Transcript Lecture - 8 Applied Enzymes Catalysisx

Dr. A.K.M. Shafiqul Islam
School of Bioprocess Engineering
22.01.10
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Four types of immobilization
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Adsorption Method
Covalent bonding
Entrapment
Encapsulation
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With – OH Group :
Supports of this type may be activated
specifically for the covalent bonding by
subjecting it to treatment with either
cyanogen bromide or triazine. The reaction
with the enzyme protein in each instance
involves the –NH2 group of lysine.
Using Supports with – OH group that are Activated
by Covalent Bonding with Cyanogen Bromide.
Using Supports with – OH group that are Activated
by Covalent Bonding with Triazine.
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With – COOH Groups :
Carboxymethyl cellulose (CMC) may be
activated either via acyl-isourea formation or
azide derivative formation.
The reaction involves the participation of
amino (– NH2) moiety present in lysine,
cysteine, serine, tyrosine — are also made
use of in the covalent bonding phenomenon.
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Immobilization of Enzymes using CMC Supports Having —
COOH with — NH2 Group or with Hydrazine (NH2–NH2) Group
via Covalent Bondage Involving Acyl Urea .
Immobilization of Enzymes using CMC Supports Having — COOH with
— NH2 Group or with Hydrazine (NH2–NH2) Group via Covalent
Bondage Involving Azide Derivative.
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With – NH2 Group :
The amino functional group containing
support material may be converted easily to
the corresponding diazonium chloride salt by
suitably treating with a mixture of sodium
nitrite (NaNO2) and diluted hydrochloric acid
(HCl) between 0-5°C (diazotization).
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Immobilization of Enzymes using Supports with
Specific —NH2 group Involving Formation of
Diazonium Chloride
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Immobilization of Enzymes using Supports
with Specific —NH2 group Involving Activation
with Glutaraldehyde.
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Entrapment refers to the phenomenon whereby
the enzyme molecules are either held or
entrapped within the appropriate fibres or gels.
This entrapment may or may not necessarily be
accomplished via covalent bonding existing
between the enzyme entities (molecules) and the
carrier matrix.
In a situation when the covalent bonding is
needed, the enzyme molecules essentially
required to be treated with synthetic reagents
e.g., acryloyl chloride, cellulose acetate etc.
The various steps involved in ‘entrapment’ are as
stated below:
1.
The enzyme(s) may be dissolved in a solution of the
polymer’s precursors.
2.
Polymers may be selected from a variety of materials
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3.
4.
a)
b)
natural gels (e.g., cellulose triacetate, alginate, agar,
gelatin) ;
synthetic gels e.g., polyacrylamide gels.
In order to check and prevent the possible leakage of
the low molecular weight enzymes from the body of
the gel, the average pore size of the gel must be
maintained as large as possible.
Two important aspects in ‘entrapment’ process,
namely:
excessive diffusion limitation, and
variability of pore size
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Polymer entrapment
liposome entrapment
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Example
Lysine residues may be prepared by
employing acryloyl chloride resulting into the
formation of the corresponding acryloyl
amides. The acryloyl amides are first
copolymerized, and secondly cross-linked
with either acrylamide and bisacrylamide to
give rise to the formation of the desired ‘gel’
which comprises of the ‘entraped enzyme’
that may be further exploited in the form of a
thin film on a solid support or as small beads.
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Example
Cellulose acetate fibres also used to entrapment
of enzymes.
Enzyme and cellulose acetate is blended together
to obtain an ‘emulsion’ in an organic solvent,
methylene chloride. The resulting emulsion is
subjected to the process of ‘extrusion’ to obtain
fibres into a solution of an aqueous precipitant.
Calcium alginate is the material used for the
entrapment of microbial, plant cells, and animal
cells.
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Encapsulation or microencapsulation or
membrane confinement is another effective
approach of enzyme immobilization.
In this method the enzyme molecules,
invariably taken up in an aqueous medium,
may be strategically confined within a
semipermeable membrane that ideally
permits an almost absolute ‘free movement’
of the enzymes in either direction to the
products and substrates but fails to allow
their migration and Escape.
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There are two enzyme entrapping methods:
1. Phase Separation:
Membranes are usually made by adopting the
process of phase-separation, that essentially
bears a close resemblance to homogenization
of water in oil.
In this particular instance one phase is
obviously not miscible with the other but
eventually gives rise to a droplet with the other
phase upon adequate mixing. Thus, ultimately
the ‘enzyme’ gets entrapped right within this
droplet,
2. Chemcical Polymerization:
The chemical polymerization aids in the
preparation of the specific water-insoluble
membrane, and thus the enzyme in
question gets duly entrapped during this
on-going phenomenon of polymerization.
2. Chemcical Polymerization:
Examples: Two type examples are as follows:
1) Semipermeable collodion or nylon membranes in
the shape of spheres (round beads) are invariably
utilized for the microencapsulation of an enzyme.
These materials are also available commercially.
2) Fibres of celluclose triacetate may also be
employed for the entrapment of enzymes within
this synthetic material. However, these fibres may
be either woven into a suitable fabric or packed
into the columns carefully.
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1.
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3.
I hope you would not mind to have a small
test today.
What is meant by enzyme specificity? Describe
lock-and-key hypothesis for enzyme specificity?
What is Lineweaver-Burk plot? How can it be used
to calculate Michaelis-Menten constant?
Define and discuss competitive and
noncompetitive inhibitor.