This document discusses the use of enzymes and microbes as catalysts for organic synthesis. It begins by defining enzymes and their properties such as being protein structures that act as catalysts. Some advantages of biocatalysts are their efficiency, selectivity, and ability to function under mild conditions. Challenges include inhibition and limited operating conditions. Techniques discussed for enzyme catalysis include using ionic liquids, enzyme immobilization, and supercritical CO2. Examples are provided of specific enzyme-catalyzed reactions using these techniques. Overall the document outlines the potentials and techniques for applying enzymes and microbes in organic synthesis reactions.

1. ORGANIC SYNTHESIS USING ENZYMES OR MICROBES
PRESENTED BY,
Aayushi kushwaha
Uttam sojitra
Shrilekha sawant
Shweta lad
Sonam sancheti

2. What Are Enzymes?
Most enzymes are Proteins (tertiary
and quaternary structures)
Act as Catalyst to accelerates a
reaction
Cofactors
•An additional non-protein molecule that is
needed by some enzymes to help the
reaction
•Tightly bound cofactors are called
prosthetic groups
•Cofactors that are bound and released
easily are called coenzymes

3. Advantages of Biocatalysts
Generally more efficient (lower concentration of enzyme needed)
More selective (types of selectivity: chemo-selectivity,
regio-selectivity, diastereo-selectivity, and enantio-selectivity)
Enzymes act under mild conditions.
Environment friendly (completely degraded in the environment)
Can be modified to increase selectivity, stability, and activity
More sustainable
Enzymes can catalyse a broad spectrum of reactions.

4. Disadvantages of Biocatalysts
Susceptible to substrate or product inhibition
Limiting operating region (enzymes typically denatured
at high temperature and pH)
Enzymes found in nature in only one enantiomeric form
Phase contact limitation

5. Potentials of Enzymes as Catalysts in Organic
Synthesis
Enzymes have unique complex three-dimensional structures and
the active site integrated therein
This enables a highly specific recognition of specific substrates
High to excellent chemoselectivity, the stereoselectivity as well as
regio-, diastereo- and enantioselectivity.
High conversion and selectivity : offer less E- factor
It is evident that enzyme catalysis requires specific suitable reaction
conditions such as pH, temperature and solvent, which have to be
considered in (bio-)process development.

7. TECHNIQUES USED FOR
ENZYME CATALYSED REACTIONS

8. Ionic liquid Technology
Ionic liquids are combinations of cations and anions that are liquid at
temperatures below 100oC
Negligible vapor pressure, ILs are often said to be “green”
The enhancement in the solubility of substrates or products
without inactivation of the enzymes, high conversion rates and high
activity and stability
For activity and stability of the enzyme within the reaction medium.
Modification in enzyme
Modification in ILs

9. Adsorption, covalent attachment, entrapment in polymeric matrixes and
cross-linking of enzyme molecules.
Modification of enzymes
Lyophilization (freeze dying without loss in activity)
Method for activating and stabilizing enzymes in non-aqueous
media.Lyophilized lipase with poly(ethylene glycol)(PEG) to prepare PEG–
lipase complexes, finding that the activity of lipase in ILs increased more
than 14-fold.(co-lyophilization)
Chemical modification
The chemical modification of enzymes with (PEG) is a well-known method (the
so-called PEGylation) for enzyme stabilization in denaturing environments.PEG
presents both hydrophilic and hydrophobic properties, so the modified
enzymes can increase their solubility in some Ils
Immobilization in a suitable support

10. Modification of the IL
reaction media
1.such as
IL coating,
additives
2.use of
microemul
-sions with
ILs
3.Introduction of
functional groups
(hydroxyl, ether,
and amide)
The advantage of this approach is that the enzyme is protected of the contact
with the solvent by a layer of water and surfactant molecules
4.Water in IL
microemulsions, or
reverse micelles (for
solubilizing
enzymes in
hydrophobic ILs.)

11. 1. Improved activity, selectivity and stability of enzymes.
2. Opens new possibilities for non-aqueous enzymology with high efficiency
3. A large variety of enzymes tolerate ILs or aqueous-IL mixtures
4. Especiality for polar non-volatile materials
5. Not all ionic liquids are suitable for enzymes. (active in ionic liquids
containing BF4, PF6, anions, but not in ionic liquids containing Cl,NO3, CF3SO3,
trifluoroacetate or acetate anions)
Advantages
Disadvantages
1. Development of green and biodegradable ILs
2. Improvements in product isolation are needed,
3. The efficient reuse and purification of ionic liquids will be essential to
maintain their ‘greenness’ for industrial applications.

13. • The synthesis of N-acetyllactosamine. Hydrolysis of the product
decreases the yield, but adding 25% ionic liquid suppressed hydrolysis
and increased yield.
Example

15. Examples
• Catalyst: candida antarctica lipase (CALB)
Enantioselective acylation of 1-phenylethylamine with 4-
pentenoic acid
• In Ionic Liquids, the enantiomeric excess is over 99%.
In a solvent-free system, only 59% ee was achieved.

16. Catalyst : PEG-lipase PS from Pseudomonas cepacia
The alcoholysis between vinyl cinnamate and benzyl alcohol
This reaction proceeds with an eight-fold higher enzyme
activity in 1-octyl-4-methylimidazolium hexafluorophospate
than in an organic solvent system.

17. Catalyst : CRL candida rugosa lipase
The esterification of 2-substituted propanoic acids with
butanol
The enantioselectivity of this reaction is enhanced by using 1-methyl-3-
octylimidazolium hexafluorophosphate
In this solvent, CRL can be recycled five times without appreciable decrease
of activity or enantioselectivity

18. In 1-ethyl-3-methylimidazolium tetrafluoroborate
and ethylpyridinium tetrafluoroborate,
high optical purity and yields are achieved.
The enzymatic resolution of a homophenylalanine ester in
Ionic Liquids
Catalyst : BL-alcalase, a commercially available serine-type
endo-proteinase from Bacillus licheniforms

19. Supercritical CO2
• Supercritical fluids- above its critical temperature and critical pressure
Supercritical carbon dioxide (critical point: 7.38 MPa, 304 K/31.1 °C)
is a nonpolar medium with large quadrupolar moment.
• Enzymes show interesting novel properties such as altered substrate
specificity and enantio-selectivity, suppression of side-reactions,
increased stability.
• It is relatively easy to control these properties as small changes in
pressure and temperature as near critical point the reactivity of
biochemical process can easily altered.

20. • As a solvent, is miscible with both fluorous and organic materials
carbon dioxide is at the maximum oxidation number of carbon
(responsible for inert behaviour).
• In Organic and supercritical media, biotechnological synthetic enzyme
reactions take place where water-insoluble substrates can be
transformed by enzymes in non-aqueous media.
• In anhydrous media they become rigid and their activity diminishes.
Enzyme itself is interested in few drops of water for maximal or full
activity.
• ScCO2 can accelerate the rate of reaction by mass transfer.
• Enzymes of terrestrial organisms need a specific amount of bound
water to be active. The activity of a majority of enzymes is decreased
upon transfer to non-aqueous solvents.

21. Enzymes in scCO2 behave as heterogeneous catalysts where
diffusion of reactants in and out of the enzyme is not rate limiting.
Advantages:
It increases catalytic activities as a result of improved mass transfer,
higher selectivities and strong suppression of side reactions.
ScCO2 as a Lewis acid is prone to react with chemical functional
groups such as amines.
Physical properties of supercritical CO2 by changing the pressure
may induce an alternative active protein state with altered enzyme
activity, specificity and stability.

22. • ScCO2 has the tendency to strip structurally relevant water from the
enzyme leading to deactivation, it is necessary to add a small amount of
water to the substrates, which can reverse the deactivation and restore
the enzyme activity.
Challenges:
• Low dielectric constant of scCO2 in the liquid state (≈1.5) might
require even higher pressures for certain classes of substances to be
efficiently dissolved.

23. The reduction of o-fluoroacetophenone 3 in scCO2 at 10 MPa was
conducted using 2-propanol as a reductant (hydrogen donor), which
afforded (S)-1-(o-fluorophenyl)ethanol (S)-4 in 81% yield (determined by
GC) after 12 h. yield increased with the reaction time, which proved that
the alcohol dehydrogenase catalyzed the reduction even under
supercritical conditions.

24. CO2 was fixed on pyrrole 5 to produce pyrrole-2-carboxylate 6 at 10
MPa and 40 °C. The yield of the carboxylation reaction in supercritical
CO2 was 12 times higher than that under atmospheric pressure.

25. Table 1. Applications of SCF in Downstream biotechnological processes

26. Fig. Solid-gas catalysis in packed bed reactors
In the packed bed, isolated enzymes ,
whole cells can be used.
For example, a solid–gas enzymatic
esterification of natural alcohols and
acids by Novozym 435 has been
transferred successfully to the industrial
scale for the production of fragrances
and aromas.
Solid – gas biocatalytic reactor:
Bio catalytic reactor:

27. Advantages:
Very high conversion yields compatible with very high production
rate for a minimal plant scale.
More efficient mass transfer
Reduced diffusion limitations due to low gas velocity and better
stability of enzymes and cofactors.
Downstream processing is simplified due to absence of solvent
phase
Scale up is simpler due to use of gaseous circulating phase
Solid-gas catalysis can be used for isolated enzymes and for whole
cells too.

28. Fig. Kanno’s beta-galactosidase promoted hydrolysis
Micro-reactor:

29. A solution of alha-galactosidase in pH 8phosphate buffer was combined
with a similarly buffered solution of p-nitrophenyl-alpha-D-galacto-
pyranoside in a 200 μm -200 μm microreactor. Here rate of reaction was
observed which was 5 folds higher than batch reaction.

30. Ultrasound Technology
1
• Sequential formation, growth and collapse of millions of
vapour bubbles of microscopic nature in liquid solution.
2
• increases the interaction between phases by cavitation
caused by the collapse of bubbles
3
• increases the interaction between phases by cavitation
caused by the collapse of bubbles
4
• Improved mixing, shearing, and mass transfer in aqueous
solutionRecently used as an efficient way of mixing lipase-catalyzed
reactions, such as transesterifications or esterificationss or
suspensions.

31. Synthesis of butyl acetate catalyzed by
MCI-CALB
The ultrasound increased the process productivity by six times,
allowing the use of higher (up to four-fold) acid concentrations,
while reducing the reaction time compared to the classical
mechanical stirring.
Immobilization of CALB (lypase B) on the styrene-divinylbenzene
support
temperature, 48.8 °C ; substratemolar ratio, 3.46:1 alcohol:acid ;
amount of biocatalyst: 7.5% and water 0.28%

32. • Microwaves (0.3–300 GHz) lie between infrared and radiofrequency
electromagnetic radiations3–5.
Microwave Technology
• Aqueous Enzymology
Necessary to use thermostable enzymes to work at higher temperatures (like 70–
90°C).
• Non-aqueous Enzymology
 The enzymes in nearly anhydrous media are extremely thermostable
 Lyophilization : making enzyme structure rigid
• Challanges
 The use of microwave reactors with temperature control
 Uncertainty of thermal effects

33. Applications of enzymes in non-aqueous media

34. Sr.
No.
Reaction Enzyme Technique Company
1 Direct
bioethanol
production
Cellulase Cellulose pre-
treated in ionic
liquid
Io-Ti-tec Ionic
Liquid
Technologies Inc.
2 Enzymatic
reactions R &
D
-- Enzymes in SC-
CO2
Natex Process
Technologies Inc.
3 Bio-oxidation
Transaminatio
n
Transesterifica
tion
Acyclase,
Dehydrogenase
, etc.
Microreactor
Technology
Novasep Services
and Technologies
4. Hydrolysis or
synthesis of
structured
lipids
Acylglycerol
lipase
Biocatalytic
Membrane
Reactor
technology
R & D Food
Industry

35. RECENT DEVELOPMENTS

36. Asymmetric aldol reactions catalyzed by the aldo–ketoreductase
enzyme
The promiscuous aldo–ketoreductase (AKR) enzyme is used as a sustainable biocatalyst for the
first time to catalyze asymmetric aldol reactions in aqueous medium.
In the absence of
enzyme at ph 5.5
No reaction took
place
While with enzyme reaction takes place with
higher yield and enantioselectivity
AKR catalyzed the aldol addition of cyclohexanone with aromatic aldehydes to give the desired
products in reasonable yields (up to 65%), enantioselectivities (up to 60% ee), and moderate to
excellent diastereoselectivities (up to 96:4, anti:syn).

37. Enzyme-catalyzed asymmetric Mannich reaction using acylase
from Aspergillus melleus
Conventional mannich
reaction
Yield= up to 83%
Less enantioselective
not diastereoselective
Enzyme catalysed
mannich reaction
Yield= up to 85%
Enantioselectivities= up
to 89%
Diastereoselectivities=u
p to 90:10
Yield = 92%
Enantioselectivity =
75%
When MeCN used as solvent

38. Reduction of C=O to CHOH Using Enzymes and Microorganisms
• Ketoreductases (KREDs) can be used to generate chiral alcohols with good
yields and excellent selectivities (often >99% ee).
• Most KREDs use either NADH or NADPH cofactors and catalyse the
reduction of carbonyl groups or the oxidation of alcohols. The
reaction starts with the binding of the NAD(P)H cofactor to the
enzyme. Next,the ketone substrate is bound to the enzyme.
Substrate binding is followed by hydride transfer from the cofact or
to the ketone to produce an alcohol. The enzyme then releases the
product alcohol.
Whole cell
Isolated enzyme
Low yield and enantioselectivity
Yield >90% and ee>98%

39. Advances in synthesis of biodiesel via enzyme catalysis: Novel
and sustainable approaches
• Lipases can effectively convert triglycerides to FAAE, thus attracting
interest in the biodiesel field.
• Feedstock oil and short chain alcohols acting as acyl acceptors
react in the presence of lipases. Lipases effectively convert
triglycerides as well as FFA to FAAE.

40. Lipase Feed stock (oil) Acyl acceptor Yield (%)
Candida sp. Glycerol Methanol 80.6
Novozyme435 Sunflower Ethyl acetate 92.7
Novozyme435 palm Isobutanol 100
Candida rugosa Soybean Methanol 87
Pseudomonas
cepacia
Soybean Methanol 90
Burkholderia
cepacia
Palm Methanol 100
Candida
antarctica
Cotton seed Methanol 97
Geotrichum sp. Waste cooking oil Methanol 85
Different Lipase with different feed stock and acyl acceptor

41. Conclusion
It is observed that use of enzymes in above mentioned techniques
provides efficient organic synthesis in terms of selectivity, yield and conversion.
At the same time overcomes drawbacks of conventional methods involving
enzymes.

43. Questions and suggestions are welcome.