To our knowledge, there are very few examples where extraction and purification of recombinant proteins from transgenic plants have been studied and quantified (Austin et al, 1994;Fuchs et al, 1993;Herbers et al, 1995;Hood et al, 1997;Kusnadi et al, 1997;Mason et al, 1996;Vandekerckhove et al, 1989;Van Rooijen and Moloney, 1995). Usually, recombinant proteins were extracted together with other seed or tissue proteins to determine accumulation levels, to establish protein functionality, and to confirm the expected amino acid sequence.…”

Section: Downstream Processing Considerationsmentioning

confidence: 99%

Production of recombinant proteins in transgenic plants: Practical considerations

Kusnadi

Nikolov

Howard³

1997

Biotechnol. Bioeng.

319

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“…2) is suffi cient to hydrolyze the cellulose and prevent staining by the cellulose dye Congo Red. Th is approach yielded a 22% conversion of Th e endoxylanase xynZ isolated from the thermophillic bacteria Clostridium thermocellum was expressed in tobacco, 48 and the plant extract demonstrated to hydrolyze purifi ed birchwood xylan to release xylose and xylobiose.…”

Section: Cell Wall Hydrolyzing Enzymesmentioning

confidence: 99%

Regulatory hurdles for transgenic biofuel crops

Lee

Nair

Chen

2009

Biofuels Bioprod Bioref

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Policy-makers have described the many potential benefi ts that biofuels can provide, but there are numerous challenges in realizing this potential. The technical hurdles to producing biofuels economically and on a scale to replace a signifi cant fraction of petroleum-based transportation fuels have been well described, along with the potential environmental concerns. The use of biotechnology can potentially address many of these technical challenges and environmental concerns, but brings signifi cant regulatory obstacles that have not been discussed extensively in the scientifi c community. This review will give an overview of some of the approaches being developed to produce transgenic biofuel feedstocks, particularly cellulosic ethanol, and the regulatory process in the United States that oversees the introduction of new transgenic plants. We hope to illustrate that the level of regulation for transgenic organisms is not proportional to their potential risk to human health or to the environment, and that while revisions to the regulatory system in the USA are currently under consideration, further modifi cations are necessary to refl ect the risk level of transgenic crops and realize their benefi ts.

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A Thermostable Xylanase from Clostridium thermocellum Expressed at High Levels in the Apoplast of Transgenic Tobacco Has No Detrimental Effects and Is Easily Purified

Cited by 107 publications

References 19 publications

The construction and characterization of two xylan‐degrading chimeric enzymes

The construction and characterization of two xylan‐degrading chimeric enzymes

Production of recombinant proteins in transgenic plants: Practical considerations

Regulatory hurdles for transgenic biofuel crops

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