Enzyme Catalysis

1. Enzymatic Catalysis in Synthesis
of fine Chemicals
Research supervisor Research student
Prof. G.D.Yadav Shrinivas A. Shete

2. _
• Synthesis of novel support
• Characterization of support
• Immobilization of lipase
• Characterization of biocatalyst
• Synthesis of hexyl acetate
Outline of project
2

3. Classification of catalysis_
Catalysis
Bio- Chemo-
Organo - Inorganic
Organo
metalic
Enzyme
3

4. Biocatalysis can be either homogeneous or
heterogeneous
Homogeneous
Heterogeneous
Chemo-
catalysis
Bio-
catalysis
Organo
Metalic
Organic
compounds
Acids and
bases
Free
enzyme
Inorganic
solids
Organic
resins
Immobilized
enzymes
Whole
cells
_
4

5.  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.
5

6. They are
soluble
catalysts
Usually
very
unstable
They may
be strongly
inhibited by
substrates
and
products
work well on
natural
substrates
and under
physiologica
l conditions
High cost
Limitations of enzyme in addition to
their excellent catalytic properties
_
6

7. 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
7

8. Development of Biocatalyst
• Factors to be considered in design of a biocatalyst.
A
Reuse of Enzyme
B
Immobilization method
C
Enzyme stability
_
Cost effectiveness & Simplicity
D
Development of Biocatalyst_
8

9. Support for enzyme immobilization
Support
Natural Synthetic Inorganic
1. cellulose
2. dextran
3. agar
4. chitin
1. polyacrylate
2. polymethacrylates
3. polyacrylamide
1. silica
2. bentonite
3. glass
_
9

10. Silica support
Porous silica
Microporous
<2nm
Mesoporous
2-50nm
Macroporous
>50nm
MCM-41 HMS SBA-15 MCF
_
10

11. Mesocellular foam [MCF]_
B
High pore volume, up to 2 ml/g
Large surface area, up to 1,000 m2/g
C
A
3D pore system
A
Connected by uniform windows (9-22 nm)
D
Large spherical cells (24-42 nm)
11

12. P123-4g + H2O-65ml + HCl-10ml
Stir at 40 ºC for 2 hours
Static at 40 ºC for 20 hours
Age at 100 ºC for 24 hours
Filter, dry & Calcination at 550 ºC
for 6 hours
TMB
TEOS
NH4F
Synthesis of MCF_

13. Characterization of MCF_
1. FT-IR
2. ASAP
3. SEM
13

14. 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
972.84
804.97
462.88
FT-IR of MCF_
14

15. ASAP of MCF_
15

16. Silica
Surface Area (m2/g) Pore volume (cm3/g) Pore
size
(nm)Single
Point
BET
BJH
Adsorption
BJH
Desorption
Single
point
BJH
Adsorption
BJH
Desorption
MCF 431.74 447.79 471.62 739.11 1.92 1.90 1.93 17.19
ASAP results_
16

17. SEM of MCF_
17

18. Advantages of Immobilization_
Immobilization
18

19. Methods of enzyme immobilization
Methods
Adsorption Entrapment
Micro-
encapsulation
Entrapment Cross linking
Covalent
binding
_
19

20. Total protein estimation
y= 0.026x
R² =0.998
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8 10 12
Absorbance
Microgram of Protein/100ul
BradfordCalibrationCurve(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
absorbance
_
20

21. Olive
oil
Tributy
rin
P-nitro
phenol
Lipase assay_
Various assay methods are used to determine the enzyme activity.

22. 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
22

23. 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
_
23

24. 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
temp
Add. of diluted enzyme
Washed three times
with buffer
Protein content &
enzyme activity was
checked
Kept in shaker for 6
hr at room temp
24

25. Covalent binding results
Immobilization
Method
Dilution
%
Immobilization
Activity
u/g
Covalent
binding
10 27 100
100 31 40
1000 80 15
_
25

26. 300mg MCF + 10ml sod.
phosphate buffer.
equilibration for 1 hour
10ml enzyme solution
Gluteraldehyde crosslinking method - I_
300mg MCF 10ml phosphate buffer
Kept it for 1hr in
incubator shaker
0.1% of Gluteraldehyde
solution
Stirred at 4ºC overnight
Centrifugation &
washing with
buffer 26

27. Immobilization
Method
Dilution
%
Immobilization
Activity
u/g
Gluteraldehyde
( Method I )
10 78 222
100 79 82
1000 100 24
Gluteraldehyde crosslinking method – I results_
27

28. Gluteraldehyde crosslinking method – II_
300mg MCF 10ml sod. Phosphate
buffer
300mg MCF + 10ml
sod. phosphate buffer.
equilibration for 1
hour
10ml enzyme solution
Vortexed for 30 sec
& then sonicated for
10sec
Kept on shaker
for 30min
0.1%
gluteraldehyde sol.
Kept on shaker
for 30min
Centrifugation &
washing with
buffer
28

29. Immobilization
Method
Dilution
%
Immobilization
Activity
u/g
Gluteraldehyd
e
( Method I )
10 97 333
100 99 198
1000 100 75
Gluteraldehyde crosslinking
method – II results
_
29

30. Comparison of results of immobilization methods
0
50
100
150
200
250
300
350
Gluteraldehyde I Gluteraldehyde II Covalant bonding
Immobilization methods
Enzymeactivityu/g
10 100 1000
_
30

31. 31

32. 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.32

33. ……..Experi
mental
Gas
Chromatography
Contd...
water bath
hexanol + vinyl acetate +
biocatalyst in organic
solvent
t0…………….t1………….tn
glass reactor
pitched blade glass
stirrer

34. Analysis

35. Effect of acyl donors
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
%conversionofhexanol
Time (min)
vinyl acetate acetic anhydride acetic acid triacetin
_
Reaction parameter
Speed of
agitation
300RPM
Temp. 50 ºC
Solvent Toluene
Enzyme
loaded MCF
20mg
hexanol:acyl
donar
1:2
35

36. Effect of speed of agitation
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
%Conversionofhexanol
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
36

37. Effect of solvents
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
%ConversionofHexanol
Time (min)
Toluene Benzene 1,4 Dioxane Acetonitrile
_
Reaction parameter
Speed of
agitation
300RPM
Temp. 50 ºC
Enzyme loaded
MCF
20mg
hexanol:vinyl
acetate
1:2
37

38. Effect of catalyst loading
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
%Conversionofhexanol
Time(min)
5mg 10mg 15mg 20mg
_
Speed of
agitation
300RPM
Temp. 50 ºC
Solvent toluene
hexanol:vinyl
acetate
1:2
Reaction parameter
38

39. Effect of temperature
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
%Conversionofhexanol
Time (min)
30ºC 40ºC 50ºC
_
Speed of
agitation
300RPM
Enzyme loaded
MCF
20mg
Solvent toluene
hexanol:vinyl
acetate
1:2
Reaction parameter
39

40. y = 0.0027x
R2 = 0.9976
y = 0.0065x
R2 = 0.9855
y = 0.0098x
R2 = 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_
40

41. -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
_
41

42. Effect of mole ratio
0
10
20
30
40
50
60
70
80
90
100
0 30 60 90 120 150
%Conversionofhexanol
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
42

43. 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
Initialrate(mols/lit.min)
Mole:ratio (hexanol:vinyl acetate)
_
Speed of
agitation
300RPM
Enzyme loaded
MCF
20mg
Solvent toluene
Temp. 50ºC
Reaction parameter
43

44. 0.00E+00
1.00E+04
2.00E+04
3.00E+04
4.00E+04
5.00E+04
6.00E+04
7.00E+04
8.00E+04
0 0.05 0.1 0.15 0.2
1/[initialrate]
1/[vinyl acetate]
5mM 15mM 20mM 10mM
Lineweaver-Burk plot
44

45. 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
Ki
B 0.013
Kinetic parameters
45

46. • 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
46

47. 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
47

48. • 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
Acknowledgm
ent
48

49. Thank You !