taller4_gajardo
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“Expresión recombinante y modificación de celobiohidrolasa
fúngica, para la producción de etanol desde residuos lignocelulósicos”
Estudiante : Iván Gajardo Hidalgo
Director de Tesis : Dra. Oriana Salazar Aguirre
Co-director de Tesis : Dr. Davor Cotoras Tadic
Universidad de Chile Facultad de Ciencias Químicas Y Farmacéuticas
Escuela de Postgrado
Programa de Magíster en Bioquímica Ambiental
“Recombinant expression of fungal cellobiohydrolase for the production
of ethanol from lignocellulosic wastes”
Student : Iván Gajardo Hidalgo
Supervisor : Dra. Oriana Salazar Aguirre
Co-supervisor : Dr. Davor Cotoras Tadic
Universidad de Chile Facultad de Ciencias Químicas y Farmacéuticas
Escuela de Postgrado
Programa de Magíster en Bioquímica Ambiental
Cellulosic ethanol
Production processes:
-Pretreatment
-Saccharification
-Fermentation
Cellulose
Cellulases
● Endo -1,4-β-D-glucanase● Cellobiohydrolase● β - glucosidase
Cellulases
Henrissat, B. (1991) A classification of glycosyl hydrolases based on amino-acid sequence similarities. Biochem. J. 280:309-316
Glycoside Hydrolases EC 3.2.1.-
Celobiohidrolasa EC 3.2.1.91
Endo-1,4-β D glucanasaEC 3.2.1.4
β-glucosidasa EC 3.2.1.21
Familias: 5 – 6 - 7 - 9 - 48
Cellulases in the Bioethanol Industry
General Purpose
●Produce efficient cellobiohydrolase in the degradation of cellulose, to be
applied to the production of second generation bioethanol.
Specific purposes
●Identify cellobiohydrolase producing fungi.
●Clone cellobiohydrolase gene selected.
●Design and develop a strategy for recombinant expression of fungal cellobiohydrolase.
●Apply methodology of directed evolution to increase the activity or thermostability of cellobiohydrolase produced by recombinant form.
Results● Identify cellobiohydrolase producing fungi.
Results● Clone cellobiohydrolase gene selected.
● Sequence a gene fragment.
Results● Clone cellobiohydrolase gene selected.
● Sequence a gene fragment.
Results● Clone cellobiohydrolase gene selected.
● Glycoside hydrolase family 6.
Results● Clone cellobiohydrolase gene selected.
● Glycoside hydrolase family 7.
Results● Clone cellobiohydrolase gene selected.
● Sequence a gene fragment.
Results● Clone cellobiohydrolase gene selected.
● Sequence a gene fragment.
97Exo7_1 :
106Exo6_1 :
114Exo7_1 :
Results● Clone cellobiohydrolase gene selected.
● Sequence a gene fragment.
Results● Clone cellobiohydrolase gene.
● Complete gene sequence.
Results● Clone cellobiohydrolase gene.
● Complete gene sequence.
PCR with degenerated
primer to the end 5'.
Results● Clone cellobiohydrolase gene selected.
● Complete gene sequence.
“Recombinant expression of fungal cellobiohydrolase for the production
of ethanol from lignocellulosic wastes”
Student : Iván Gajardo Hidalgo
Supervisor : Dra. Oriana Salazar Aguirre
Co-supervisor : Dr. Davor Cotoras Tadic
Universidad de Chile Facultad de Ciencias Químicas y Farmacéuticas
Escuela de Postgrado
Programa de Magíster en Bioquímica Ambiental
“Expresión recombinante y modificación de celobiohidrolasa
fúngica, para la producción de etanol desde residuos lignocelulósicos”
Estudiante : Iván Gajardo Hidalgo
Director de Tesis : Dra. Oriana Salazar Aguirre
Co-director de Tesis : Dr. Davor Cotoras Tadic
Universidad de Chile Facultad de Ciencias Químicas Y Farmacéuticas
Escuela de Postgrado
Programa de Magíster en Bioquímica Ambiental