2 Technology transfer - Espace INRS
1
INTRODUCTION The Laccase enzyme: p-diphenol: oxygen oxidoreductase(EC 1.10.3.2) Multicopper enzyme belongs to blue oxidases Active site contains four copper ions (Cu 2+ ) per molecule Broad substrate specificity, High oxidation capacity, Uses oxygen as final electron acceptor. Source: Majority of laccase have been isolated from higher fungi – Ascomycetes, Deuteromycetes and Basidiomycetes; especially white-rot Basidiomycete. Applications : In pulp & paper, textile and cosmetic industries, for detoxification and decolouration of sewage In organic synthesis, for degradation of xenobiotics & bioremediation to create antimicrobial compositions In production of wood-fiber plates, wood-blocks & cardboard without using toxic linkers In detergents production In elaboration of biosensors and cathodes of biofuel cells. Current production technologies & their limitations: Most filamentous fungi produce several isoforms of laccase in high amounts. However, an efficient production system at bioreactor level is still lacking. Our approach: Over-expression of laccase in a suitable host Reducing the cost of laccase production by optimizing the fermentation strategy. Yeast are suitable host for heterologous protein production because of high capacity for growth, easy manipulation of unicellular organism & a eukaryotic organization enables post- translational modification. Pichia is a well described & widely applied production system well known for high cell densities fermentation & hence higher productivity could be expected. RESULTS FURTHER PLANS Experimental work is planned to achieve: complete characterization of recombinant protein. optimization of operation strategies to reduce the overall process time. an integrated approach for simultaneous fermentation & (semi) continuous product recovery. improved stability of recombinant product. minimal down-stream processing cost. Laccase activity was observed on induction with methanol. Maximum activities were observed on day 3 of the process on BMMGy medium (in shake flask) Activity observed in the range of 100 IU/L ( with ABTS as substrate). Laccase activity was fairly stable up to 100 h when stored at 4C Scale up was carried with a bench scale fermenter (5L) in a fed-batch mode Total production of 1200 IU/L was obtained (fig 1). Final biomass concentration was 270 g/L WCW (fig 1). Overall productivity of process was 300 IU/L.D Productivity with recombinant culture was 1.3 folds higher than the native fungus. Temperature stability of laccase: Laccase showed biphasic trend of heat stability (fig 2). Effect of copper on laccase stability at room temperature No significant effect of Cu 2+ was observed on the stability of laccase with respect to control (Fig 3) when added in the supernatant. Process Development Media optimization Parameters optimization Process optimization Scale-up Up-stream Down-stream Process Development Media optimization Parameters optimization Process optimization Scale-up Technology transfer Strain development Up-stream Down-stream Approach Half Life of Laccase enzyme at different temperatures,Phase-1 y = -0.2478x R 2 = 1 y = -0.0136x R 2 = 1 y = -0.0997x R 2 = 1 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 0 5 10 15 20 Time of Incubation (min) Ln (Et/Eo) 70°C 50°C 60°C Half Lifeof Laccase enzyme at different te mpe rature s ,Phas e -2 y = -0.014x + 0.0556 R 2 = 0.9635 y = -0.0111x - 1.3525 R 2 = 0.9382 y = -0.0421x - 3.2266 R 2 = 0.9679 -7 -6 -5 -4 -3 -2 -1 0 0 10 20 30 40 50 60 70 Time of incubation (min.) Ln (Et/Eo) 70°C 50°C 60°C Fig 2 : Effect of different temperatures on Laccase stability (above); biphasic trend of temperature stability (below) -0.2 0 0.2 0.4 0.6 0.8 1 0 10 20 30 40 50 60 70 Time of incubation (min.) Residual Activity (IU/ml) At 50C At 60 C At 70 C Fig 3:Effect of Cu 2+ on the stability of the protein Fig 1:Process Chart 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 0 5 10 15 20 25 30 35 40 45 50 Incubation Time (h) Activity(IU/ml) Activity(control) Activity(0.25mM) Activity(0.5 mM) Activity(0.1 mM) 0 100 200 300 400 500 600 700 800 900 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 Culture Age (h) Total Biomass (g) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 Activity (IU/ml) Total Biomass Laccase Activity Protease Activity M e O H F e e d i n g HIGH-CELL DENSITY FERMENTATION FOR PRODUCTION OF LACCASE ENZYME USING METHYLOTROPIC YEAST – PICHIA PASTORIS Lalit Kumar 1,2 , Ashwani Gautam 1 , Rishabh Gangwar 1 , Saroj Mishra 1 ,Vikram Sahai 1 , R.D. Tyagi 2 1 Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India 2 Institut national de la recherche scientifique, Université du Québec, Québec, Canada G1K 9A9 Corresponding author- [email protected] Master Plate Sub-culture Plate Pre-Culture- I Pre-Culture- II Fermentor Fig 4.Schematic diagram of fermentation process with P. pastoris Complete cDNA encoding laccase from Cyathus buleri was cloned, sequenced & expressed in Pichia pastoris under the influence of AOX1 promoter. Laccase secreted into the medium under the control of α-factor secretion signal of Saccharomyces cerevisiae Cultivation of the developed Pichia strain has been done at shake flask level on different media - YPD and BMMGy. WORK DONE SO FAR
Transcript of 2 Technology transfer - Espace INRS
INTRODUCTION The Laccase enzyme: p-diphenol: oxygen oxidoreductase(EC 1.10.3.2)
Multicopper enzyme belongs to blue oxidases
Active site contains four copper ions (Cu2+) per molecule
Broad substrate specificity, High oxidation capacity, Uses
oxygen as final electron acceptor.
Source: Majority of laccase have been isolated from higher fungi –
Ascomycetes, Deuteromycetes and Basidiomycetes; especially white-rot
Basidiomycete.
Applications :
In pulp & paper, textile and cosmetic industries, for
detoxification and decolouration of sewage
In organic synthesis, for degradation of xenobiotics &
bioremediation to create antimicrobial compositions
In production of wood-fiber plates, wood-blocks &
cardboard without using toxic linkers
In detergents production
In elaboration of biosensors and cathodes of biofuel cells.
Current production technologies & their limitations:
Most filamentous fungi produce several isoforms of laccase in
high amounts.
However, an efficient production system at bioreactor level
is still lacking.
Our approach:
Over-expression of laccase in a suitable host
Reducing the cost of laccase production by optimizing the
fermentation strategy.
Yeast are suitable host for heterologous protein production
because of high capacity for growth, easy manipulation of
unicellular organism & a eukaryotic organization enables post-
translational modification.
Pichia is a well described & widely applied production system
well known for high cell densities fermentation & hence higher
productivity could be expected.
RESULTS
FURTHER PLANSExperimental work is planned to achieve:
complete characterization of recombinant protein.
optimization of operation strategies to reduce the overall process
time.
an integrated approach for simultaneous fermentation & (semi)
continuous product recovery.
improved stability of recombinant product.
minimal down-stream processing cost.
Laccase activity was observed on induction with methanol.
Maximum activities were observed on day 3 of the process on
BMMGy medium (in shake flask)
Activity observed in the range of 100 IU/L ( with ABTS as
substrate).
Laccase activity was fairly stable up to 100 h when stored at
4 C
Scale up was carried with a bench scale fermenter (5L) in a
fed-batch mode
Total production of 1200 IU/L was obtained (fig 1).
Final biomass concentration was 270 g/L WCW (fig 1).
Overall productivity of process was 300 IU/L.D
Productivity with recombinant culture was 1.3 folds
higher than the native fungus.
Temperature stability of laccase:
Laccase showed biphasic trend of heat stability (fig 2).
Effect of copper on laccase stability at room temperature
No significant effect of Cu2+ was observed on the stability of
laccase with respect to control (Fig 3) when added in the
supernatant.
Process Development
Media optimization
Parameters optimization
Process optimization
Scale-up
Technology transfer
Strain development
Up-stream Down-stream
Process Development
Media optimization
Parameters optimization
Process optimization
Scale-up
Technology transfer
Strain development
Up-stream Down-stream
Approach
Half Life of Laccase enzyme at different
temperatures,Phase-1
y = -0.2478x
R2 = 1
y = -0.0136x
R2 = 1
y = -0.0997x
R2 = 1
-4
-3.5
-3
-2.5
-2
-1.5
-1
-0.5
0
0 5 10 15 20
Time of Incubation (min)
Ln
(E
t/E
o)
70°C
50°C
60°C
Half Lifeof Laccase enzyme at different
temperatures,Phase-2
y = -0.014x + 0.0556
R2 = 0.9635
y = -0.0111x - 1.3525
R2 = 0.9382
y = -0.0421x - 3.2266
R2 = 0.9679-7
-6
-5
-4
-3
-2
-1
0
0 10 20 30 40 50 60 70
Time of incubation (min.)
Ln
(E
t/E
o)
70°C
50°C
60°C
Fig 2 : Effect of
different temperatures
on Laccase stability
(above); biphasic trend
of temperature stability
(below)
-0.2
0
0.2
0.4
0.6
0.8
1
0 10 20 30 40 50 60 70
Time of incubation (min.)
Resid
ual
Activ
ity (
IU
/ml)
At 50C At 60 C At 70 C
Fig 3:Effect of Cu 2+ on
the stability of the
protein
Fig 1:Process Chart
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0 5 10 15 20 25 30 35 40 45 50
Incubation Time (h)
Activ
ity(IU
/ml)
Activity(control) Activity(0.25mM) Activity(0.5 mM) Activity(0.1 mM)
0
100
200
300
400
500
600
700
800
900
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102
Culture Age (h)
Total B
iom
ass (
g)
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Ac
tiv
ity
(IU
/ml)
Total Biomass Laccase Activity Protease Activity
M
e
O
H
F
e
e
d
i
n
g
HIGH-CELL DENSITY FERMENTATION FOR PRODUCTION OF LACCASE ENZYME USING
METHYLOTROPIC YEAST – PICHIA PASTORIS
Lalit Kumar1,2
, Ashwani Gautam1, Rishabh Gangwar
1, Saroj Mishra
1,Vikram Sahai
1, R.D. Tyagi
2
1Indian Institute of Technology Delhi, Hauz Khas, New Delhi-110016, India
2Institut national de la recherche scientifique, Université du Québec, Québec, Canada G1K 9A9
Corresponding author- [email protected]
Master
PlateSub-culture
Plate Pre-Culture- I Pre-Culture- II Fermentor
Fig 4.Schematic diagram of fermentation process with P. pastoris
Complete cDNA encoding laccase from Cyathus buleri was cloned, sequenced & expressed in Pichia pastoris under the influence of AOX1 promoter.
Laccase secreted into the medium under the control of α-factor secretion signal of Saccharomyces cerevisiae
Cultivation of the developed Pichia strain has been done at shake flask level on different media - YPD and BMMGy.
WORK DONE SO FAR
