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….”

RELATIONSHIP

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

Punto de referencia para determinar actividad.20uM

16h.

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

DISCUSSION

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

MAPA SERGIO

MAPA LAURA

GRACIAS!!!