The document discusses enzymes being less active in organic solvents than in water. It provides several reasons for this, including differences in diffusion, substrate desolvation, and conformational mobility between organic solvents and water. The document also outlines various methods for using enzymes in organic solvents, including immobilization techniques and modifying the enzyme or solvent. It gives examples of industrial applications of enzymes in organic solvents such as producing intermediates for herbicides and pharmaceuticals.

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1
PRESENTED BY: BALVEER KAUR
M.Sc. II BT {SEM III}
130181118
PRESENTED TO : PARVEEN PAHUJA

2.  Introduction
 why are enzymes less active in organic
solvents than in water?
 Modes of using enzymes in organic solvents
 Fundamentals of non aqueous enzymology
 Properties of enzymes in organic solvents
 Advantages
 Disadvantages
 Applications
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3.  Enzymes used in their natural aqueous media
for production of chemicals & polymers
 Most of such compounds are insoluble in
water, water frequently give unwanted side
reactions & degrades organic reagents
 Such reactions possible only in organic solvents
 Thermodynamic equilibria of mostly these
processes are unfavorable in water
 Technological utility of enzymes enhanced
greatly in organic solvents
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4.  For eg: the proteases α-chymotrypsin & subtilisin
have activities 104-105-times lower in anhydrous
octane than in water; the two enzymes are less
active still in most other organic solvents.
 Reasons:
 Diffusion & accessibility factors
 Structural changes
 Substrate desolvation & transition state energy
 Conformational mobility
 ph situation
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 Natural enzymes with organic solvent-tolerance
are useful for employing in organic solvents.
 To find organic solvent tolerant enzymes,
screening for microorganisms is done.
 First reported organic solvent-tolerant lipolytic
enzyme from an organic solvent-tolerant
bacterium, Pseudomonas aeruginosa.
 Then reported an organic solvent-tolerant
proteolytic enzyme from an organic solvent
tolerant bacterium, P. aeruginosa

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SOLUBILIZED ENZYME
PREPARATIONS
SOLID STATE
PREPARATIONS

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PEGPPyethylene glycol
A mono methoxy- PEG was allowed to react with cyan uric chloride so
that 2 PEG molecules were bound to each cyan uric chloride residue .
Amino groups on enzymes made a nucleophilic attack in the third
activated position . In this way 2 PEG chains linked/ amino group
modified
Polyacrylates
A polymer formed using acrylic acid, methyl methacrylate &
2- ethoxy ethyl methacrylate

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NON COVALENTLY
MODIFIED
COMPLEXES
Enzyme
surfactants
complexes eg:
didodecyl
glucosyly
glutamate and
Aerosol OT
Enzyme polymer
complexes eg:
ethlycellulose, poly
vinyl butyral &
polyethylene glycol
Surfactant coated
Nano granules eg;
Aerosol OT as
surfactant & range
of org. solvents
soluble in
nanogranules are
toluene, acetone &
ethanol

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ENZYMES IN
MICROEMULSIONS
Surfactants &
solvents eg: aerosol
OT , CTAB
chloroform
Spectroscopic
studies eg:
Fluorescence & CD
Detergent less
micro emulsions
Eg:
Water, hexane &
isopropanol

12.  ENZYMES IMMOBILIZED ON SUPPORTS
 Immobilization method
 Mass transfer limitations
 Influence of pore size
 Direct effects of the support on the enzyme
 Effect of additives
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13.  Inorganic supports eg: controlled pore glass
& diatomaceous earth (celite)
 Synthetic polymers eg: polyethene,
polypropene, ion exchange resins, cross-linked
polystyrene etc.
 Polysaccharide supports eg: Agarose gels,
alginate gels, chitin etc.
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14.  Enzyme powders- lyophilization eg: Resolution of
racemic mixtures using hydrolytic enzymes (
Lipase)
 pH control
 Inactivation during lyophilization eg: sorbitol (
lyoprotectants)
 Enzyme crystals : crosslinking with
glutaraldehyde & stability increased towards the
dimethoxyethane.
 Active site quantification eg: lyophilized
chymotrypsin and subtilisin show that about 65%
active site were accessible
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15.  WATER : Amount of water associated with the enzyme –
key determinant of the properties of enzymes.
Effect of water on enzyme activity
 Water content in typical non aqueous enzyme system is
usually as low as o.o1% . Small variation in water content
changes the enzyme activity.
 Amount of water required for catalysis – dependent on
enzyme eg: lipases are highly active when few molecules are
associated
 subtilisin & chymotrypsin - < 50 molecules of water/
enzyme molecule
 making an enzyme more hydrophobic by chemical
modification can reduce the requirement of water for
enzyme
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Effect of water on protein mobility
 Water acts as plasticizer to increase the
flexibility – polarizability increases – mobility
also increases.
 Active site mobility increases upon addition of
water eg: For subtilisin the increase in active
site flexibility – increases active site polarity

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 SOLVENT : solvent not only directly or
indirectly affects the enzyme activity &
stability but also changes the specificity.
Effect of solvent on enzyme active centers:
 Solvent can affect the activity by disrupting the
total number of active sites.
 Active site conc. of chymotrypsin in water not
affected by addition of 3 dipolar solvents: 32%
dioxan,14% acetone & 13% acetonitrile but only
2/3 of this is catalytically active in dry octane .
 Eg: active site of chymotrypsin in organic
media is disrupted around 42%

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Effect of solvent on substrates & products
 Solvents can also effect the conc. of substrates
& products in aqueous layer around the
enzyme & then affect the enzyme activity.

19.  Substrate specificity .
 Enantio selectivity
 Chemo selectivity
 Regioselectivity
 Rigidity
 Enhanced substrate stability
 Ligand induced enzyme memory
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20.  Binding energy of an enzyme with substrate
determined by the difference btw energy of ES
complex & energy of enzyme & substrate in solution,
binding is always influenced by solvent eg: substrate
specificity of α-chymotrypsin, esterase, & subtilisin
changed upon replacement of reaction medium with
an organic solvent.
 The reversal of specificity in solvents was due to lack of
hydrophobic interaction in non aqueous media.
 In fact, the substrate specificity of α-chymotrypsin in
octane was reversed compared to that in water.
 Similar results with PEG modified chymotrypsin
,trypsin & subtilisin in benzene.
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21.  Enzymatic enantio- and prochiral selectivities can
be greatly influenced, and sometimes reversed in
organic solvents
Example : The enantioselectivity of α- chymotrypsin
in the transesterification of methyl 3-hydroxy-2-
phenylpropionate with propanol has been studied.
The enzyme strongly prefers the S-enantiomer of the
substrate in some solvents, the R-enantiomer is more
reactive in others.
 Few methods exist that affect the enantio
selectivity of enzymatic reactions : site directed
mutagenesis, use of enantio selective inhibitors,
coenzyme analogs, temperature & water miscible
co solvents.
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22.  Enantio selectivity of the enzyme was lower in
the solvents with higher hydrophobicity.
 Eg: Enantio selectivity of subtilisin, elastase,
trypsin & α-chymotrypsin were lower in
organic solvents different from that in water.
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23.  Ability to discriminate btw chemically distinct
functional groups.
 Eg. Aspergillus niger lipase catalyzed acylation of 6-
amino – 1- hexanol proceeded with preference for
hydroxyl group .
 This unexpected selectivity allowed the authors to
produce monoesters of amino alcohols in good yield.
 Chemo selectivity of the enzyme affected by the
reaction medium eg: the chemo selectivity of
pseudomonas sp. Lipase in the acylation of N-α-
benzoyl-L-lysinol with trifluoroethyl butyrate varied
from 1.1 in tertbutyl alcohol to 21 in 1,2-dichloroethane
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24.  Few studies of effect of media on regioselectivity of
enzymes.
 Rudio et.al reported that the reaction rates of P. cepacia
lipase catalyzed transesterification of 9 with butanol in
organic solvents differed significantly.
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25.  Organic solvents lack water’s ability to engage
in multiple hydrogen bonds,& have lower
dielectric constants, leading to stronger
intraprotein electrostatic interactions leading to
rigidity.
 Addition of small quantities of water or
glycerol or ethylene glycol helps increase
flexibility.
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26.  Reason for enhanced thermo stability-
Rigidity of molecules.
Covalent processes such as deamination, peptide
hydrolysis & cysteine decomposition require
water.
 Ex:- porcine pancreatic lipase, lysozyme,
chymotrypsin, mitochondrial cytochrome
oxidase & ATPase.
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27.  Subtilisin lyophilized from aqueous solution
containing various competitive inhibitors was
100 times more active in anhydrous solvents
than the enzyme lyophilized in the absence of
ligands
 Ligand-induced enzyme memory disappears
when the enzyme is re-dissolved in water
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 One of the imp property 'molecular memory'
effect that leads to high conformational
rigidity in organic solvents
 For example, lyophilized -chymotrypsin first
dissolved in water and then diluted 100-fold
with t-amyl alcohol has a specific activity of
greater magnitude that of the same
lyophilized enzyme directly suspended in that
solvent containing the same 1% of water. As
extra water is added to this suspension,
presumably erasing the memory

30.  When substrates have greater solubility in organic
solvents
 Reduced risk of microbial growth
 Enhanced thermo-stability
 Relative ease of product recovery from organic
solvents
 More energy efficient downstream processing when
volatile solvents are used
 Ability to carry out new reactions impossible in water
because of kinetic or thermodynamic restrictions
 Insolubility of enzymes in organic media
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31.  Inactivation of enzymes.
 Labour & cost-intensive preparation of
biocatalysts in covalently modified
systems.
 Mass-transfer limitations in case of
heterogeneous systems or viscous
solvents.
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32.  PRODUCTION OF INTERMEDIATES
OF HERBICIDES &
PHARMACEUTICALS.
 PRODUCTION OF ESTER FUELS.
 PRODUCTION OF POLYPHENOLS.
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33.  Enantiopure 2-chloro- and 2-bromo-propionic
acids , used as intermediates for the synthesis
of phenoxypropionic herbicides and of some
pharmaceuticals have been obtained from yeast
lipase catalysed enantioselective butanolysis in
anhydrous solvents.
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34.  Production from coal-derived alcohols and
fatty acids
 Phenolic tars from coal gasification wastes
were converted to alcohol by treating with
ethylene oxide and the intermediate alcohols
were esterified with the fatty acids in a
nonaqueous lipase system. Phenoxyethyl esters
thus formed could be substituted for diesel
fuels
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35.  Deals with peroxidase- catalyzed
polymerization of phenols.
 Polyphenols thus formed are used as
conventional phenol- formaldehyde resins as
adhesives
 Also as laminates and photographic developers
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36.  Ionic liquids can be defined as salts that do not
crystallize at room temperature
 Ionic liquids are possible “green” replacements
for organic solvents because have no vapour
pressure and, therefore, may be easier to
efficiently reuse than organic solvents
 Ionic liquids are widely investigated for
applications in organo-metallic catalysis
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37.  Enzyme activities in ionic liquids are generally
comparable or sometimes higher than those
observed in organic solvents
 In ionic liquids enhanced thermal and operational
stabilities and regio- or enantioselectivities have
been observed
 Ionic liquids permit to carry out enzyme-catalyzed
reactions in non-aqueous media on polar
substrates such as peptides, sugars, nucleotides,
and biochemical intermediates
 A serious drawback of ionic liquids is represented
by the fact that product isolation is more complex,
especially for non-volatile materials
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38.  Gupta, M. N. (1992) Enzyme function in organic
solvents.
 A.M Klibanov, A. M. (1988) Enzymatic Catalysis in
Non-aqueous Solvent.
 NET sources:
 http://biowiki.ucdavis.edu/Biochemistry/Catalysis/E
NZYME_CATALYSIS_IN_ORGANIC_SOLVENTS
 users.unimi.it/ScDotChi/documents/lezioni/riva_ser
gio/Riva%20_Organic%20solvents%20_%207_%20fund
amentals.pdf
 syncozymes.com/chinese/bioresource/Enzyme
Immobilization-Papers/trends biotechnol,1997,15,97-
101.pdf
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