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Efficient extracellular secretion of an endoglucanase and a b-glucosidase in
E. coli
Shefali Gupta, Nidhi Adlakha, Syed Shams Yazdani
Laura OrtizSergio Ramírez
Medicine studentsMolecular Biology
INTRODUCTION
Bioethanol is a renewable alternative promising to fossil fuels to meet the transportation needs of energy
Raw materials
exhausted deposits
This led to an extensive search for an alternative fuel source
Lignocellulosic biomass
INTRODUCTION
The enzymatic hydrolysis step is considered crucial impediment to the commercialization of bioethanol from lignocellulosic biomass
enzymes are responsible for hydrolysis
Escherichia coli
• Important microorganism• Studies due easy manipulation• Butanol, fatty acids, biodiesel and alkanes / alkenes
INTRODUCTION
This microorganism is ideal for the production of recombinant protein but its secretion to extracellular medium is difficult. If E. coli to secrete recombinant proteins would be efficient, such as cellulolytic enzymes which hydrolyze the lignocellulosic biomass to generate monomeric sugars, and then fermented into bioethanol
INTRODUCTION
Paenibacillus sp produce a series of cellulolytic enzymes
β-1 ,4-endoglucanase
β-glucosidase
Through plasmids want to obtain recombinant E.coli expected if these cellulolytic enzymes are synthesized and secrete
BIOETHANOL
GENERALITIES
Escherichia coli has lately been a favorite microorganism forresearchers in the biofuel area because of its easy genetic manipulation
Is considered one of the most appropriate hosts for the production of recombinant proteins. However, its usage is undermined by its inability to secrete proteins into the extracellular medium
ENZYMES
Cellulose degradation is catalyzed by the cellulases
β-1,4-ENDOGLUCANASE is one of this type enzyme activity. This immediately cut the cellulose chains at different sites, possibly at random, producing different sized oligosaccharides. β-GLUCOSIDASE is a cellulose too, that’s why hydrolyzes glycosidic bonds to generate smaller carbohydrates.
TRANSCRIPTION
Is the process responsible for the synthesis of an RNA molecule from the genetic information contained in a template sequence of one strand of DNA. This nucleotide sequence is complementary to the gene from which it was transcribed, so it has the same information contained in the gene itself
OPERON
Is a functional unit that regulates the transcription of mRNA, is controlled by a sequence adjacent to the start site of transcription is known as the operator
“…to reduce production costs, we constructed a synthetic operon containing genes for OsmY–Endo5A and OsmY–Gluc1C….”
OBJECTIVE
Determine the genetic and environmental parameters for the optimal secretion of enzymes β-1,4-endoglucanase (Endo5A) and β-glucosidase (Gluc1C) to the culture medium from recombinant E. coli to see its capacity to be a source of bioethanol
MATERIALES Y MÉTODOS
La E. coli es una bacteria gram negativa, que posee un cromosoma circular, compuesto ͠ 3 millones de pares de bases. Este microorganismo puede crecer rápidamente en un medio que contenga compuestos de carbono como azucares
E. coli DH5αEs usada ampliamente en
tecnología de ADN recombinante
BLR(DE3) Es usada para sobreexpresar
proteínas
MATERIALES Y MÉTODOS
• Plásmido: Son pequeños elementos elementos genéticos extracromosómicos, que pueden transferirse entre bacterias y pueden replicarse dentro de una cell hospedera
pQE30 Endo5A
pQE30 Gluc 1C
pET28a
MATERIALES Y MÉTODOS
EXPRESIÓN Y RECOMBINANTES
E. Coli transformada con varios plásmidos fue usada para la expresión recombinante de OsmY–Endo5A, OsmY–Gluc1C. Las células fueron inducidas a crecimiento en un medio LB y diferentes concentraciones de IPGT
MATERIALES Y MÉTODOS
• ENDOGLUCANASA
• Midiendo la cantidad de la reducción de azúcar liberado durante la incubación con carboximetilcelulosa como sustrato
• 1U de actividad enzimática : cantidad de enzima que libera 1mol de azúcar reductor por minuto
• B-GLUCOSIDASA
• Midiendo la cantidad de p-nitrofenol liberado durante la incubación con p-nitrofenil-β-D-glucopiranósido como sustrato
• 1U de actividad : cantidad de enzima requerida para liberar 1mol de p-nitrofenol por minuto
MATERIALES Y MÉTODOS
Uno de los métodos de electroforesis más comúnmente aplicado para proteínas es el que emplea geles de poliacrilamida (PAGE = polyacrylamide gel electrophoresis) en presencia del detergente aniónico dodecilsulfato sódico (SDS)
RESULTADOS
Concentraciones y actividad.*16h despues ˃ extracelular*Antes de 16h = extracelular y citoplasma
RESULTADOS
GEL SDS PAGE
Celulas no inducidas e inducidas con gen:Endo5A, Gluc1C, Operon sintetico, Quimera EG3
*Endo5A**Gluc1C***Quimera
RESULTADOS
ZIMOGRAMA
Celulas no inducidas e inducidas con gen:Endo5A, Gluc1C, Operon sintetico, Quimera EG3
SOLO endogluconasa- gel4 metilumbelifenil Celubiosa
RESULTADOS
SOLO glucosidasa- gel4 metilumbelifenil Glucosido
Proteinas a un PM ˃ a lo esperado oligomerizacion o efecto de la carga de migracion
CONCLUSIONS
OsmY (hyperosmotically inducible periplasmic protein) is very important in efficient
extracellular secretion of enzymes endogluconase and β-
glucosidase
It has to follow doing many studies about bioethanol production from E.coli.
CONCLUSIONS
The synthetic operon is more effective than the quimera.
The plasmid is essential to obtain good results
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