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Expression of Trichoderma reesei (&)#223;-Mannanase in Tobacco Chloroplasts and its Utilization in Lignocellulosic Woody Biomass Hydrolysis

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Date Issued:
2011
Abstract/Description:
Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. ?-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, man1 gene encoding ?-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed the site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds showed inheritance of transgenes into the progeny without Mendelian segregation. Expression of the endo-?-mannanase gene for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-?-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-?-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40 (&)deg;C to 70 (&)deg;C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6-7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the paper, oil, pharmaceutical, coffee and detergent industries.
Title: Expression of Trichoderma reesei (&)#223;-Mannanase in Tobacco Chloroplasts and its Utilization in Lignocellulosic Woody Biomass Hydrolysis.
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Name(s): Agrawal, Pankaj, Author
Daniell, Henry, Committee Chair
Kolattukudy, Pappachan, Committee Member
Self, William, Committee Member
, Committee Member
University of Central Florida, Degree Grantor
Type of Resource: text
Date Issued: 2011
Publisher: University of Central Florida
Language(s): English
Abstract/Description: Lignocellulosic ethanol offers a promising alternative to conventional fossil fuels. One among the major limitations in the lignocellulosic biomass hydrolysis is unavailability of efficient and environmentally biomass degrading technologies. Plant-based production of these enzymes on large scale offers a cost effective solution. Cellulases, hemicellulases including mannanases and other accessory enzymes are required for conversion of lignocellulosic biomass into fermentable sugars. ?-mannanase catalyzes endo-hydrolysis of the mannan backbone, a major constituent of woody biomass. In this study, man1 gene encoding ?-mannanase was isolated from Trichoderma reesei and expressed via the chloroplast genome. PCR and Southern hybridization analysis confirmed the site-specific transgene integration into the tobacco chloroplast genomes and homoplasmy. Transplastomic plants were fertile and set viable seeds. Germination of seeds showed inheritance of transgenes into the progeny without Mendelian segregation. Expression of the endo-?-mannanase gene for the first time in plants facilitated its characterization for use in enhanced lignocellulosic biomass hydrolysis. Gel diffusion assay for endo-?-mannanase showed the zone of clearance confirming functionality of chloroplast-derived mannanase. Endo-?-mannanase expression levels reached up to 25 units per gram of leaf (fresh weight). Chloroplast-derived mannanase had higher temperature stability (40 (&)deg;C to 70 (&)deg;C) and wider pH optima (pH 3.0 to 7.0) than E.coli enzyme extracts. Plant crude extracts showed 6-7 fold higher enzyme activity than E.coli extracts due to the formation of disulfide bonds in chloroplasts, thereby facilitating their direct utilization in enzyme cocktails without any purification. Chloroplast-derived mannanase when added to the enzyme cocktail containing a combination of different enzymes yielded 20% more glucose equivalents from pinewood than the cocktail without mannanase. Our results demonstrate that chloroplast-derived mannanase is an important component of enzymatic cocktail for woody biomass hydrolysis and should provide a cost-effective solution for its diverse applications in the paper, oil, pharmaceutical, coffee and detergent industries.
Identifier: CFE0004467 (IID), ucf:49309 (fedora)
Note(s): 2011-12-01
M.S.
Medicine, Molecular Biology and Microbiology
Masters
This record was generated from author submitted information.
Subject(s): Mannanase
Biofuel
Genetically modified plants
Biomass degrading enzymes
Persistent Link to This Record: http://purl.flvc.org/ucf/fd/CFE0004467
Restrictions on Access: campus 2013-06-15
Host Institution: UCF

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