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In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane.
- Source :
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Plant molecular biology [Plant Mol Biol] 2017 Mar; Vol. 93 (4-5), pp. 465-478. Date of Electronic Publication: 2016 Dec 22. - Publication Year :
- 2017
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Abstract
- Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105 °C and only residual catalytic activity at temperatures below 70 °C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35 °C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity and stability of the in planta produced Xyl10B with xylobiose as a prominent degradation product. These findings will contribute to advancing consolidated processing of lignocellulosic sugarcane biomass.
- Subjects :
- Biocatalysis
Blotting, Southern
Blotting, Western
Cellulose metabolism
Disaccharides metabolism
Endo-1,4-beta Xylanases metabolism
Enzyme Stability
Enzyme-Linked Immunosorbent Assay
Gene Expression Regulation, Enzymologic
Reverse Transcriptase Polymerase Chain Reaction
Substrate Specificity
Xylans metabolism
Endo-1,4-beta Xylanases genetics
Hot Temperature
Plants, Genetically Modified genetics
Saccharum genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1573-5028
- Volume :
- 93
- Issue :
- 4-5
- Database :
- MEDLINE
- Journal :
- Plant molecular biology
- Publication Type :
- Academic Journal
- Accession number :
- 28005227
- Full Text :
- https://doi.org/10.1007/s11103-016-0573-5