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 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
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 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
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)
L n ( 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 (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 70Time of incubation (min.)
R e s id u al A c t ivi t y ( I U /m l) 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) A c ti vi ty(I U /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) T ot al B iom as s ( g ) 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 A c ti v it y ( IU /m l)
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
21
Indian 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- lalit20.iitd@gmail.com
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.
