APPLICATIONS OF 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)
MODIFIED ENZYMES
CHEMICALLY MODIFIED
ENZYMES
Surface modified enzymes
Bioimprinting
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
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
,-DICARBOXYL-PEG
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
GENETICALLY MODIFIED
ENZYMES
CATALYTIC ANTIBODIES
ABZYMES
Enzymes possessing
an active site but no
chemical operators
for catalytic activity
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
[(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
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 )
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