The document summarizes a study on efficiently secreting endoglucanase and beta-glucosidase enzymes extracellularly in E. coli. The researchers constructed a synthetic operon containing genes for OsmY–Endo5A and OsmY–Gluc1C enzymes. They transformed E. coli with plasmids expressing these enzymes individually and as a synthetic operon. Results showed the synthetic operon was more effective at secreting the enzymes extracellularly than a chimeric construct, as measured by enzyme activity assays and SDS-PAGE gels. The study demonstrates the potential for E. coli to efficiently secrete cellulolytic enzymes for the production of bioethanol from lignocellulosic biomass

1. Efficient extracellular secretion of an
endoglucanase and a b-glucosidase in
E. coli
Shefali Gupta, Nidhi Adlakha, Syed Shams
Yazdani
Laura Ortiz
Sergio Ramírez
Medicine students
Molecular Biology

2. INTRODUCTION
Bioethanol is a renewable
alternative promising to fossil fuels
to meet the transportation needs of
energy This led to an extensive search
for an alternative fuel source
Raw materials
Lignocellulosic biomass
exhausted deposits

3. INTRODUCTION
The enzymatic hydrolysis step is
enzymes are responsible
considered crucial impediment to the
for hydrolysis
commercialization of bioethanol from
lignocellulosic biomass
Escherichia coli
• Important microorganism
• Studies due easy manipulation
• Butanol, fatty acids, biodiesel
and alkanes / alkenes

4. 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

5. INTRODUCTION
produce a series of
Paenibacillus sp
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

6. GENERALITIES
Escherichia coli has lately been a favorite microorganism for
researchers 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

7. 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.

8. 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

9. 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….”

10. RELATIONSHIP

11. 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

12. 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 DH5α BLR(DE3)
Es usada ampliamente en Es usada para sobreexpresar
tecnología de ADN recombinante proteínas

13. 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

14. 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

15. MATERIALES Y MÉTODOS
• ENDOGLUCANASA • B-GLUCOSIDASA
• Midiendo la cantidad de la reducción • Midiendo la cantidad de p-nitrofenol
de azúcar liberado durante la liberado durante la incubación con p-
incubación con carboximetilcelulosa nitrofenil-β-D-glucopiranósido como
como sustrato sustrato
• 1U de actividad enzimática : • 1U de actividad : cantidad de enzima
cantidad de enzima que libera 1mol requerida para liberar 1mol de p-
de azúcar reductor por minuto nitrofenol por minuto

16. 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)

17. RESULTADOS
Punto de referencia para
determinar actividad.
20uM
16h.

18. RESULTADOS
Concentraciones y
actividad.
*16h despues ˃ extracelular
*Antes de 16h =
extracelular y citoplasma

19. RESULTADOS
GEL SDS PAGE
Celulas no inducidas e
inducidas con gen:
Endo5A, Gluc1C, Operon
sintetico, Quimera EG3
*Endo5A
**Gluc1C
***Quimera

20. RESULTADOS
ZIMOGRAMA
Celulas no inducidas e
inducidas con gen:
Endo5A, Gluc1C, Operon
sintetico, Quimera EG3
SOLO endogluconasa-
gel4 metilumbelifenil
Celubiosa

21. RESULTADOS
SOLO glucosidasa- gel4
metilumbelifenil
Glucosido
Proteinas a un PM ˃ a lo
esperado oligomerizacion o
efecto de la carga de
migracion

22. DISCUSSION

23. CONCLUSIONS
OsmY (hyperosmotically
OsmY (hyperosmotically
inducible periplasmic protein)
inducible periplasmic protein)
is very important in efficient
is very important in efficient
extracellular secretion of
extracellular secretion of
enzymes endogluconase and β-
enzymes endogluconase and β-
glucosidase
glucosidase
It has to follow doing many
It has to follow doing many
studies about bioethanol
studies about bioethanol
production from E.coli.
production from E.coli.

24. CONCLUSIONS
The synthetic operon is more
The synthetic operon is more
effective than the quimera.
effective than the quimera.
The plasmid is essential to
The plasmid is essential to
obtain good results
obtain good results

25. MAPA SERGIO

26. MAPA LAURA

27. GRACIAS!!!