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Article ; Online: Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Brunecky, Roman / Knott, Brandon C / Subramanian, Venkataramanan / Linger, Jeffrey G / Beckham, Gregg T / Amore, Antonella / Taylor, Larry E / Vander Wall, Todd A / Lunin, Vladimir V / Zheng, Fei / Garrido, Mercedes / Schuster, Logan / Fulk, Emily M / Farmer, Samuel / Himmel, Michael E / Decker, Stephen R

The Journal of biological chemistry

2024 Volume 300, Issue 3, Page(s) 105749

Abstract: Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically ... ...

Abstract	Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.
MeSH term(s)	Aspergillus oryzae/enzymology ; Aspergillus oryzae/genetics ; Cellulose 1,4-beta-Cellobiosidase/chemistry ; Cellulose 1,4-beta-Cellobiosidase/classification ; Cellulose 1,4-beta-Cellobiosidase/genetics ; Cellulose 1,4-beta-Cellobiosidase/metabolism ; Enzyme Assays ; Genome, Fungal/genetics ; Mutation ; Protein Engineering/methods ; Substrate Specificity ; Talaromyces/enzymology ; Talaromyces/genetics ; Trichoderma/enzymology ; Trichoderma/genetics ; Trichoderma/metabolism ; Biocatalysis
Chemical Substances	Cellulose 1,4-beta-Cellobiosidase (EC 3.2.1.91) ; lignocellulose (11132-73-3)
Language	English
Publishing date	2024-02-13
Publishing country	United States
Document type	Comparative Study ; Journal Article
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1016/j.jbc.2024.105749
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Article: Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass

Inoue, Hiroyuki / Decker, Stephen R / Sawayama, Shigeki / Taylor, Larry E / Yano, Shinichi

Biotechnology for biofuels. 2014 Dec., v. 7, no. 1

2014

Abstract: BACKGROUND: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the ... ...

Abstract	BACKGROUND: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. RESULTS: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. CONCLUSIONS: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.
Keywords	Acremonium ; beta-glucosidase ; biomass ; cellulose ; cellulose 1,4-beta-cellobiosidase ; corn stover ; endo-1,4-beta-glucanase ; enzymatic hydrolysis ; fungi ; hydrolysis ; lignocellulose ; sugars ; synergism ; Talaromyces ; xylanases
Language	English
Dates of publication	2014-12
Size	p. 151.
Publishing place	BioMed Central
Document type	Article
ZDB-ID	2421351-2
ISSN	1754-6834
ISSN	1754-6834
DOI	10.1186/s13068-014-0151-5
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Article: Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass.

Inoue, Hiroyuki / Decker, Stephen R / Taylor, Larry E / Yano, Shinichi / Sawayama, Shigeki

Biotechnology for biofuels

2014 Volume 7, Issue 1, Page(s) 151

Abstract: Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the ... ...

Abstract	Background: Enzymatic hydrolysis of pretreated lignocellulosic biomass is an essential process for the production of fermentable sugars for industrial use. A better understanding of fungal cellulase systems will provide clues for maximizing the hydrolysis of target biomass. Talaromyces cellulolyticus is a promising fungus for cellulase production and efficient biomass hydrolysis. Several cellulolytic enzymes purified from T. cellulolyticus were characterized in earlier studies, but the core enzymes critical for hydrolysis of lignocellulosic biomass remain unknown. Results: Six cellulolytic enzymes critical for the hydrolysis of crystalline cellulose were purified from T. cellulolyticus culture supernatant using an enzyme assay based on synergistic hydrolysis of Avicel. The purified enzymes were identified by their substrate specificities and analyses of trypsin-digested peptide fragments and were classified into the following glycosyl hydrolase (GH) families: GH3 (β-glucosidase, Bgl3A), GH5 (endoglucanase, Cel5A), GH6 (cellobiohydrolase II, Cel6A), GH7 (cellobiohydrolase I and endoglucanase, Cel7A and Cel7B, respectively), and GH10 (xylanase, Xyl10A). Hydrolysis of dilute acid-pretreated corn stover (PCS) with mixtures of the purified enzymes showed that Cel5A, Cel7B, and Xyl10A each had synergistic effects with a mixture of Cel6A and Cel7A. Cel5A seemed to be more effective in the synergistic hydrolysis of the PCS than Cel7B. The ratio of Cel5A, Cel6A, Cel7A, and Xyl10A was statistically optimized for the hydrolysis of PCS glucan in the presence of Bgl3A. The resultant mixture achieved higher PCS glucan hydrolysis at lower enzyme loading than a culture filtrate from T. cellulolyticus or a commercial enzyme preparation, demonstrating that the five enzymes play a role as core enzymes in the hydrolysis of PCS glucan. Conclusions: Core cellulolytic enzymes in the T. cellulolyticus cellulase system were identified to Cel5A, Cel6A, Cel7A, Xyl10A, and Bgl3A and characterized. The optimized mixture of these five enzymes was highly effective for the hydrolysis of PCS glucan, providing a foundation for future improvement of the T. cellulolyticus cellulase system.
Language	English
Publishing date	2014-10-09
Publishing country	England
Document type	Journal Article
ZDB-ID	2421351-2
ISSN	1754-6834
ISSN	1754-6834
DOI	10.1186/s13068-014-0151-5
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Article: Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins.

Yarbrough, John M / Mittal, Ashutosh / Katahira, Rui / Mansfield, Elisabeth / Taylor, Larry E / Decker, Stephen R / Himmel, Michael E / Vinzant, Todd

Current biotechnology

2016 Volume 6, Issue 2, Page(s) 128–138

Abstract: Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Colorimetric assays for general glycoside hydrolase activities showed distinct differences in enzyme binding ... ...

Abstract	Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Colorimetric assays for general glycoside hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native cellulase preparations demonstrated low binding of endo- and exocellulases, high binding of xylanase, and moderate binding for β-D-glucosidases. Engineered cellulase formulations exhibited low binding of exocellulases, very strong binding of endocellulases and β-D-glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of β-D-glucosidase activities. Bound and unbound activities were correlated to general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated to binding of β-D-glucosidase activity. Whereas β-D-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated to xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between these three classes of cellulases preparations indicates that it is possible to alter the binding of specific glycoside hydrolase activities during the enzyme formulation process. It remains unclear whether or not loss of endocellulase activity to lignin binding is problematic for biomass conversion.
Language	English
Publishing date	2016-12-23
Publishing country	Netherlands
Document type	Journal Article
ISSN	2211-5501
ISSN	2211-5501
DOI	10.2174/2211550105666160916170630
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Article: A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina.

Linger, Jeffrey G / Taylor, Larry E / Baker, John O / Vander Wall, Todd / Hobdey, Sarah E / Podkaminer, Kara / Himmel, Michael E / Decker, Stephen R

Biotechnology for biofuels

2015 Volume 8, Page(s) 45

Abstract: Background: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is ... ...

Abstract	Background: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. Results: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in 'fast' transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. 'Slow' transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. Conclusions: The elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.
Language	English
Publishing date	2015-03-18
Publishing country	England
Document type	Journal Article
ZDB-ID	2421351-2
ISSN	1754-6834
ISSN	1754-6834
DOI	10.1186/s13068-015-0230-2
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Article ; Online: Heterologous protein expression in Hypocrea jecorina: a historical perspective and new developments.

Singh, Arjun / Taylor, Larry E / Vander Wall, Todd A / Linger, Jeffrey / Himmel, Michael E / Podkaminer, Kara / Adney, William S / Decker, Stephen R

Biotechnology advances

2015 Volume 33, Issue 1, Page(s) 142–154

Abstract: Hypocrea jecorina, the sexual teleomorph of Trichoderma reesei, has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or ... ...

Abstract	Hypocrea jecorina, the sexual teleomorph of Trichoderma reesei, has long been favored as an industrial cellulase producer, first utilizing its native cellulase system and later augmented by the introduction of heterologous enzymatic activities or improved variants of native enzymes. Expression of heterologous proteins in H. jecorina was once considered difficult when the target was an improved variant of a native cellulase. Developments over the past nearly 30 years have produced strains, vectors, and selection mechanisms that have continued to simplify and streamline heterologous protein expression in this fungus. More recent developments in fungal molecular biology have pointed the way toward a fundamental transformation in the ease and efficiency of heterologous protein expression in this important industrial host. Here, 1) we provide a historical perspective on advances in H. jecorina molecular biology, 2) outline host strain engineering, transformation, selection, and expression strategies, 3) detail potential pitfalls when working with this organism, and 4) provide consolidated examples of successful cellulase expression outcomes from our laboratory.
MeSH term(s)	Cellulase/genetics ; Cellulase/metabolism ; Drug Resistance, Fungal/genetics ; Fungal Proteins/genetics ; Fungal Proteins/metabolism ; Genes, Fungal ; Genetic Loci ; Hypocrea/genetics ; Hypocrea/metabolism ; Industrial Microbiology ; Phylogeny ; Promoter Regions, Genetic ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Trichoderma/genetics ; Trichoderma/metabolism
Chemical Substances	Fungal Proteins ; Recombinant Proteins ; Cellulase (EC 3.2.1.4)
Language	English
Publishing date	2015-01
Publishing country	England
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S. ; Review
ZDB-ID	47165-3
ISSN	1873-1899 ; 0734-9750
ISSN (online)	1873-1899
ISSN	0734-9750
DOI	10.1016/j.biotechadv.2014.11.009
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Article ; Online: Analysis of transgenic glycoside hydrolases expressed in plants: T. reesei CBH I and A. cellulolyticus EI.

Brunecky, Roman / Baker, John O / Wei, Hui / Taylor, Larry E / Himmel, Michael E / Decker, Stephen R

Methods in molecular biology (Clifton, N.J.)

2012 Volume 908, Page(s) 197–211

Abstract: Plant cell walls are composed of three basic structural biomolecules: cellulose, hemicellulose, and lignin with cellulose being the most abundant biopolymer on earth. Cellulose is composed of cellodextrins, which are linear polymers of glucose, and ... ...

Abstract	Plant cell walls are composed of three basic structural biomolecules: cellulose, hemicellulose, and lignin with cellulose being the most abundant biopolymer on earth. Cellulose is composed of cellodextrins, which are linear polymers of glucose, and considered to be microcrystalline in structure. The conversion of cellulose to free glucose is one of the primary steps in the fermentative conversion of biomass to fuels and chemicals. However, the crystalline nature of this complex, noncovalent structure is highly resistant to enzymatic hydrolysis. Thus, the substantial cost currently associated with biomass saccharification primarily represents the cost of biomass degrading enzymes. Despite the fact that the microbial cellulose hydrolytic "machinery" for the recycling of carbon from plant biomass already exists in nature, the natural enzymatic degradation of plant material is typically a slow and complex process. Thus, if commercial biofuels production is to become a reality, it must be more cost-effective. One method proposed for achieving this objective is to express all or some of the requisite cellulolytic enzymes in planta, thus reducing both enzyme and thermochemical pretreatment costs.
MeSH term(s)	Actinomycetales/enzymology ; Biofuels ; Biomass ; Biotechnology/methods ; Blotting, Western ; Chemistry Techniques, Analytical/methods ; Fluorescent Dyes/metabolism ; Glycoside Hydrolases/genetics ; Glycoside Hydrolases/metabolism ; Lignin/metabolism ; Nicotiana/chemistry ; Nicotiana/metabolism ; Trichoderma/enzymology ; Zea mays/chemistry ; Zea mays/metabolism
Chemical Substances	Biofuels ; Fluorescent Dyes ; lignocellulose (11132-73-3) ; Lignin (9005-53-2) ; Glycoside Hydrolases (EC 3.2.1.-)
Language	English
Publishing date	2012-07-29
Publishing country	United States
Document type	Journal Article ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-61779-956-3_18
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Article: A constitutive expression system for glycosyl hydrolase family 7 cellobiohydrolases in Hypocrea jecorina

Linger, Jeffrey G / Taylor, Larry E, II / Baker, John O / Vander Wall, Todd / Hobdey, Sarah E / Podkaminer, Kara / Himmel, Michael E / Decker, Stephen R

Biotechnology for biofuels. 2015 Dec., v. 8, no. 1

2015

Abstract: BACKGROUND: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is ... ...

Abstract	BACKGROUND: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. RESULTS: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo- and other cellulase activity and greatly simplifying purification of the recombinant protein. Coupling this system to a Δcbh1 strain of H. jecorina ensures that only the recombinant Cel7A protein is produced. Two distinct transformant colony morphologies were observed and correlated with high and null protein production. Production levels in ‘fast’ transformants are roughly equivalent to those in the native QM6a strain of H. jecorina, typically in the range of 10 to 30 mg/L when grown in continuous stirred-tank fermenters. ‘Slow’ transformants showed no evidence of Cel7A production. Specific activity of the purified recombinant Cel7A protein is equivalent to that of native protein when assayed on pretreated corn stover, as is the thermal stability and glycosylation level. Purified Cel7A produced from growth on glucose demonstrated remarkably consistent specific activity. Purified Cel7A from the same strain grown on lactose demonstrated significantly higher variability in activity. CONCLUSIONS: The elimination of background cellulase induction provides much more consistent measured specific activity compared to a traditional cbh1 promoter system induced with lactose. This expression system provides a powerful tool for the expression and comparison of mutant and/or phylogenetically diverse cellobiohydrolases in the industrially relevant cellulase production host H. jecorina.
Keywords	Trichoderma reesei ; biofuels ; biomass ; biotechnology ; cellulose ; cellulose 1,4-beta-cellobiosidase ; corn stover ; depolymerization ; endo-1,4-beta-glucanase ; fermenters ; fungi ; gene expression ; glucose ; glycosylation ; lactose ; mutants ; phosphopyruvate hydratase ; phylogeny ; protein synthesis ; recombinant proteins ; secretion ; signal peptide ; thermal stability
Language	English
Dates of publication	2015-12
Size	p. 230.
Publishing place	Springer-Verlag
Document type	Article
ZDB-ID	2421351-2
ISSN	1754-6834
ISSN	1754-6834
DOI	10.1186/s13068-015-0230-2
Database	NAL-Catalogue (AGRICOLA)

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Article: High temperature pre-digestion of corn stover biomass for improved product yields.

Brunecky, Roman / Hobdey, Sarah E / Taylor, Larry E / Tao, Ling / Tucker, Melvin P / Himmel, Michael E / Decker, Stephen R

Biotechnology for biofuels

2014 Volume 7, Issue 1, Page(s) 170

Abstract: Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification ... ...

Abstract	Introduction: The efficient conversion of lignocellulosic feedstocks remains a key step in the commercialization of biofuels. One of the barriers to cost-effective conversion of lignocellulosic biomass to sugars remains the enzymatic saccharification process step. Here, we describe a novel hybrid processing approach comprising enzymatic pre-digestion with newly characterized hyperthermophilic enzyme cocktails followed by conventional saccharification with commercial enzyme preparations. Dilute acid pretreated corn stover was subjected to this new procedure to test its efficacy. Thermal tolerant enzymes from Acidothermus cellulolyticus and Caldicellulosiruptor bescii were used to pre-digest pretreated biomass at elevated temperatures prior to saccharification by the commercial cellulase formulation. Results: We report that pre-digestion of biomass with these enzymes at elevated temperatures prior to addition of the commercial cellulase formulation increased conversion rates and yields when compared to commercial cellulase formulation alone under low solids conditions. Conclusion: Our results demonstrating improvements in rates and yields of conversion point the way forward for hybrid biomass conversion schemes utilizing catalytic amounts of hyperthermophilic enzymes.
Language	English
Publishing date	2014-12-03
Publishing country	England
Document type	Journal Article
ZDB-ID	2421351-2
ISSN	1754-6834
ISSN	1754-6834
DOI	10.1186/s13068-014-0170-2
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Article ; Online: Cellulose-inducible xylanase Xyl10A from Acremonium cellulolyticus: Purification, cloning and homologous expression.

Kishishita, Seiichiro / Yoshimi, Miho / Fujii, Tatsuya / Taylor, Larry E / Decker, Stephen R / Ishikawa, Kazuhiko / Inoue, Hiroyuki

Protein expression and purification

2014 Volume 94, Page(s) 40–45

Abstract: Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction ...

Abstract	Cellulose-inducible endo-β-1,4-xylanase (Xyl10A) from the mesophilic fungus Acremonium cellulolyticus was purified, characterized, and expressed by a homologous expression system. A. cellulolyticus CF-2612 produces a high level of xylanase upon induction by Solka-Floc cellulose. To identify this xylanase, the major fraction showing xylanase activity was purified from the CF-2612 culture supernatant, and its gene was identified from the genome sequence. Amino acid sequence homology of Xyl10A revealed that the purified xylanase, designated Xyl10A, exhibited significant homology to family 10 of the glycoside hydrolases (GH10), possessing a cellulose-binding module 1 in the C-terminal region. The xyl10A gene was cloned and expressed in A. cellulolyticus under the control of a glucoamylase promoter. Two recombinant Xyl10As (rXyl10A-I, 53kDa, and rXyl10A-II, 51kDa) were purified that have slightly different molecular weights based on SDS-PAGE. The rXyl10As had the same physicochemical and enzymatic properties as wtXyl10A: high thermostability (Tm 80.5°C), optimum pH 5.0 and specific activity 232-251U/mg for birchwood xylan. The molecular weights of N-deglycosylated rXyl10As were consistent with that of wild-type Xyl10A (wtXyl10A, 51kDa).
MeSH term(s)	Acremonium/enzymology ; Cellulose/chemistry ; Cloning, Molecular ; Endo-1,4-beta Xylanases/biosynthesis ; Endo-1,4-beta Xylanases/chemistry ; Endo-1,4-beta Xylanases/genetics ; Endo-1,4-beta Xylanases/isolation & purification ; Gene Expression Regulation, Fungal ; Glucan 1,4-alpha-Glucosidase/genetics ; Promoter Regions, Genetic ; Sequence Homology, Amino Acid ; Xylans/chemistry ; Xylans/metabolism
Chemical Substances	Xylans ; Cellulose (9004-34-6) ; Glucan 1,4-alpha-Glucosidase (EC 3.2.1.3) ; Endo-1,4-beta Xylanases (EC 3.2.1.8)
Language	English
Publishing date	2014-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1055455-5
ISSN	1096-0279 ; 1046-5928
ISSN (online)	1096-0279
ISSN	1046-5928
DOI	10.1016/j.pep.2013.10.020
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