shrinivas colloquium 18_06_10
DESCRIPTION
Enzyme CatalysisTRANSCRIPT
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Enzymatic Catalysis in Synthesis of fine
Chemicals
Research supervisor Research student Prof. G.D.Yadav Shrinivas A. Shete
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_
• Synthesis of novel support
• Characterization of support
• Immobilization of lipase
• Characterization of biocatalyst
• Synthesis of hexyl acetate
Outline of project
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Classification of catalysis_
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Biocatalysis can be either homogeneous or heterogeneous
Homogeneous Heterogeneo
us
Chemo-
catalysis
Bio-
catalysis
Organo Metalic
Organic compounds
Acids and
bases
Free enzyme
Inorganic solids
Organic resins
Immobilized enzymes
Whole cells
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Stability in organic solvents
Mild reaction conditions
Do not require cofactors
Eco friendly catalyst
Higher reaction rates
Possess broad substrate specificity
Exhibit high enantioselectivity
Lipase (3.1.1.3)_
Lipase can be employed in the production of pharmaceuticals, cosmetics, leather,
detergents, foods, perfumery and other organic synthetic materials.
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They are soluble
catalysts
Usually very
unstable
They may be strongly
inhibited by substrates and
products
work well on natural
substrates and under
physiological conditions
High cost
Limitations of enzyme in addition totheir excellent catalytic properties
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Engineering of enzymes from biological to chemical industry
• Screening of enzymes with suitable properties• Improvement of enzyme properties via
techniques of molecular biology• improvement of enzyme properties via reaction
and reactor engineering
_
• Improvement of enzyme properties via immobilization
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Development of Biocatalyst
• Factors to be considered in design of a biocatalyst.
AReuse of Enzyme
B Immobilization method
C
Enzyme stability
_
Cost effectiveness & SimplicityD
Development of Biocatalyst _
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Support for enzyme immobilization
1. cellulose 2. dextran 3. agar 4. chitin
1. polyacrylate 2. polymethacrylates 3. polyacrylamide
1. silica 2. bentonite 3. glass
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Silica support_
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Mesocellular foam [MCF]_
BHigh pore volume, up to 2 ml/g
Large surface area, up to 1,000 m2/g C
A3D pore system
A
Connected by uniform windows (9-22 nm)
DLarge spherical cells (24-42 nm)
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P123-4g + H2
O-65ml + HCl-10ml
Stir at 40 ºC for 2 hours
Static at 40 ºC for 20 hours
Filter, dry & Calcination at 550 ºC for 6 hours
TMB
TEOS
NH4F
Synthesis of MCF_
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Characterization of MCF_
1. FT-IR
2. ASAP
3. SEM
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4000.0 3000 2000 1500 1000 400.0
-1.5
5
10
15
20
25
30
35
40
45
50
55
61.9
cm-1
%T
3465.76
1652.48
1086.56
972.84804.97
462.88
FT-IR of MCF_
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ASAP of MCF_
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Silica
Surface Area (m2/g) Pore volume (cm3/g) Poresize(nm)Single
PointBET
BJHAdsorption
BJHDesorption
Singlepoint
BJHAdsorption
BJHDesorption
MCF 431.74 447.79 471.62 739.11 1.92 1.90 1.93 17.19
ASAP results_
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SEM of MCF_
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Advantages of Immobilization_
Reusability
Stability
Product Recovery
Continuous use
Economic
Immobilization
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Methods of enzyme immobilization_
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Total protein estimation
y = 0.026xR² = 0.998
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8 10 12
Abso
rban
ce
Microgram of Protein/100ul
Bradford Calibration Curve (Microassay) Bradford Calibration Curve (Macroassay)
y = 0.007x
R2 = 0.9912
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 20 40 60 80 100 120
micro gram protein/100ul
abso
rban
ce
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Olive oil
Tributyrin
P-nitro phenol
Lipase assay_
Various assay methods are used to determine the enzyme activity.
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Tributyrin method_
188µl tributyrin + 1062µl of
0.1M phosphate
buffer + 250µl enzyme
Mix thoroughly on cyclomixer for 1 min
Kept on shaker for 14 min
Released acid titrated with 0.05N NaOH
Reac
tion
stop
ped
by m
etha
nol
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0.2 g of Calcined MCF + 20 ml ethanol
Appropriate amount of APTES
Mixture reflux for 850C for 8 h.
Wash with DI water & ethanol
Filter white solid & Dry at 600C for 24 h
Covalent binding
Functionalization with APTES
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Procedure_
300mg FMCF + 10ml sod.
phosphate buffer. equilibration for 1
hour 10ml of 0.1% gluteraldehyde
soln
Kept in shaker for 1 hr at room
tempAdd. of diluted enzyme
Washed three times with
buffer
Protein content & enzyme
activity was checked
Kept in shaker for 6 hr at room
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Covalent binding results
ImmobilizationMethod Dilution
%Immobilization
Activity u/g
Covalentbinding
10 27 100
100 31 40
1000 80 15
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300mg MCF + 10ml sod. phosphate
buffer. equilibration for 1
hour10ml enzyme solution
Gluteraldehyde crosslinking method - I
_
300mg MCF
10ml phosphate buffer
Kept it for 1hr in incubator
shaker0.1% of
Gluteraldehyde solution
Stirred at 4ºC overnight
Centrifugation & washing with buffer 26
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ImmobilizationMethod Dilution
%Immobilization
Activityu/g
Gluteraldehyde( Method I )
10 78 222
100 79 82
1000 100 24
Gluteraldehyde crosslinking method – I results
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Gluteraldehyde crosslinking method – II
_
300mg MCF
10ml sod. Phosphate
buffer
300mg MCF + 10ml sod. phosphate
buffer. equilibration for
1 hour10ml enzyme solution
Vortexed for 30 sec & then
sonicated for 10sec
Kept on shaker for
30min
0.1% gluteraldehyd
e sol.Kept on
shaker for 30min
Centrifugation &
washing with buffer
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Immobilization
MethodDilution
%Immobilizatio
n
Activity u/g
Gluteraldehyde
( Method I )
10 97 333
100 99 198
1000 100 75
Gluteraldehyde crosslinking method – II results
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Comparison of results of immobilization methods
0
50
100
150
200
250
300
350
Gluteraldehyde I Gluteraldehyde II Covalant bonding
Immobilization methods
En
zym
e ac
tivi
ty u
/g
10 100 1000
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Synthesis of hexyl acetate
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Reaction scheme_
CH3 OH +
CH2
O
O CH3
CH3 O O
CH3
+ CH2
OH
CH3 O
vinyl alcohol
vinyl acetate
hexanol hexyl acetate
acetaldehyde
Lipase
Hexylacetate is a significant green note flavor and widely used in food industry.
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……..Experimental
Gas Chromatography
Contd...
water bathhexanol + vinyl acetate + biocatalyst in organic solvent
t0…………….t1………….tn
glass reactor
pitched blade glass stirrer
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Analysis
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Effect of acyl donors
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
% c
on
vers
ion
of
hex
ano
l
Time (min)
vinyl acetate acetic anhydride acetic acid triacetin
_
Reaction parameterSpeed of
agitation300RPM
Temp. 50 ºC
Solvent Toluene
Enzyme loaded MCF
20mg
hexanol:acyl donar
1:2
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Effect of speed of agitation
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
% C
on
vers
ion
of
hex
ano
l
Time ( min )
200 RPM 300 RPM 400 RPM 500 RPM
_
Temp. 50 ºC
Solvent Toluene
Enzyme loaded MCF
20mg
hexanol:vinyl acetate
1:2
Reaction parameter
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Effect of solvents
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
% C
on
vers
ion
of
Hex
ano
l
Time (min)
Toluene Benzene 1,4 Dioxane Acetonitrile
_
Reaction parameterSpeed of
agitation300RPM
Temp. 50 ºC
Enzyme loaded MCF
20mg
hexanol:vinyl acetate
1:2
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Effect of catalyst loading
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
% C
on
vers
ion
of
hex
ano
l
Time(min)
5mg 10mg 15mg 20mg
_
Speed of agitation
300RPM
Temp. 50 ºC
Solvent toluene
hexanol:vinyl acetate
1:2
Reaction parameter
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Effect of temperature
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
% C
onve
rsio
n of
hex
anol
Time (min)
30ºC 40ºC 50ºC
_
Speed of agitation
300RPM
Enzyme loaded MCF
20mg
Solvent toluene
hexanol:vinyl acetate
1:2
Reaction parameter
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y = 0.0027xR2 = 0.9976
y = 0.0065xR2 = 0.9855
y = 0.0098xR2 = 0.994
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 30 60 90 120 150
ln((2
-Xa)/2
*(1-
Xa))
Time (min)
30ºC 40ºC 50ºC
Second order plot_
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-6
-5.5
-5
-4.5
-4
-3.5
-3
0.00305 0.0031 0.00315 0.0032 0.00325 0.0033 0.00335
ln (k
)
1/T×103 (1/K)
Arrhenius plot
Activation energy
= 52.6KJ/mol
=12.58Kcal/mol
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Effect of mole ratio
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
% C
on
vers
ion
of
hex
ano
l
Time (min)
1:01 1:05 1:02 01:02.5 1:03
_
Speed of agitation
300RPM
Enzyme loaded MCF
20mg
Solvent toluene
Temp. 40ºC
Reaction parameter
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Substrate study
0.00E+00
1.00E-05
2.00E-05
3.00E-05
4.00E-05
5.00E-05
6.00E-05
7.00E-05
8.00E-05
9.00E-05
1.00E-04
Initi
al ra
te (m
ols/l
it. m
in)
Mole:ratio (hexanol:vinyl acetate)
_
Speed of agitation
300RPM
Enzyme loaded MCF
20mg
Solvent toluene
Temp. 50ºC
Reaction parameter
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0.00E+001.00E+042.00E+043.00E+044.00E+045.00E+046.00E+047.00E+048.00E+04
0 0.05 0.1 0.15 0.2
1/[in
itial
rate
]
1/[vinyl acetate]
5mM 15mM 20mM 10mM
Lineweaver-Burk plot
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Parameters Values refined by polymath
Vmax (mol/lit.min)0.00057
KmA (mol/lit) 0.065
KmB (mol/lit) 0.533
KiA (mol/lit) 0.083
KiB 0.013
Kinetic parameters
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Conclusion…
• MCF is the best support for enzyme immobilization
• Gluteraldehyde cross-linking method II (ship-in-a-bottle- approach) is the best method for lipase immobilization
• Selective biocatalyst for hexyl acetate synthesis
• Economic process as compared to other reported methods
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Future plan………
• Functionalization of MCF for effective immobilization
of Enzyme
• Enhancement of thermo stability of enzyme by
immobilization method
• Carry out reactions with packed bed reactor
• Synthesis of chiral MCF
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• Prof. G. D. Yadav• Prof. A. M. Lali• DBT Govt. of India• Novo Nordisk• Dr. Reddy’s lab.• Chem. Engg. Dept. ICT, Mumbai• Lab mates
Acknowledgment
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Thank You !