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Enzymology

STB3142
Lecture 11

Applications of enzymes
Enzyme immobilization
Usage of enzyme in a technological process depends
on their expense
Recombinant technology makes cheaper cost of
producing enzymes
Enzymes are catalytic molecules (not directly used up
by the processes) so they can be reused when possible
The solution is to separate the enzyme and product
during the reaction in a two phase system
Enzyme is imprisoned in its phase allowing reuse or
continuous use
Also prevent enzyme from contaminating the product
Methods of immobilization
a) Adsorption
b) Covalent bonding
c) Entrapment
d) Membrane confinement
Adsorption
Very simple method of wide applicability and
capable of high enzyme loading (1 g of protein
per 1 g of matrix)
Involves simple mixing of enzyme with suitable
adsorbent, under appropriate pH and ionic
strength after a sufficient incubation period
Combination of hydrophobic effects and
formation of several salt links immobilize enzyme
molecules
Examples of suitable absorbents are ion-exchange
matrices, porous carbon, clays, hydrous metal
oxides glasses and polymeric aromatic resins
Adsorption limit of enzyme

Support type
DEAE-Sephadex CM-Sephadex
% bound at
anion exchanger cation exchanger
pH 2.5 0 100
pH 4.7 100 75
pH 7.0 100 34
Covalent bonding of enzymes
Enzymes are covalently coupled to insoluble matrices
Only small amount of enzymes may be immobilized
(0.02 g per 1 g of matrix)
Strength of binding is very strong with very little
leakage
Lysine residues are found to be most useful for
covalent bonding of enzymes to insoluble matrices
Example: Sepharose, activated by cyanogen bromide
Disadvantage of this method is high cost of Sepharose
Covalent bonding of enzymes
Covalent bonding of enzymes
Effects of covalently bound enzymes
Entrapment of enzymes
A convenient method when involving low
molecular weight substrates
More than 1 g of enzyme per g of matrix can
be entrapped
May involve purely physical caging of enzyme
or covalent binding
Difficult for high molecular weight substrates
to approach enzymes
Example: Enzymes in cellulose acetate fibres
Membrane confinement
Utilizes the semipermeable nature of the membrane
Membrane must confine enzyme while allowing free
passage to the reaction products and substrates
Simplest form is by placing enzyme on one side of the
semipermeable membrane while reactant and product
stream is on the other side
Easy to use for wide variety of enzymes but expensive
method
Enzymes can also be encapsulated in droplets or
liposomes (example: enzyme in aqueous solution of
1,6-diaminohexane)
Comparison of different enzyme
immobilization techniques
Covalent Membrane
Characteristics Adsorption Entrapment
binding confinement

Preparation Simple Difficult Difficult Simple


Cost Low High Moderate High
Binding force Variable Strong Weak Strong
Enzyme leakage Yes No Yes No
Applicability Wide Selective Wide Very wide
Running Problems High Low High High
Matrix effects Yes Yes Yes No
Large diffusional barriers No No Yes Yes
Microbial protection No No Yes Yes
Enzyme reactors
Stirred tank batch reactor (STR)
Generally consist of a tank and a stirrer
Can be use for soluble and immobilized enzymes
Batch membrane reactor (MR)
Mainly consist of membrane to separate between enzymes
and substrate/products
A simplified example of this membrane is dialysis
membrane
Packed bed reactor (PBR)
Also called plug-flow reactor
The substrate flows at the same velocity parallel to the
reactor axis
Application of immobilized enzymes
Industrial use of immobilized enzyme
Lactases in dairy industry
Lactose represent 4.7% w/v in milk
Its presence in milk makes it unsuitable fpr
majority of adult population (lactose intolerant)
There is a need for low-lactose milk for lactose
intolerant children and adults suffering from
protein-calorie malnutrition
Hydrolysis of lactose to glucose would prevent
problems
Industrial use of immobilized enzyme
Lactases in dairy industry
Lactases are also used in the production of ice
cream and condensed milks
Lactose is hydrolyzed giving a sweeter product
which will not crystallize in condensed or frozen
Improves handling and creaminess of ice cream
products
Industrial use of immobilized enzyme
Production of penicillin
Penicillin can be produced from 6-
aminopenicillanic acid (6-APA) which are produced
by fermentation (Penicillium fungi)
Partially purified penicillin amidase form E.coli
trapped into cellulose triacetate fibers can be used
for production of penicillin
Industrial use of enzymes in solution
Enzyme in detergent
More than half of all detergents utilize enzymes
Dirt comes in many forms includes proteins, starches
and lipids
Use of enzymes allows lower temperatures to be
employed and shorter periods of agitation during
washing
Enzymes are used in small amounts in most detergent
(only 0.4% - 0.8% of weight)
Enzymes mostly originate from bacteria (Bacillus
licheniformis) including proteases, alcalase, esperase
and amylase
Enzymes in detergent
Composition of enzyme detergent
Constituent Composition (%)
Sodium tripolyphosphate (water softener, loosens dirt)a 38.0
Sodium alkane sulphonate (surfactant) 25.0
Sodium perborate tetrahydrate (oxidising agent) 25.0
Soap (sodium alkane carboxylates) 3.0
Sodium sulphate (filler, water softener) 2.5
Sodium carboxymethyl cellulose (dirt-suspending agent) 1.6
Sodium metasilicate (binder, loosens dirt) 1.0
Bacillus protease (3% active) 0.8
Fluorescent brighteners 0.3
Foam-controlling agents Trace
Perfume Trace
Water to 100%
Industrial use of enzymes in solution
Enzymes in starch hydrolysis
Starch is the most common storage of
carbohydrate
Starch from all plants are stored in the form of
granules differ in size and characteristics from
species to species
Acid hydrolysis of starch has had widespread use
in the past but now largely replaced by enzyme
processes
Enzymes used in starch hydrolysis
Enzyme EC number Source Action
Only a-1,4-oligosaccharide links are
cleaved to give a-dextrins and
Bacillus amyloliquefaciens
predominantly maltose (G2), G3, G6 and
G7 oligosaccharides
Only a-1,4-oligosaccharide links are
cleaved to give a-dextrins and
a-Amylase 3.2.1.1 B. licheniformis
predominantly maltose, G3, G4 and G5
oligosaccharides
Only a-1,4 oligosaccharide links are
cleaved to give a-dextrins and
Aspergillus oryzae,A. niger
predominantly maltose and G3
oligosaccharides
Only a-1,4-oligosaccharide links are
Saccharifyinga-
3.2.1.1 B. subtilis(amylosacchariticus) cleaved to give a-dextrins with maltose,
amylase
G3, G4 and up to 50% (w/w) glucose
Only a-1,4-links are cleaved, from non-
b-Amylase 3.2.1.2 Malted barley reducing ends, to give limit dextrins andb-
maltose
a-1,4 and a-1,6-links are cleaved, from the
Glucoamylase 3.2.1.3 A. niger
nonreducing ends, to give b-glucose
Only a-1,6-links are cleaved to give
Pullulanase 3.2.1.41 B. acidopullulyticus
straight-chain maltodextrins
Enzymes used in starch hydrolysis
Enzymes used in starch hydrolysis
Enzymes in starch hydrolysis
There are 3 stages in conversion of starch:
Gelatinization: dissolution of nanogram-sized starch granules
to a viscous suspension
Liquefaction: partial hydrolysis of the starch with
concomitant loss in viscosity
Saccharification: production of glucose and maltose by
further hydrolysis
In the starch and glucose syrup industry, dextrose
equivalent (DE) unit is used to represent the
percentage of hydrolysis (glucose is DE 100, maltose is
50 and starch is 0)
Revision
Protein Engineering
Directed evolution
Rational Designs
Production of recombinant protein
Site-directed mutagenesis
Protein purification
Industrial enzymes