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Enzymes in food industry
Basics
Dr Otmar Höglinger
Dr.
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Classification and Numbering of Enzyms
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Enzymes are divided into six main classes according to the type of
reaction catalyzed.
y
They are assigned code numbers, prefixed by E.C., which contain
four elements separated by points and have the following meaning:
1. the number first indicates to which of the six classes the
enzyme belongs,
2. the second indicates the subclass
3. the third number indicates the sub-subclass, and
4 the ffourth is the serial number off the enzyme in the sub4.
subclass
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The six classes are distinguished in the following manner
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1. Oxidoreductases
This class encompasses all enzymes that catalyze redox reactions
reactions.
The recommended name is dehydrogenase whenever possible, but
reductase can also be used. Oxidase is used only when O2 is the
acceptor for reduction. The systematic name is formed according to
d
donor:
acceptor
t oxidoreductase.
id d t
2. Transferases
Transferases catalyze the transfer of a specific group, such as
methyl,
th l acyl,
l amino,
i
glycosyl,
l
l or phosphate,
h
h t ffrom one substance
b t
tto
another. The recommended name is normally acceptor
grouptransferase or donor grouptransferase. The systematic name
is formed according to donor: acceptor grouptransferase
grouptransferase.
3. Hydrolases
Hydrolases catalyze the hydrolytic cleavage of C-O, C-N, C-C, and
some other bonds
bonds. The recommended name often consists simply of
the Substrate name with the suffix -ase. The systematic name
always includes hydrolase.
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The six classes are distinguished in the following manner
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4. Lyases
Lyases catalyze the cleavage of C-C, C-O, C-N, and other bonds by
elimination. The recommended name is,, for example,
p ,
decarboxylase, aldolase, dehydratase (e-limination of CO2,
aldehyde, and water, respectively). The systematic name is formed
according to Substrate group-lyase.
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5. Isomerases
Isomerases catalyze geometric or structural rearrangements within a
molecule.
l
l Th
The diff
differentt ttypes off iisomerism
i
llead
d tto th
the names
racemase, epimerase, isomerase, tautomerase, mutase, or
cyloisomerase.
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6. Ligases
Ligases catalyze the joining of two molecules, coupled with the
hydrolysis of a pyrophosphate bond in ATP or another nucleoside
triphosphate.
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Catalytic Activity of Enzymes
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The theory of enzyme-catalyzed reactions proposed by Michaelis
Menten is based on the assumption
p
that the enzyme
y
( E ) and the
substrate (S) form a complex (ES) by a reversible reaction.
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The complex is then converted into the product (P) with the reaction
rate k2, when practically no product is present.
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Under commonly used conditions off enzyme activity measurement,
cES can be considered to be sufficiently constant during the
observed reaction period
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Catalytic Activity of Enzymes
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By using a term for the total concentration of enzyme
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O obtains
One
bt i
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Or
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By introducing the Michaelis constant KM
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The reaction rate as a function of cES
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Reaction rate as a function of substrate concentration
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The maximum reaction rate V, which is reached, when all of the enzyme is
satureted whit substrate (cES = CEt)
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The Michalis-Menten equation is
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In the case of frequently occuring two-substrate reaction, a similar
derivation leads to the formula:
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Reaction rate as a function of substrate concentration
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Factors Governing Catalytic Activity-Temperature
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The temperature dependence of enzyme-catalyzed reactions exhibits an
optimum because the thermodynamic increase of reaction rate is followed
by a steep drop caused by thermal denaturation of the enzyme. The
optimum is generally between 40-60°C. Some temperature insensitive
enzymes may exhibit an optimum at almost 100°C.
100 C.
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Pressure
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Yields of enzymatic processes can also be influenced by changing the
pressure.
Ad
Advantage
t
can be
b taken
t k off the
th stability
t bilit off many enzymes ttowards
d hi
high
h
pressure in order to carry out reactions in supercritical fluids.
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For example, cholesterol oxidase catalyzes the oxidation of cholesterol to 4cholesten-3-one at 123 bar and 31°C in supercritical CO2,giving a 100%
conversion in one hour.
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This procedure has several advantages: cholesterol is 50 times soluble in
p
CO2 as in water;; the solvent is inert,, nontoxic,, and
supercritical
nonflammable, and the enzyme, being insoluble, is easily recovered.
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Aqueous versus organic media
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In commun with other reactions, enzymatic processes are also
influenced by
y the nature of solvent.
The nature of the solvent can dramatically effect the substrate
specifity, activity, stability, and regio- and stereoselectivity of
an enzymatic transformation
transformation.
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The use of enzymes in organic media offers several advatages:
– Enzymes are generally more thermally stable in nonaqueous media
(PPL is inactivated virtuelly instantaneously at 100°C
100 C while in
hydrocarbons it has a half-life of tens of hours at this temperature)
– Water lubricates enzyme molecules, rendering them
conformationally flexible.
flexible Dehydration causes enzyms to adopt a
more rigid structure. They remain properly folded in organic media
even at high temperatures and resistant to denaturation.
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Aqueous versus organic media
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Another advantage of using enzymes in nonaqueous media is that it allows
reactions
ti
to
t be
b carried
i d outt which
hi h are nott feasible
f
ibl iin water
t d
due tto
thermodynamically unfavorable equilibria (eg esterifications or peptide
formation)
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Performing enzymatic transformations in organic solvents renders them
more compatible with organic synthesis, this is important because the
majority off compounds off interest to organic chemists are insoluble in water.
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Value of pH
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All enzymes have an optimum pH range for activity. The optimum depends
not only on pH but also on ionic strength and type of buffer
buffer.
It max be influenced by temperature, substrate, and coenzyme
concentration.
F mostt enzymes, the
For
th pH
H optimum
ti
lilies iin th
the range 5 tto 7
7.
Extreme values of 1,5 and 10,5 have been found for pepsin and for alkine
phosphatase.
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Activation
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In addition to substrates and coenzymes many enzymes require nonprotein
or, in some cases, protein compounds to be fully active
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The activating ion may be involved directly in the reaction by
complexing
p
g the coenzyme
y
or cosubstrate ((eg
g Fe ions bound to flavin
or the ATP-Mg complex).
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In other cases,
cases the ion is part of the enzyme and either acts as a
stabilizer for the active conformation (eg Zn ions in alkine
phosphatase) or participates directly at the active sit (eg Mn ions in
isocitrate dehydrogenase)
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Inhibition-Irreversible Inhibition
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An irreversible inhibitor frequently forms a stable compound with the
enzyme
y
byy covalent bonding
g with an aminoacid residue at the active side.
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For example, diisopropyl fluorophosphat (DIFP) reacts with the serine
residue at the active site of acetylcholinesterase
y
to form an inactive
diisopropylphosporyl enzyme.
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Alkylating
y
g reagents,
g
, such as iodoacetamid,, inactivate enzymes
y
with
mercapto groups at their acive sites by modifying cysteine.
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Reversible Inhibition
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Reversible inhibition in contrast, is characterized by an equilibrium between
enzyme und inhibitor. Several main groups of reversible inhibitory
mechanisms can be differentiated
differentiated.
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Competitive Inhibition: The inhibitor competes with the substrate or
coenzyme for the binding site on the active center by forming an enzyme
inhibitor complex EI. Inhibition can be made ineffective by excess substrate,
as is the case for inhibition of succinate dehydrogenase by malonate.
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Noncompetitive Inhibition: The inhibitor decreases the catalytic
activity of an enzyme without influencing the binding relationship between
substrate and enzyme.
This means that inhibitor and substrate can bind simultaneously to an
enzyme
y
molecule to form ES,EI, or ESI complexes.
p
Noncompetitive
p
inhibition is dependent solely on the inhibitor concentration and is not
overcome by high substrate concentration. An example is the blocking of an
essential cysteine residue by such heavy metals as copper or mercury.
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Inhibition
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Uncompetitive Inhibition: The inhibitor reacts only with the intermediary
enzyme-substrate
b t t complex.
l
A
An example
l iis th
the reaktion
kti off azide
id with
ith th
the
oxidized form of cytochrome oxidase.
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Competitive inhibition:
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Noncompetitive inhibition:
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Uncompetive inhibition:
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Inhibiton
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Substrate Inhibition: High concentration of substrate may decrease the
catalytic acivity of an enzyme. Examples are the action of ATP on
phosphofructokinase or of urea on urease.
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product inhibition: In manyy multienzyme
y
systems,
y
, the end product
p
of
End p
the reaction sequence may act as a specific inhibitor of an enzyme at or
near the beginning of the sequence. This type of inhibition is also called
feedback inhibition or retroinhibition.
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Lineweaver Burk graphs of reversibly inhibited enzyme reactions
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A) Competitive
C
titi iinhibition
hibiti
B) Noncompetitive inhibition
C)) Uncompetitive
p
inhibition
----- Uninhibited reaction
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Inhibition
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Competitive inhibition
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Noncompetitive inhibition
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Uncompetitive inhibition
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Allostery
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Cosubstrates with central role in the metabolism, such as acetyl-CoA,
ATP, or AMP, may also influence the rate or reaction sequences by
allosteric regulation
regulation.
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For example, phosphofructokinase, the first enzyme in the energy-supplying
Embden Meyerhof
Meyerhof-Parnass
Parnass pathway,
pathway is inhibited by a high concentration of
ATP.
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The binding of the effector modifies the conformation of the subunit and
ist active center, which then affects modifies the conformation of the subunit
and ist active center, which then affects the conformation and hence the
catalytic
y activityy or the entire molecule.
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Allosteric enzymes ussually do not show the classical Michaelis-Menten
kinetic relationship of cs,V and KM.
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Biogenic Regulation of Acitvity
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In principle, enzyme activity also be controlled by regulating the amount of
enzyme in the cell.
cell This can be accomplished by regulating the biosynthesis
of induvidual enzymes or of several functionally related enzymes by
induction or repression, or by specific attack of proteolytic enzymes.
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Biogenic regulation of activity
Activity of an allosteric enzyme as a function of substrate concentration
in absence and p
presence of an allosteric activator or inhibitor.
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Quality Evaluation of Enzym preparation-Quality Criteria
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The quality of enzyme preparations is characterized by activity, purity,
stability, formulation, and packing.
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Specific Activity:
– One of the most important
p
q
quality
y criteria of an enzyme
y
preparation
p p
is ist specific
p
activity, i.e. the catalytic activity related to ist protein content. Specific acivity is
ussually expressed as units per milligram or, for less purified products, units per
gram.
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Protein Determination:
– Ultraviolet Absorption: Because of their content of aromatic amino acids,
proteins exhibit an absorption maximum at 270-280 nm
– Biuret Method: The reaction of peptide bonds with copper ions in alkaline
solution yields a purple complex which can be determined photometrically.
– Lowry Method: The Lowry method combines the biuret reaction of proteins with
reduction of the Folin
Folin-Ciocalteu
Ciocalteu phenol reagent by tyrosin and tryptophan
tryptophan.
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Advantages and Limitations
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Advantages:
Very efficient catalysis
Milf conditions
No organic
g
solvent needed
Wide range of acitvity
High substrate selectivity, chemoselecitvity, regioselectivity, and
steroselectivity
t
l ti it
Often fewer steps
Inexpensive source of complex chiral ligands
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Advantages and Limitations
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Limitations
A il bilit and
Availability
d price
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Operational stability and lack of flexibility
Substrate and/or product inhibition
Not all types of reaction accessible
Cofactor regeneration sometimes needed
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Whole Cells Versus Isolated Enzymes in Organic Synthesis
Wh l cellll
Whole
I l t d enzyme
Isolated
Advantages
Cheap
Cofactors present
Simpler equipment
Simpler work up
Less contamination from
other enzymes
Disadvantages
High dilution
Complex work up
Side reactions caused by
other enzymes present
Expensive
Addition of cofactors
necessary where
required
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Immobilization-Carrier Binding
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Enzyme proteins have amino acid residues containing chemically reactive
groups, ionic
i i groups, and/or
d/ hydrophobic
h d h bi groups, as wellll as h
hydrophobic
d h bi
domains.
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These amino acid residue and the hydrophobic domains can participate in
the immobilization of enzymes through covalent linkage, ionic binding, or
physical adsorption.
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Covalent Binding: Amino acid residues that are not involved in the active
g site of the enzymes
y
to be immobilized can be used
site or substrate-binding
for covalent binding with supports.
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These are the εε-amino
amino group of lysine
lysine, the mercapto group of cysteine
cysteine, the
β-carboxyl group of aspartic acid, the γ-carboxylgroup of glutamatic acid,
the phenolic hydroxyl group of tyrosine, or the hydroxyl groups of serine and
threonine.
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Examples of immobilized enzymes used in major commercial
processes
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Prinziples of enzyme immobilization
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1) Si
Sie kkennen d
das N
Nomenklatursysteme
kl t
t
fü
für E
Enzyme und
d kö
können es anwenden.
d
2) Sie kennen die Klassifizierung von Enzymen, können die einzelnen Enzymklassen beschreiben
3) Sie können die Michaelis-Menten Gleichung ableiten, sie verstehen die Anwendung dieser
g können die dazugehörigen
g
g Diagramme
g
zeichnen
Gleichung,
4) Kennen die Faktoren die die Enzymaktivität beeinflussen und können diese an Beispielen
beschreiben.
5) Kennen die Regulationsmechanismen von Enzymen, wissen welche Inhibitorentypen es gibt
und diese auch Inhaltlich beschreiben
beschreiben. Sie können die einzelnen Typen auch grafisch darstellen
darstellen.
6) Sie kennen auch allosterische Hemmtypen und biogene Mechanismen der Enzymregulation.
7) Wie erfolgt die Bestimmung von Proteinen, was versteht man unter spezifischer Aktivität
8)) Welche vor- und Nachteile bringt
g der Einsatz von Enzymen.
y
9) Welche Vor- und Nachteile bringen ganze Zellen vs. Enzymen
10) Wie können Träger für Enzyme unterteilt werden
11) Welche prinzipiellen Möglichkeiten der Immobilisierung gibt es
12) Sie können einzelne Methoden beschreiben wie Adsorbtion, Entrappment, Microverkapseln,
kovalente Bindungen
13) Welche vor- und Nachteile hat die Immobilisierung
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Advantages/Disadvantages
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Enzymes iimmobilized
E
bili d b
by covalent
l t bi
binding
di h
have th
the ffollowing
ll i
advantages:
(1) because of the tight binding, they do not leak or detach from
supports during utilization
(2) immobilized enzymes can easily come into contact with
substrates because the enzymes are localized on the surface of
supports
(3) an increase in heat stability is often observed because of the
strong interaction between enzyme molecules and supports
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Disadvantages: (1) active structures of enzyme molecules are liable to be
destroyed by partial modification
(2) strong interaction between enzyme molecules and supports often
hi d
hinders
th
the ffree movementt off enzyme molecules,
l
l
resulting
lti iin d
decreased
d
enzyme activity.
(3) optimal conditions of immobilization are difficult to find
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(4) this method is not suitable for immobilization of cells
(5) supports, in general, are not renewable
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Functional groups in proteins relevant for immobilization
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Properties of matrices relevant for enzyme immobilization
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Carriers
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The different types of carriers used may be classified according either to the
basic material,, origin
g or source,, or to their structure.
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Inorganic
Organic from natural sources; and
Organic synthetic materials
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Inorganic carriers exhibit high pressure stability, but mayundergo
g
materials from natural sources in
abrasion in stired vessels;; organic
most cases offer favorable compatibility with proteins; organic
synthetic carriers in general exhibit high chemical stability.
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Synthetic polymers
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Ion exchange materials have proven to be an economic and
technically
y appropriate
pp p
solution, as awide range
g of carriers is
currently available offering good capacity for enzyme immobilization,
as well as proporties that have relevant to industrial scale
processing.
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Glucose isomerase
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High fructose corn syrup is currently produced at a scale ofabout 10
million tons per year,
year using about 1500 tons of immobilized enzyme
enzyme.
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Glucose isomerase is used for the isomerization reaction.
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Scheme of procedures for immobilization of glucose isomerase,
Novo process,
process crosslinking of cells
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Glucose isomerase
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Scheme of procedures for immobilization of glucose isomerase, Novo
process, crosslinking of cells
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Glucose isomerase
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Genencor, prinziple of isolation of crystalline enzyme
Adsorption onto a composite ion
ion-exchange
exchange matrix formed by
extrusion with 50% polystyrene, 20% TIO2, 30% DEAE cellulose
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Entrapment
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Entrapped biocatalysts are classified according to the following
different types:
1) Lattice type: biocatalysts entrapped in gel matrices prepared
from polysacharides, proteins, or synthetic polymers
2) Micorcapsule type: biocatalysts entrapped in microcapsules of
semipermeable synthetic polymers
3) Liposome type: biocatalysts entrapped within liquid membranes
prepared from phospholipids
4) Hollow fiber type: biocatalysts separeted from the environment
b h
by
hollow
ll
fib
fibres
5) Membrane type: biocatalysts separeted from the spent reaction
solution by
y ultrafiltration membranes
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Polyacrylamide Gel
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In a typical procedure, acrylamide and N,N`-methylenebisacrylamide are
mixed with biocatalysts and polymerized in the presence of an initiator
initiator.
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Alginate Gel
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Several natural polysaccharides, such as alginate, agar and carrageenan
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Carrageenan Gel
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Methods of immobilization of microorganisms
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Immobilization in alginate (Syring method, jet cutter method)
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Applications of technical enzymes
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Industrial applications of enzymes
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Applications of technical enzymes
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Optimierung einer rekombinanten mikrobiellen Transglutaminase
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Die mikrobielle
Di
ik bi ll T
Transglutaminase
l t i
aus St
Streptomyces
t
mobaransis
b
i wird
i d bi
bisher
h
überwiegend in der Lebensmitteltechnologie angewendet, da durch
Vernetzung von Proteinen die Textureigenschaften zB Wurstprodukten zu
verbessern.
erbessern
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Transglutaminasen sind Enzyme,die einen Acyl-Transfer zwischen den γCarboxyamid-Gruppe eines proteingebundenen Glutamins und primären
Aminen katalysieren.
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Neuere Technologien beschäftigen sich mit dem Einsatz der mikrobiellen
Transglutaminase bei der Herstellung von bioabbaubaren Polymeren, wie
beispielsweise
p
Filme und Folien aus Proteinen wie Zein und Casein.
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Optimierung einer rekombinanten mikrobiellen Transglutaminase
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Für Anwendungen
Fü
A
d
i d
in
der L
Lebensmittelindustrie
b
itt li d t i wäre
ä es vorteilhaft,
t ilh ft eine
i
Enzymvariante zur Verfügung zu haben, die bei niedrigen Temperaturen
eine hohe Aktivität aufweist, sich aber bei höheren Temperaturen schnell
inakti ieren lässt
inaktivieren
lässt.
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Random Mutagenese des MTG Gens
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Die Random Mutagenese wurde mit dem GeneMorphII EZClone Domain
Mutagenisis Kit durchgeführt. Die Plasmide die die Mutationen enthielten
wurden, wurden schließlich in E.coli Zellen transformiert.
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Screening auf thermostabile Varianten
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Die Klonbibliothek wurde auf Varianten untersucht, die eine erhöhte
Restaktivität nach Vorinkubation bei höheren Temperaturen aufwiesen
aufwiesen.
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Dazu wurde der aktivierte Rohenzymextrakt in 96 well bei 55°C im
W
Wasserbad
b d vorinkubiert.
i k bi t N
Nach
h 30 min
i wurde
d auff 20°C abgekühlt.
b kühlt
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Anschließend wurde die Aktivität der Varianten mittels kalorimetrischen
Hydroxamattests gemessen.
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Mutanten
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Inaktivierungsverhalten ausgewählter thermostabiler Varianten
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