Marek Bryjak
Część II
Zakład Materiałów Polimerowych i Węglowych
Bud. H6, pokój 105
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
5
Co to jest enzym?
•
Białko
(Struktura 3 i 4
rzędowa)
• Katalizator
(przyspiesza
reakcje)
8
Induced Fit
• A change in the
shape
of an
enzyme’s active
site
• 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
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
Ener
gy
req
uired
(n
o
cat
al
ysi
s)
Ener
gy
decr
eases
(u
nd
er
cat
al
ysi
s)
T = Transition state
Adapted from Alberts et al (2002) Molecular Biology of the Cell (4e) p.166
Active Site Is a Deep Buried Pocket
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
Enzyme Active Site Is Deeper than Binding Site
Adapted from Nelson & Cox (2000) Lehninger Principles of Biochemistry (3e) p.252
H
O
H
Acid-Base Catalysis
Induced to transition state
C
O
=
N
H
H
C
H
N
H
+
C
-
O
O
H
O
H
-
d
+
d
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
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
Check for
C-terminal
Site for
specificity
Active
site
Substrate
peptide
chain
R
N
C
N
C
COO
-
O
-
C
+
Zn
Ju
a
n
g
RH
(20
0
4
)
B
Cba
sics
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
Specificity
Site
Catalytic Site
Juang RH (2004) BCbasics
Specificity of Ser-Protease Family
COO
-
C
Asp
Active Site
Trypsin
Chymotrypsin
Elastase
cut at Lys, Arg
cut at Trp, Phe, Tyr
cut at Ala, Gly
Non-polar
De
ep
and
nega
tiv
ely
ch
ar
ged
poc
ke
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
Ju
a
n
g
RH
(20
0
4
)
B
Cba
sics
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
b
b
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
Ju
a
n
g
RH
(20
0
4
)
B
Cba
sics
1
2
Increa
se
Sub
strate
Concen
trat
ion
2
1
3
4
5
6
7
8
0
0 2
4
6 8
Substrate (
m
mole)
Prod
uc
t
80
60
40
20
0
S
+
E
↓
P
(in a
fixed
period
of
time)
Juang RH (2004) BCbasics
Essential of Enzyme Kinetics
E
S
+
P
+
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
Conc
en
tra
tio
n
Juang RH (2004) BCbasics
v =
V
max
[S]
K
m
+
[S]
(
v
)
E + S
ES
E + P
k
2
k
1
k
3
For [substrate] low,
k
3
=
k
cat
Problem: Adsorption on solid surfaces has to be avoided!
M. Santore et al., Langmuir 2002, 18 (3), 706.
Adsorption on solid surface
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
a
0
20
40
60
80
100
0
3
6
9
pH
a
kt
y
w
n
o
ść
Aktywność enzymu immobilizowanego
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)
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
CH
CH2
COO-
CH
CH2
SO
3
-
+
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.
UF
low ionic strength
high ionic strength
Wittemann et al., J. Am. Chem. Soc. 2005, 127, 9688.
Cryogenic transmission electron microscopy (Cryo-TEM)
Localisation of adsorbed protein cont‘d
Ribonuclease A
Bovine hemoglobin
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
„Nanoplant“
Cascade reactions:
Possible system:
-Amylase:
starch
maltose
b
-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
Antibiotics
