Enzymatic Catalysis in Synthesis of fine

Chemicals

Research supervisor Research student Prof. G.D.Yadav Shrinivas A. Shete

_

• Synthesis of novel support

• Characterization of support

• Immobilization of lipase

• Characterization of biocatalyst

• Synthesis of hexyl acetate

Outline of project

2

Classification of catalysis_

3

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

_

4

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

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

_

6

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

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 _

8

Support for enzyme immobilization

1. cellulose 2. dextran 3. agar 4. chitin

1. polyacrylate 2. polymethacrylates 3. polyacrylamide

1. silica 2. bentonite 3. glass

_

9

Silica support_

10

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)

11

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_

Characterization of MCF_

1. FT-IR

2. ASAP

3. SEM

13

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_

14

ASAP of MCF_

15

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_

16

SEM of MCF_

17

Advantages of Immobilization_

Reusability

Stability

Product Recovery

Continuous use

Economic

Immobilization

18

Methods of enzyme immobilization_

19

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

_

20

Olive oil

Tributyrin

P-nitro phenol

Lipase assay_

Various assay methods are used to determine the enzyme activity.

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

22

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

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

temp24

Covalent binding results

ImmobilizationMethod Dilution

%Immobilization

Activity u/g

Covalentbinding

10 27 100

100 31 40

1000 80 15

_

25

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

ImmobilizationMethod Dilution

%Immobilization

Activityu/g

Gluteraldehyde( Method I )

10 78 222

100 79 82

1000 100 24

Gluteraldehyde crosslinking method – I results

_

27

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

28

Immobilization

MethodDilution

%Immobilizatio

n

Activity u/g

Gluteraldehyde

( Method I )

10 97 333

100 99 198

1000 100 75

Gluteraldehyde crosslinking method – II results

_

29

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

_

30

Synthesis of hexyl acetate

31

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

……..Experimental

Gas Chromatography

Contd...

water bathhexanol + vinyl acetate + biocatalyst in organic solvent

t0…………….t1………….tn

glass reactor

pitched blade glass stirrer

Analysis

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

35

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

36

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

37

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

38

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

39

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_

40

-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

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

42

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

43

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

44

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

45

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

46

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

• 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

48

Thank You !