BIOTRANSFORMATIONS

APPLICATIONS OF ENZYMES

MODIFIED AND ARTIFICIAL ENZYMES

 Dream of a chemical biologist has always been to

harness the enzymatic efficiency for the needs of
chemical synthesis and if possible to synthetically
mimic enzymatic action using synthetic catalysts.
Two approaches have been taken.
Artificial enzyme mimics
Modified enzymes

ARTIFICIAL ENZYMES
Also called biomimetics, articial enzymes (synzymes) are
created de novo from a nonproteinogenic articial scaffold,
such a cyclodextrin, dendrimer or polymer, which is endowed
with a rationally designed catalytic site.
The catalytic machinery represents a simplied chemical
model of the chemical operators found in enzymes
Cofactor (cytochrome, pyridoxal or pyridoxamine
phosphate)
Catalytic metal as Lewis acid (Zn)
Catalytic metal as mediator in a redox reaction (Cu, Co,
Mn, Fe)

 Good catalytic activity but very low catalytic efficiency and

poor selectivity
Used to study the multiple subtle contributions which add up
in a synergistic fashion and thereby make enzymes as efcient
as they are

MODIFIED ENZYMES

Modied enzymes are created by leaving their protein scaffold

basically intact but altering some of their properties by
chemical or genetic methods, which yields altered and/or
improved enzymes for synthetic purposes

CHEMICALLY MODIFIED
ENZYMES
Surface modified enzymes

Bioimprinting

Chemical modification of active site

SURFACE MODIFIED
ENZYMES
Enzymes acting in nearly anhydrous organic solvents always
give rise to heterogeneous systems
In order to turn them into homogeneous systems, proteins can
be modied to make them soluble in lipophilic organic
solvents.
Achieved by covalent attachment of the amphipathic polymer
polyethylene glycol (PEG) onto the surface of enzymes

ADVANTAGES AND DISADVANTAGES

They dissolve in organic solvents such as toluene or
chlorinated hydrocarbons, such as chloroform, 1,1,1trichloroethane, or trichloroethylene.
Due to their solubility in various organic solvents,
spectroscopic studies on the conformation of enzymes are
simplied .
Their properties such as stability, activity and specicity may
be altered.

MONOMETHYL PEG

Mol Wt 5 kDa
Linkage of the polymer chains onto the enzymes surface may be achieved
by reactions involving the -amino groups of lysine residues preferably
located on the surface of the enzyme.
Typical linker used is cyanuric chloride .
Nucleophilic substitution followed by alkylation

 The cyanuric chloride/PEG method used for all classes of

enzymes, including hydrolases (lipases, proteases,
glucosidases) and redox enzymes (dehydrogenases, oxidases).
The residual activities are usually high (5080%), and for most
enzymes about ve to ten PEG chains per enzyme molecule are
sufcient to render them soluble in organic solvents.
Extensive modication leading to deactivation has to be
avoided.
PEG-modied enzymes may be recovered from a toluene
solution by precipitation upon the addition of a hydrocarbon
such as petroleum ether or hexane

,-DICARBOXYL-PEG

Allows simple recovery of the enzyme from solution using magnetic

forces.
,-dicarboxyl-PEG is exposed to a mixture of ferric ions and hydrogen
peroxide to form ferromagnetic magnetite particles (Fe 3O4) which are
tightly adsorbed to the carboxylate.
The free end of the magnetic modier is then covalently linked to the
enzyme via succinimide activation.
Used in synthesis of esters, polyesters, amides and peptides

LIPIDS
Various lipids, such as simple long-chain fatty acids or
amphiphilic compounds can be attached onto its surface by
mild adsorption.
About 150 lipid molecules/protein molecule
Lipid-coating used for lipases, phospholipases, glycosidases
and catalase.

BIOIMPRINTING
Used to understand chiral recognition process

CHEMICAL MODIFICATION OF
ACTIVE SITE
To modify physico chemical properties and catalytic activity
of enzymes
Modication of nucleophilic OH- or SH-residues in Ser- or
Cys-hydrolases, such as subtilisin or papain, respectively into
their selenium analogs via chemical activation followed by
nucleophilic substitution by HSe Seleno-subtilisin showed a 700-fold enhanced rate for
aminolysis versus hydrolysis in comparison to native subtilisin

 SeH group may be reversibly oxidized to its seleninic acid

analog (subtilisin-SeOH) by hydrogen peroxide or organic
hydroperoxides giving rise to a semisynthetic peroxidase

Articial redox enzyme avopapain can be obtained from

papain (a hydrolase) by attaching a avin-type redox cofactor
to the nucleophilic thiol group in the active site of papain.

 Replacement of the central metal ion while leaving the overall

protein intact has been used to convert carbonic anhydrase (a
lyase) into an artificial peroxidase (oxidoreductase)

 Central Zn2+ from the active site was removed by chelation

and the apoenzyme reconstituted with Mn2+
The modified enzyme now catalyzed the asymmetric
epoxidation of styrene-type alkenes at the expense of
hydrogen peroxide as oxidant
Reaction rate and stereoselectivity was comparable to HRP,
but its stability was 100 times lower

GENETICALLY MODIFIED
ENZYMES

Changing substrate specificity

Altering cofactor requirements
Inverting stereochemistry
Engineering catalysis

CATALYTIC ANTIBODIES

ABZYMES
Enzymes possessing
an active site but no
chemical operators
for catalytic activity

ADVANTAGES AND DISADVANTAGES

They offer a unique access to articial tailor-made enzyme-like catalysts and are
able to catalyze reactions which have no equivalent counterpart within the
diverse natural enzymes.
The construction of abzymes having an opposite stereochemical preference is
possible.
The catalytic efciency is low in comparison to that of natural enzymes, mainly
because the transition-state mimics which are used to elicit the antibody only act
as a template for the true transition state.
The production of abzymes on a substantial scale is tedious and preparativescale reactions are still in the micromolar range. Furthermore, to avoid the
occurrence of catalytically active enzyme impurities, the purity of the abzymes
must be very high.
The high binding energy for the substrate (up to 20 kcal/mol) which is required
for efcient catalysis remember, that catalytic antibodies do not have a
chemical operator like enzymes also results in tight binding of the product.
Thus, product inhibition, which limits the overall performance, is an inherent
disadvantage of catalytic antibodies

HYDROLYSIS
Mechanism of hydrolysing enzymes are similar- attack by a
nucleophilic group from the enzyme active site.
For serine hydrolases, we have Ser, His, Asp triad that forms
the catalytic core. The special arrangement of the triad reduces
the pKa of serine which can then act as an effective
nucleophile to form acyl-enzyme intermediate
Specific chirality in substrate is recognised and hence specific
chiral product is formed which is determined by the kinetics of
the reaction pathway

Ser-His-Asp TRIAD

NUCLEOPHILIC ATTACK

ENA
NTIO
FACE
DIFF
ERE
NTIA
TION

ENA
NTIO
TOPI
C
DIFF
ERE
NTIA
TION

DESYM
METRIS
ATON
OF
mesoSUBST
RATES

 Hydrolytic reactions done in aqueous conditions are

irreversible
When a prochiral or meso substrate is converted into two
different enantiomeric products at different rates, k1 and k2,
the selectivity () is governed by the ratio k1/k2 which is
independent of conversion and hence remains constant
throughout the reaction time.
Thus optical purity (ee) is not dependent on extent of
conversion and cannot be improved by stopping the reaction at
different times but only by changing the environment of
reaction

[(k1+k2)/(k3+k4)]
has a major impact
on the chemical
yield of P +Q
symmetry of the
selectivities
[(k1>k2, k3>k4 or
k1>k2, k4>k3)]
determines the
optical purity of
the product

 Due to the chiral nature of the enzyme active site, one

enantiomer fits better than the other and is hence converted at
a higher rate comopared to its mirror image and the racemic
substrate is hence kinetically resolved during enzymatic
transformation. This is known as Enantiomeric
differentiation

AMIDE HYDROLYSIS
Essentially used to produce enantiomerically pure amino acids
in their natural L-configuration (additives for animal feed, for
infusion solutions and as enantiopure starting materials for the
synthesis of pharma- and agrochemicals or articial
sweeteners) or unnatural D-configuration (D-phenylglycine
and its p-hydroxy derivative are used for the synthesis of
antibiotics such as ampicillin and amoxicillin, respectively,
and D-valine is an essential component of the insecticidal
synthetic pyrethroid uvalinate )

ENZYMATIC ROUTES TO PURE AMINO ACIDS

METHODS OF AMIDE
HYDROLYSIS
Esterase method
Amidase method
Acylase method (to produce D-amino
acids)
Hydantoinase method (to produce Damino acids)
Lactamase method

ESTERASE METHOD