CHEM-E3140 Bioprocess technology II
Lecture 1
27.10.2015
Tero Eerikäinen
Course program
Date
Time
Tue 27.10. 14:15-15:45
Fri 30.10. 10:15-11:45
Tue 3.11. 14:15-15:45
Fri 6.11. 10:15-11:45
Tue 10.11. 14:15-15:45
Thu 12.11. 10:15-11:45
Fri 13.11. 10:15-11:00
Tue 17.11. 14:15-15:45
Thu 19.11. 10:15-11:45
Fri 20.11. 10:15-11:45
Tue 24.11. 14:15-15:45
Thu 26.11. 10:15-11:45
Fri 27.11. 10:15-11:45
Tue 1.12. 14:15-15:45
Thu 3.12. 10:15-11:45
Fri 4.12. 10:15-11:45
Place Topic
KE4
Introduction, Industrial enzymes, enzyme kinetics (Tero Eerikäinen)
KE4
Enzyme discovery and engineering (Ossi Turunen)
KE4
Enzyme reactions and mechanisms (Ossi Turunen)
KE4
Industrial immobilized enzyme, sugar conversions (Tero Eerikäinen)
KE4
Heterogeneous reactions (Tero Eerikäinen)
Class 2&3 Reaction kinetics exercises (Tero Eerikäinen)
KE4
Enzyme reactors design and operations1- (Tero Eerikäinen)
KE4
Enzyme reactors design and operations2 - (Tero Eerikäinen)
Class 2&3 Heterogenic reactions exercises (Tero Eerikäinen)
KE4
Development of pretreatment technology and enzymatic hydrolysis for biorefineries (Anne Kallioinen)
KE4
Enzymes in non-conventional environments: solvents, inhibitor solutions, ionic liquids, DES (Ossi Turunen)
Class 2&3 Inspection of enzymes from 3D perspective; Computer class 2 (Ossi Turunen)- Group I
KE4
Optimization of enzyme reactor operation (Tero Eerikäinen)
KE4
Effect of lignin structure on enzymatic hydrolysis of plant residues (Mika Sipponen)
Class 2&3 Inspection of enzymes from 3D perspective; Computer class 2 (Ossi Turunen)- Group II
KE4
Lignin-modifying enzymes: Solid-state production and application in dye decolorization and enzymatic lignocellulose hydrolysis (Ulla Moilanen)
Student
presentation
1
2
3
4
5
6
Course material
- Course e-book:
Problem Solving in Enzyme Biocatalys
-lecture slides
-additional articles and texts
-exercises
Student presentations:
-4 member per group
-4 pages summary
-15 min presentation 10 min discussion
-Article numbers correspond
presentation date in course program
Participants
Ayyanar Ramachandran
Büscher
Didierjean
Isaza Ferro
Kataja
Kaustara
Koponen
Kruus
Kurula
Leivo
Mughal
Multia
Nai
Neumann
Pastinen-Kurula
Plagnat
Qian
Roman
Sartori
Tajuddin
Tan
Wang
Wegelius
Praniesh
Niclas
Laura
Estefania
Kim Mikael
Sanna Katariina
Tino M
Satu Maria
Simo Ilmari
Jimi Juho Waltteri
Muhammad Irfan
Evgen
An Ni
Eric André
Hilkka Ilona Emilia
Laura Marie
Chun-Man Franco
Veronica Lorena
Nicolas
Ibrahim
Hui En
Yang
Oskar Enri Matias
Introduction: enzymes
http://blogs.scientificamerican.com/guest-blog/enzymes-the-little-molecules-that-bake-bread/
Reactions of metabolic networks in living organism
are catalyzed by enzymes
https://physics.aps.org/articles/v4/63
http://science.jrank.org/article_images/ep201102/science/science4253.png
http://cdn.phys.org/newman/gfx/news/hires/2012/1-promiscuouse.jpg
Genetic and environmental regulation
http://pubs.rsc.org/en/content/articlehtml/2013/mb/c3mb25489e
Highly evolved and complex
http://www.weizmann.ac.il/Biological_Chemistry/scientist/Tawfik/
http://phys.org/news/2013-03-insight-biochemical-methane-production.html
http://scitechdaily.com/artificial-enzyme-reveals-a-never-before-seen-structure/
Specific, efficient, precise
https://naribiochemwiz007.wordpress.com/2013/04/11/about-enzymes/
http://www.jayreimer.com/TEXTBOOK/ebook/products/0-13-115516-4/sb4189f1.png
Active site: small portion of the structure
/
https://rhodopsinreader.wordpress.com/2013/04/13/enzymes
http://alevelnotes.com/Enzymes/144?tree=
Collision theory, activation energy
• The collision theory states that
chemical reactions can occur when
atoms, ions, and molecules collide
• Activation energy is needed to
disrupt electronic configurations
• Reaction rate is the frequency of
collisions with enough energy to bring
about a reaction.
• Reaction rate can be increased by
enzymes or by increasing
temperature or pressure
From Pearson Education, Inc.
Enzymes
•Apoenzyme: protein portion of an
enzyme without cofactor
•Cofactor: Nonprotein component, help
catalyze by forming a bridge between
the enzyme and the substrate
•Coenzyme: Organic cofactor,
NAD+NADP+FAD Coenzyme A, may
assist the enzymatic reaction by
accepting atoms removed from the
substrate or by donating atoms
required by the substrate, electron
carriers
•Holoenzyme: Apoenzyme + cofactor
(coenzyme)
From
Pearson
Figure
5.3 Education, Inc.
Natural vs. process environment
• Biological catalysts separated from the cellular environment
• How about performance under nonphysiological conditions?
• Specificity, reactivity, stability
• Complex, labile
• Stabilization strategies
Applications
• Wide spectrum from industrial processes to molecular biology
• Chemical and clinical analysis, diagnostic kits, sensors
• Therapy, precise and efficient removal of unwanted metabolites
• Waste treatment and bioremediation.
• Cost factors
• Biotechnological research and development. Thermostable
polymerases, restriction enzymes.
Enzyme Classification
6 classes, named for type of chemical reaction they catalyze
• Oxidoreductase
• Transferase
• Hydrolase
• Lyase
• Isomerase
• Ligase
Oxidation-reduction
reactions
Transfer functional groups
Hydrolysis
Addition or removal of groups
without hydrolysis
Rearrangement of molecules
Joining of molecules, uses ATP
General classification of enzymes based on
their catalytic functions
EC numbers by IUBMB (INTERNATIONAL UNION OF
BIOCHEMISTRY AND MOLECULAR BIOLOGY)
Sarrouh et al., J Bioproces Biotechniq 2012, S:4
http://dx.doi.org/10.4172/2155-9821.S4-002
Examples of different types of enzymes used in industry
http://nptel.ac.in/courses/103103026/module3/lec35/4.html
List of commercial enzymes from genetically modified
microorganisms used in food industry
Sarrouh et al., J Bioproces
Biotechniq 2012, S:4
http://dx.doi.org/10.4172/21
55-9821.S4-002
Enzymes involved in lignocellulose degradation
and their mode of action
Sarrouh et al., J Bioproces
Biotechniq 2012, S:4
http://dx.doi.org/10.4172/21
55-9821.S4-002
Industrial enzyme
production (technical +
food and beverage) in
2014 worth 3,1 G€ (23 G
DKK). Growth from 2013
about 5 %
Other enzymes:
about 1,8 G€
10/27/2015
Source: Novozymes/Annual Report 2014
20
Enzyme kinetics (homogeneous system)
• Catalytic potential: activity
• Reduction of energy barrier magnitude
• Maxiumum catalytic potential (Reaction rate)  Initial rate
• Rate decrease due to:
• Substrate desaturation, enzyme inactivation, equilibrium displacement,
product inhibition
• Enzyme activity can be determined e.g. by substrate (S) consumption
or product (P) generation (or some more detectable analyte or
coenzyme):
Energy Requirements of a Chemical Reaction
Figure 5.2
From Pearson Education, Inc.
Rate measurement
• Initial rate
• Adjust the enzyme concentration
• Linearity over a reasonable sampling time
• Exceptions with chemically ill-defined substrates, e.g.:
• Cellulase enzyme complex includes endo- and exoglucanase activities
• In the beginning amorphous portion of the cellulose complex is “easier” than
crystalline portion of cellulose at a later stage of reaction
•  filter paper activity assay
Enzyme activity units and measurement
• The Enzyme Commission (EC) of the International Union of Biochemistry
recommends using international units (IU).
• One IU = µmol/min
• amount of enzyme that catalyzes the transformation of one micromole of substrate
per minute under:
• standard pH and T, compromise between activity and stability
• optimal substrate conc., at least 5 times Km-value
• SI units expect moles per second  katal=mol/s
• Disadvantage: numerical values are very small
• Reaction rates from: the product, the substrate, a cofactor, a coupled
analyte.
• economic considerations, accuracy, and simplicity.
• spectrophotometer, fluorometer, polarimeter/RI detector, viscometers, manometes
Basics of kinetics
• Quantitative evaluation of all factors that condition enzyme activity
• Most important factors:
• concentrations of active enzyme, substrates and inhibitors
• pH, and temperature
• Kinetics needed for
• understand the molecular mechanisms of enzyme action
• design of enzyme reactors and for
• performance evaluation
• Determine initial rates of reaction
• Determine the quantitative effect of the important factors
Activity proportional to the concentration of active enzyme
Enzyme kinetics
S+E
k1
k2
ES
k
P+E
dS dP
=
= k ⋅[ ES ] = k cat ⋅[ ES ]
v=−
dt
dt
kE0 S Vmax S
=
v=
k2
+S K +S
k1
Michaelis - Menten
Vmax S
kE0 S
=
v=
k2 + k
+S KD +S
k1
Henri kinetics
K1 dissoc. const ES
K2 dissoc. const EP
P=0 or insignificant
Rapid equilibrium
K= equilibrium constant
The binding step in equilibrium and
much faster than the conversion step
Briggs - Haldane
Steady state hypothesis
KD = dissociation constant
Briggs-Haldane apparent steady state
After a very short transient state the enzyme–substrate complex reaches steady state, so that its
concentration remains constant throughout the reaction
(
[
E0 ]−[ES ])[S ]
[ES ]=
v = k [ES ]
KD
ES formation = k1 [E ][S ]
ES dissociation = (k 2 + k )[ES ]
In steady state:
ES formation = ES dissociation ⇒
k1 [E ][S ]= (k 2 + k )[ES ]
[ES ]=
[E ][S ]
(k 2 + k ) k1
[E ]= [E0 ]−[ES ]
d [ ES ]
=0
dt
[
E ][S ]
=
KD
KD is the dissociation constant of the ES complex into E and S
[ES ]K M + [ES ][S ]= [E0 ][S ]
[
E0 ][S ]
[ES ]=
K D + [S ]
[
E0 ][S ]
[
S]
=Vmax
v=k
K D + [S ]
K D + [S ]
Notation in
course book:
[E0]=e
[ES]=c
[S]=s
when S >> K D →
v =Vmax = k [E0 ] (0 order reaction kinetics)
when S << K D →
v =Vmax
[S ]
KD
(1st order reaction kinetics)
KM and Vmax
Whatever the hypothesis, the rate equation is expressed in terms of two parameters:
KM is “Michaelis constant,” (not being dependent on either enzyme or substrate concentration)
Vmax is a lumped parameter containing the enzyme concentration (e or [E0]) and the catalytic rate constant (k or kcat).
(dimension of k or kcat will be determined by the enzyme concentration dimension)
When defining kinetic parameter, the value of the determined parameter KM should be in the midpoint of that
range. KM corresponds the substrate concentration in which reaction rate in half of the maximum.
Irreversible
reaction
18,0
16,0
14,0
v (g/l s)
12,0
Km =10
v = (Vmax*s)/(Km+s)
Vmax = 20
10,0
8,0
6,0
4,0
Km
2,0
0,0
0,0
20,0
40,0
s (g/l)
60,0
With small substrate concentration 1st
order kinetics
With large substrate concentration zero
order kinetics
Single substrate reactions
• If reversibility is important (e.g. isomerization) the steady-state
hypothesis should be used to develop kinetic expressions:
Try to verify this from the previous
equations and following parameter
definitions:
Enzyme inhibition
• Competive
• Noncompetive
• Mixed-type
• Uncompetive
• Kinetic parameters apparent, dependent on the
inhibitor concentration
Factors Influencing Enzyme Activity
• Competitive inhibition – inhibitors fill the active site of
an enzyme and compete with the normal substrate for
the active site
FromFigure
Pearson
Education,
Inc.
5.7a,
b
Factors Influencing Enzyme Activity
Ex – PABA is an essential nutrient of many bacteria
in the synthesis of folic acid. Sulfanilamide binds to
the enzyme that converts PABA to folic acid,
bacteria cannot grow
From Pearson Education, Inc.
Factors Influencing Enzyme Activity
• Noncompetitive inhibition- inhibitor interacts with
another part of the enzyme
• Allosteric inhibitor –inhibitor binds to a site other than
the substrate binding site and cause the active site to
change shape making it non-functional
FromFigure
Pearson
Education,
Inc.
5.7a,
c
Factors Influencing Enzyme Activity
• Feedback inhibition –
a series of enzymes
make an end product
that inhibits the first
enzyme in the series,
this shuts down the
entire pathway when
sufficient end product
has been made
From Pearson
Inc.
FigureEducation,
5.8
Apparent kinetic parameters
• Rate equations of
inhibition in the
parametric form of
the Michaelis–
Menten equation:
Factors Influencing Enzyme Activity
• Enzymes can be denatured by temperature and pH
From Pearson
Inc.
FigureEducation,
5.6
Effect of T and pH on enzyme kinetics
• Enzymes are polyionic polymers, pH
• affects the ionization stage in aqueous media
• has no meaning in nonaqueous media
• Temperature
•
•
•
•
affects enzyme activity and enzyme stability
affects the rate of any chemical reaction
affects the rate of enzyme inactivation
Balance needed
pH and kinetics
• The active site of the enzyme exists in
three ionic configurations of successive
numbers of charges
• Only the intermediate ionic species is
active, being able to bind and transform
the substrate
• n denotes the number of charges at the
active site (arbitrarily set as negative) and
Ki are the ionic equilibrium constants of
the free enzyme (E) and the enzyme–
substrate complex (ES)
• Plotting log VmaxAP versus pH (log[h+])
and log(KMAP /VmaxAP) versus pH, all
kinetic parameters be determined
Temperature and kinetics
•
•
•
•
•
•
The temperature that maximizes activity is harmful for the stability
Normally enzyme must be active for an extended period of time
Determination of optimum process temperature is a complex task
KMAP can be obtained as temperature-explicit function
Catalytic rate constants from the semi-empirical Arrhenius equation
Similar equations can be derived for inhibition reaction
Factors Influencing Enzyme Activity
• Temperature
pH
From Pearson
Inc.
FigureEducation,
5.5a
Multiple substrate reactions
• Sequential,
oscillatory or
pingpong mechanism
• Expressing the rate
with respect to
substrate A,
parameters are a
function of the
concentration of B
and vice versa.
Multi-enzyme reactions
one-pot reaction
intermediates do not require isolation or purification
downstream operation costs are reduced significantly
conditions that are not optimal for any of the enzymes but are the result of
a compromise between them all
• (a) the cascade scheme (b) parallel reactions (c) the network pattern
• Due to nonlinearity solution usually with numerical methods
•
•
•
•
Parameter determination
• Series of measurements of initial reaction rates at different concentrations
of substrates and effectors
• Linearization of the rate data or by nonlinear regression
• A geometric progression of substrate concentration
• Use a substrate concentration range that contains the value of the
dissociation constants
• The kinetic parameters to be determined by linearization of the integrated
equation
Linearization methods
• Mechanism of inhibition be detected with a doublereciprocal plot (Lineweaver-Burk)
• When using varying inhibitor concentrations:
• competitive inhibition will yield a common intercept
in the y-axis
• noncompetitive inhibition will yield a common
intercept in the x-axis,
• mixed-type inhibition will yield lines intersecting at
some point in quadrant II,
• Uncompetitive inhibition will yield parallel lines.
• The double reciprocal plot is unreliable for the
determination of enzyme kinetic parameters
• Use non-linear estimation or other linearization forms
like Eadie-Hofstee, Langmuir, etc.
[S ] = [S ]
v
v max
Km
+
v max
v = v max − K m
v
[S ]
Langmuir
Eadie − Hofstee
http://www.slideshare.net/sandipayan/sandipayanenzyme-inhibitionseminar
K 1
1
1
=
+ m
v v max v max [S ]
Lineweaver − Burk