Transcript Immobilization of enzymes

Enzymes in Food Industries
By Lydia ETCHEBEST
Frédéric RIVAL
Immobilization of enzymes
Why it is important to choose a method of attachment prevent
loss of enzyme activity?
- To avoid reaction
- The structure is retained in the enzyme through hydrogen
bonding or the formation of electron transition complexes:
prevent vibration of the enzyme and increase thermal stability
The different methods are:
Carrier-Binding: the binding of enzymes to water-insoluble carriers
Cross-linking: intermolecular cross-linking of enzymes by bi-functional or
multi-functional reagents.
Entrapping: incorporating enzymes into the lattices of a semi-permeable
gel or enclosing the enzymes in a semi-permeable polymer membrane
 Carrier-Binding
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The oldest immobilization technique for enzymes
Some of the most commonly used carriers for enzyme
immobilization are polysaccharide derivatives such as
cellulose, dextran, agarose, and polyacrylamide gel.
The selection of the carrier depends on the nature
of the enzyme itself, as well as the:
- Particle size
- Surface area
- Molar ratio of hydrophilic to hydrophobic groups
- Chemical composition
the carrier-binding method can be further sub-classified into:
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Physical Adsorption
Covalent Binding
Ionic Binding
1-1 : Physical Adsorption
Of enzyme protein on the surface of waterinsoluble carriers.
Advantages : no reagents and only a minimum of
activation steps are required
Disadvantages : the adsorbed enzyme may leak
from the carrier during use due to a weak
binding force between the enzyme and the
carrier. Moreover, the adsorption is nonspecific, further adsorption of other proteins
or other substances
1-2 : Covalent Binding
Based on the binding of enzymes and water-insoluble carriers by
covalent bonds
The functional groups that may take part in this binding are Amino
group,
Carboxyl
group,
Sulfhydryl
group,
Hydroxyl
group, Imidazole group, Phenolic group, Thiol group, Threonine
group,Indole group
Disadvantages : covalent binding may alter the conformational
structure and active center of the enzyme, resulting in major loss
of activity and/or changes of the substrate
Advantages : the binding force between enzyme and carrier is so
strong that no leakage of the enzymes occurs, even in the
presence of substrate or solution of high ionic strength.
1-3 : Ionic Binding
Of the enzyme protein to water-insoluble
carriers containing ion-exchange residues
Polysaccharides and synthetic polymers having
ion-exchange centers are usually used as
carriers
Advantages : the enzyme to carrier linkages is
much stronger for ionic binding
Disadvantages : the binding forces between
enzyme proteins and carriers are weaker than
those in covalent binding
 Cross-Linking
Either to other protein molecules or to functional
groups on an insoluble support matrix
It is used mostly as a means of stabilizing
adsorbed enzymes and also for preventing leakage
from polyacrylamide gels
The most common reagent used for
cross-linking is glutaraldehyde
Disadvantages : Cross-linking reactions are
carried out under relatively severe conditions.
These harsh conditions can change the
conformation of active center of the enzyme; and
so may lead to significant loss of activity.
 Entrapping Enzymes
 Entrapping Enzymes
continuation
Based on the localization of an enzyme within the lattice
of a polymer matrix or membrane
It can be classified into lattice and micro capsule types.
This method differs from the covalent binding and cross
linking in that the enzyme itself does not bind to the gel
matrix or membrane. This results in a wide applicability
Disadvantages : The conditions used in the chemical
polymerization reaction are relatively severe and result
in the loss of enzyme activity.
Enzymes That Aid Beverages
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Enzymes perform many functions in beverages
They can help to form nutrients for the fermentation
process, facilitate processing, and affect the color,
flavor and clarity of the finished product
They are Biological catalysts based mainly on protein,
they remain unchanged at the completion of the reaction
Enzymes are typically named for the reactions they
catalyze. They fall into six major categories:
oxidoreductases, hydrolases, lysases, transferases,
ligases and isomerases. Of these types, hydrolases play
the most important role in the beverage industry.
Enzymes That Aid Beverages
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Most enzymes catalyze highly specific reactions (alpha-14-glucan glucanhydrolase) but not all enzymes show the
same degree of specificity (papain)
A number of factors affect enzyme activity : T°C, pH,
concentration, contact time with the substrate, trace
metals, salt and salt ions, and oxidizing agents.
enzymes in beverages can occur naturally in the
ingredients used to formulate the beverage. Most fruits
contain low levels of pectinase, and the malting process
produces significant levels of amylase. In other cases, a
product designer can add an enzyme preparation to
achieve a specific goal or to supplement or standardize
naturally occurring enzymes.
 The brew crew
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crucial role in the production of beer and other types of
malted liquor, such as whiskey
three major functions: the formation of sugars to be
used during fermentation; viscosity control; and, in beer,
"chill-proofing."
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The additional enzymes can help make up for the lack
of amylases in the grains used and increase the level
of fermentable sugars.
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The beta-glucans and pentosans are left intact : they
absorb high levels of water, increasing the viscosity.
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Protein precipitation can cause the “Chill haze” in
beer : addition of papain
 The brew crew
continuation
Enzymes
Uses
Amylases
Starch breakdown
Β glucanases
Improved filtering ability
Proteases
Prevent “chill haze”
 The Vine
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Even if wine-making generally relies on the
natural enzymes present in the grape or formed
as a product of fermentation, added enzymes
could help in several areas.
Added pectinase can aid in pressing and
clarification. Particularly helpful during during
the mash process, since higher temperatures
mean increased levels of pectin in the juice
 Juices
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Enzymes are used to extract juice from fruits and prepare a
finished product. For non-citrus juices, such as apple, grape
and berry, processors add enzymes at the beginning of the
mash stage.
The cell walls of fruits consist of cellulose, hemicelluloses,
pectin and proteins. To extract a larger amount of juice and to
facilitate pressing, structures must be break down
the
viscosity of the juice decreases and the size of the particles
is increased. The combination of those two elements causes a
floc that settles out, and the clarified juice can be removed
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The most prevalent enzymes used in juice processing are
pectinases
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Enzymes in fruit juice also affect the color and flavor of the
juice
Enzymes
Uses
Amyloglucosidase
Starch breakdown in early season fruit
Cellulase
Liquefaction of fruit
Esterase
Aroma development
Lipoxygenase
Aroma development
Pectinesterase
Clarification of juice
Polygaclacturonase
Clarification of juice
Polyphenoloxidase
Color and flavor
Dairy doses
(milk and other fluid dairy products)
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The enzymatic process of interest is the
hydrolysis of lactose, for the lactose-
intolerant
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Lactase, catalyzes the hydrolysis of the
beta-D-galactoside, converting it into
glucose and galactose.
Starch & sugar Industries
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Considerable quantities of the sweeteners used
throughout the world are derived from starch as
opposed to cane or beet sugar
The treatment of starch with enzymes results in
a variety of sweet syrups
Starch & sugar Industries
continuation
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Three stages can be identified in starch modification:
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amylases liberate "maltodextrin" by the liquefaction
process; they are not very sweet as they contain dextrins
and oligosaccharides.
The dextrins and oligosaccharides are further hydrolysed
by enzymes such as pullulanase and glucoamylase in a
process known as saccharification. Complete
saccharification converts all the limit dextrans to
glucose, maltose and isomaltose. The resulting syrups are
moderately sweet and are frequently modified further.
Treatment of glucose/maltose syrups with glucose
isomerase converts a large proportion of the glucose to
fructose which is sweeter than glucose, process of
isomerisation (50 % fructose and 50 % glucose: High
Fructose Syrups )
Breadmaking
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Important in human nutrition: half of
their required carbohydrates and about
one-third of their protein from bread
Process uses:
Endogenous enzymes
Exogenous enzymes (more efficience)
Breadmaking - Amylase
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Purposes:
to improve or control dough-handling properties
to improve product quality
Activity: hydrolisation of starch
Origins:
Fungal
Bacterial: cheaper but thermostable (excess of
activity)
Breadmaking - Proteinase
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Purposes:
destruction of gluten protein cohesiveness
improve elasticity and handling properties
of doughs (good volume)
Over - Activity: Decomposition of bread
structure
Breadmaking Pentosanase
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Purposes (rye flour):
a less tough dough
improve volume
a softer crumb
better storage properties
Meat tenderization
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Characteristics of good meat:
juiciness
good chewability
firm texture
color
taste
Meat tenderization Enzymes
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Main Enzymes:
Papain
Bromelain
calpain
Meat tenderization Enzymes
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Characterisics of calpain:
The protease must be endogenous to
skeletal muscle cells
The protease must have the ability to
reproduce post-mortem changes in
myofibrils in vitro
The protease must have access to
myofibrils in tissue
Meat tenderization Enzymes
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Mechanism of tenderization (calpain):
proteolysis of key myofibrillar proteins
resistance of myofibrillar proteins to
calpains
regulation and stability of calpains in
muscles
Meat tenderization Enzymes
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Calpains attack certain proteins of the
Z-line proteolysis of key myofibrillar
proteins
cathepsins attack myosin and actin
Cheese making
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a way of preserving the nutrients of
milk
More than 1000 varieties of cheese
Cheese making
Process:
 clotting
 enzymatic conversion of k-casein into
para-k-casein
 coagulation of the micelles of
paracasein
 aging
Cheese making
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Inside the raw milk, there are
endogenous enzymes
Pasteurized milk includes inactivated
enzymes
Food industry must use exogenous
enzymes
Cheese making
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Bacteria convert lactose to lactic acid.
This acid environment would eventually
coagulate the protein.
Using of rennet
Split the k-casein
One part is extremly soluble and the
other part remains in micelles
Micelles coagule
Cheese making
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lipases
Fat hydrolysis
Flavour development
13-galactosidase and lactase (dairy
industry)
increase sweetness
prevent crystallization
Conclusion:
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Endogenous enzymes are used
traditionally
Exogenous enzymes are more and more
used in food industry:
Control the process
Homogenization of the production
More efficient
That’s all !
Thank you!