1
Marek Bryjak
Część II
Zakład Materiałów Polimerowych i Węglowych
Bud. H6, pokój 105
Procesy enzymatyczne
Procesy mikrobiologiczne
Procesy polimeryzacji
Procesy separacyjne (membranowe)
Procesy przygotowania surowców
Procesy wydzielania produktu/ów
Biotechnologia
Inż. chemiczna
Procesy biotechnologiczne
Procesy enzymatyczne
4
Co to jest enzym
Co to jest enzym
?
?
•
Białko
(
(
Struktura 3 i 4
Struktura 3 i 4
rzędowa)
rzędowa)
• Katalizator
(przyspiesza
reakcje)
6
Enzymes
Enzymes
• Are specific for
what they will
catalyze
catalyze
• Are
Reusable
Reusable
• End in –
ase
ase
-
-
Sucrase
Sucrase
-
-
Lactase
Lactase
-
-
Maltase
Maltase
2
8
Induced Fit
Induced Fit
• A change in the
shape
shape
of an
enzyme’s active
site
•
•
Induced
Induced
by the
substrate
Stickase
Substrate
If enzyme just binds substrate
then there will be no further reaction
Transition state
Product
Enzyme not only recognizes substrate,
but also induces the formation of transition state
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
X
The Nature of Enzyme Catalysis
●
●
Enzyme provides a catalytic surface
Enzyme provides a catalytic surface
●
●
This surface stabilizes transition state
This surface stabilizes transition state
●
●
Transformed transition state to product
Transformed transition state to product
B
B
A
Catalytic surface
A
Juang RH (2004) BCbasics
Enzyme Stabilizes Transition State
S
P
ES
ES
T
EP
S
T
Reaction direction
Energy change
E
n
er
g
y
r
eq
u
ir
ed
(
n
o
c
at
al
ys
is
)
E
n
er
g
y
d
ec
re
as
es
(
u
n
d
er
c
at
al
ys
is
)
What’s the difference?
T = Transition state
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
Active Site Is a Deep Buried Pocket
Why energy required to reach transition state
is lower in the active site?
It is a magic pocket
(1) Stabilizes transition
(2) Expels water
(3) Reactive groups
(4) Coenzyme helps
(2)
(3)
(4)
(1)
CoE
+
-
Juang RH (2004) BCbasics
3
Enzyme Active Site Is Deeper than Ab Binding
Instead, active site on enzyme
also recognizes substrate, but
actually complementally fits the
transition state and stabilized it.
Ag binding site on Ab binds to Ag
complementally, no further reaction
occurs.
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
X
H
O
H
Acid-Base Catalysis
A
d
a
p
te
d
f
ro
m
N
e
ls
o
n
&
C
o
x
(2
0
0
0
)
L
e
h
n
in
g
e
r
P
ri
n
c
ip
le
s
o
f
B
io
c
h
e
m
is
tr
y
(
3
e
)
p
.2
5
2
Induced to transition state
C
O
=
N
H
H
C
H
N
H
+
C
-
O
O
H
O
H
-δ
+δ
H
O
H
C
O
=
N
H
H
C
H
C
O
=
N
H
H
C
H
C
O
=
N
H
H
C
H
Slow
Fast
Fast
Very Fast
Acid-base
Catalysis
Base
catalysis
Acid
catalysis
Both
A
d
a
p
te
d
f
ro
m
A
lb
e
rt
s
e
t
a
l
(2
0
0
2
)
M
o
le
c
u
la
r
B
io
lo
g
y
o
f
th
e
C
e
ll
(4
e
)
p
.1
6
7
N
H
+
C
-
O
O
H
O
H
Specific
Concerted Mechanism of Catalysis
1
2
3
4
5
O
-
H
+
H
COO
-
(270)
Glu
(248)
Tyr
O
-
H
His
(196)
His (69)
Glu
(72)
+
Arg (145)
Carboxypeptidase A
C-terminus
ACTIVE
SITE
ACTIVE
SITE
Check for
C-terminal
Site for
specificity
Active
site
Substrate
peptide
chain
R
N
C
N
C
COO
-
O
-
C
+
Zn
J
u
a
n
g
R
H
(
2
0
0
4
)
B
C
b
a
s
ic
s
O
O
N–C–C–
N–C–C
N–C–C
–N–C–C
R
H
R’
Chymotrypsin Has A Site for Specificity
O
-
C
Ser
Active Site
Active Site
Specificity
Site
Specificity
Site
Catalytic Site
Juang RH (2004) BCbasics
Asp102
His57
Ser195
Catalytic Triad
Catalytic Triad
H
H
Chymotrypsin Catalytic Mechanism A1
N
C
C
N
[
HOOC]
H
O
C
C
N
C
C
[
NH
2
]
C
C
O
Check substrate specificity
Asp102
His57
Ser195
H
H
Chymotrypsin Catalytic Mechanism A2
N
C
C
N
[HOOC]
H
O
C
C
N
C
C
[NH
2
]
C
C
O
First Transition State
4
H
H
Chymotrypsin Catalytic Mechanism A3
N
C
C
N
[HOOC]
H
O
C
C
N
C
C
[NH
2
]
C
C
O
Acyl-Enzyme Intermediate
H
Chymotrypsin Catalytic Mechanism D1
N
-H
C
C
N
[HOOC]
H
O
C
C
N
C
C
[NH
2
]
C
C
O
H
O
H
Acyl-Enzyme Water Intermediate
H
Chymotrypsin Catalytic Mechanism D2
O
O
C
C
N
C
C
[NH
2
]
C
C
H
Second Transition State
O
H
H
Chymotrypsin Catalytic Mechanism D3
O
C
C
N
C
C
[
NH
2
]
C
C
O
O
H
Deacylation
H
Specificity of Ser-Protease Family
COO
-
C
Asp
COO
-
C
Asp
Active Site
Trypsin
Chymotrypsin
Elastase
cut at Lys, Arg
cut at Trp, Phe, Tyr
cut at Ala, Gly
Non-polar
D
e
e
p
a
n
d
n
e
g
a
ti
v
e
ly
c
h
a
rg
e
d
p
o
c
k
e
t
Shallow and
non-polar
O O
–C–
N–C–C
–
N–
C
C
C
C
NH
3
+
O O
–C–
N–C–C
–
N–
C
O O
–C–
N–C–C
–
N–
CH
3
J
u
a
n
g
R
H
(
2
0
0
4
)
B
C
b
a
s
ic
s
24
Two examples of Enzyme
Two examples of Enzyme
Inhibitors
Inhibitors
a.
a.
Competitive
Competitive
inhibitors
inhibitors
:
:
are chemicals
that
resemble
resemble
an
enzyme
enzyme
’
’
s
s
normal
normal
substrate
substrate
and
compete
compete
with
it for the
active site
active site.
Enzyme
Competitive inhibitor
Substrate
5
25
Inhibitors
Inhibitors
b.
b.
Noncompetitive
Noncompetitive
inhibitors
inhibitors
:
:
Inhibitors that
do
do
not enter the
not enter the
active
active
site
site,
but
bind to
bind to
another part
another part
of the
enzyme
enzyme
causing the
enzyme
enzyme
to
change its
change its
shape
shape
, which in turn
alters the active
alters the active
site
site.
Enzyme
active site
altered
Noncompetitive
Inhibitor
Substrate
Invertase (IT)
IT
Sucrose
Non-reducing sugar
Reducing
sugars
Glucose
Fructose
Reducing Power
+
HOCH
2
O
OH
1
2
3
4
5
6
6
5
4
3
2
1
1
2
3
4
5
6
HOCH
2
O
OH
O
HOCH
2
HOCH
2
OH
H
2
O
O
HOCH
2
HOCH
2
HO
O
HOCH
2
O
HOCH
2
HOCH
2
O
β
β
CHO
H-C-OH
HO-C-H
H-C-OH
H-C-
OH
H
2
-C-OH
H
2
C-OH
C=O
HO-C-H
H-C-OH
H-C-
OH
H
2
-C-OH
J
u
a
n
g
R
H
(
2
0
0
4
)
B
C
b
a
s
ic
s
1
2
In
cr
e
a
se
S
u
b
s
tra
te
C
o
n
ce
n
tra
tio
n
2
1
3
4
5
6
7
8
0
0 2 4 6 8
Substrate (µmole)
P
ro
d
u
ct
80
60
40
20
0
S
+
E
↓
P
(in
a
fix
ed
p
er
io
d o
f ti
m
e)
Juang RH (2004) BCbasics
Essential of Enzyme Kinetics
E
S
+
P
+
Steady State Theory
Steady State Theory
In steady state, the production and consumption of
the transition state proceed at the same rate. So the
concentration of transition state keeps a constant.
S
E
E
Juang RH (2004) BCbasics
Constant ES Concentration at Steady State
S
P
E
ES
Reaction Time
C
o
n
ce
n
tra
tio
n
Juang RH (2004) BCbasics
6
v
o
=
V
max
[S]
K
m
+
[S]
(
v
o
)
E + S
ES
E + P
k
2
k
1
k
3
For [substrate] low,
k
3
=
k
cat
7
Problem: Adsorption on solid surfaces has to be avoided!
M. Santore et al., Langmuir 2002, 18 (3), 706.
Adsorption on solid surface
Denaturation of proteins
by adsorption on solid
surfaces
- strong attraction by
van der Waals or
hydrophobic interaction
⇒
Loss of biological
function
8
Czynniki wiążące
•
Gr funk nośnika
Gr funk białka
Czynniki wiążący
•
-COOH
-NH
2
karbodiimid
•
-COOH
- -COOH
izocyjanki
•
-COOH
-CH
2
, -SH
azydek
•
-OH
NH
2
bromocyjan
•
-NH
2
-NH
2
aldehyd glutarowy
•
-NH
2
-COOH
karbodimid
•
-NH
2
-COOH
izocyjanki
•
-NH
2
--NH
2
triazol
•
Grupy oksiranowe
-NH
2
, -OH
•
-OH
- -NH
2
, -OH
diwinylosulfon
•
-OH
NH
2
hydrazyna
•
-OH
NH
2
karbodiimid
•
-OH
NH
2
nadjodan sodu
Aktywność immobilizowanego enzymu
Enzym sieciowany i kryształy
a
0
20
40
60
80
100
0
3
6
9
pH
a
k
ty
w
n
o
ść
Aktywność enzymu immobilizowanego
9
Immobilizacja enzymów w objętości bioreaktora
inkludowanie
związanie z nośnikiem
sieciowanie
-
w sieci żelu
- adsorpcyjnie
rozpuszczonego białka -
- otoczkowanie
- koordynacyjnie
- agregatów białkowych
- w mikro(makro)kapsułkach
- specyficznie
- kryształów białka
- w mikro(makro)emulsjach
- kowalencyjnie
-(w materiale membrany)
- (
na powierzchni membrany)
Nierozpuszczalne w wodzie
Przykład
Immobilization of proteins on colloidal carriers
„bionanoparticles“
Colloidal particles
• provide large surfaces
• large amount of immobilized
biomolecules
Enzymes can be used
as catalysts for
technical applications
substrate
bound
enzymes
product
PS
R
L
CH
CH2
COO-
CH
CH2
SO
3
-
•
Long charged polyelectrolytes attached to colloidal particles
weak
polyelectrolyte
strong
polyelectrolyte
Can be used as
carrier particles
for proteins
Spherical Polyelectrolyte Brush (SPB)
10
Confinement of counterions inside brush layer
PS
R
L
CH
CH 2
SO
3
-
Confined counterions
• high osmotic pressure inside brush
• chains strongly stretched
⇒
Properties of the particles determined
by the confinement of the counterions
PS
R
L
+
negatively charged
negatively charged
carrier
protein
Adsorption on the
„wrong side“: pH > pI
Double trouble:
Electrostatic repulsion + steric
repulsion
? ?
Protein adsorption on Spherical Polyelectrolyte Brushes ?
Protein adsorption: Experimental procedure
Wittemann et al., Phys. Chem. Chem. Phys. 2003, 5, 1671.
Ultra-
filtration
PS
PS
PS
Mixing
Protein solution
Brush latex
Protein coated
brush latex
Protein coated brush
latex + dissolved
proteins
A certain amount of protein remains
adsorbed after exhaustive ultrafiltration!
Main driving force:
Counterion release force
Uptake of protein leads to release of counterions
• strong driving force for protein adsorption even at „wrong“
side of the IEP
High
osmotic
pressure
partially
relieved by
multi-valent
counterions
+
+
+
+
-
-
-
+
+
+
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
--
--
N
+
N
-
-
-
-
+
+
+
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
--
--
2N
+
- N
-
released counterions
+
+
+
+
+
+
+
+
--
--
--
+
+
+
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
--
--
--
--
--
--
--
--
--
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
--
--
N
+
N
-
--
--
--
+
+
+
+
+
+
--
--
--
--
--
--
--
--
--
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
--
--
+
+
+
+
+
+
+
+
+
+
+
--
--
+
+
+
--
--
2N
+
- N
-
released counterions
Polyelectrolyte Mediated Protein Adsorption (PMPA)
Review on the PMPA:
Wittemann, A.; Ballauff, M.
Phys. Chem. Chem. Phys. 2006.
Theoretical description:
Leermakers, F.A.M.; Ballauff,
M.; Borisov, O.V.
Langmuir, in
press.
low ionic strength
high ionic strength
Wittemann et al., J. Am. Chem. Soc. 2005, 127, 9688.
Cryogenic transmission electron microscopy (Cryo-TEM)
Osmotic brush:
confined counterions,
c
s
>
c
a
Salted brush: c
s
=
c
a
11
Localisation of adsorbed protein cont‘d
Ribonulease A
Bovine hemoglobin
Adsorption onto SPB consisting of strong polyelectrolytes
Activity of
enzyme preserved
[ ]
[ ]
S
K
S
v
v
M
+
=
max
0
Activity of bound glucoamylase: Michaelis-Menten analysis
Neumann et al., Macromol. Biosci. 2004, 4, 13; Haupt et al., Biomacromolecules 2005, 6, 948.
-1/K
m
PS
Aim:
„Nanoplant“
Cascade reactions:
Possible system:
α
-Amylase:
starch → maltose
β
-Glucosidase:
maltose → glucose
Glucose Oxidase:
glucose → H
2
O
2
enzyme A
enzyme B
end product
PS
R
L
CH
CH2
COO-
CH
CH2
SO
3
-
protein
„Nanoreactor“
Carrier
particles
for
proteins
Confined counterions
Polyelectrolyte-
protein complexes
Polyelectrolyte-mediated
protein adsorption
• both can take place on the „wrong side“ of the IEP
• both can be traced back to patches of opposite charge
However:
PMPA
leads to
stronger protein binding
because
of the
Donnan effect
and a much
stronger correlation of
the counterions
Synthesis: photoemulsion polymerization
Guo et al., Macromolecules 1999, 32, 6043.
r.t., h
ν
acrylic
acid
PS
PAA
PS
photo-
initiator
70°C
PS
Step 1:
PS latex
Step 2
Photoinitiator layer
Step 3
Shell composed of
linear polyelectrolytes
12
SPB: decisive parameters
R:
core radius
L:
hydrodynamic brush
layer thickness
L
C
:
contour length of the
poly(acrylic acid) chains
σ
σ
σ
σ
:
grafting density of the
tethered chains
D:
average distance of the
grafting points
PS
PAA
R
L
C
D
L
Guo et al., Langmuir 2000, 16, 8719.
id
Π
Π
=
φ
kT
n
R
id
=
Π
Osmotic coefficient φ: Fraction of „free“ counterions
Measurement of
the osmotic
pressure of dilute
salt-free
suspensions
ca. 95 % of
counterions
confined in brush
as predicted for
osmotic limit
water
membrane
∆
p <
0
pressure
solution
Antibiotics
13