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BIOPROCESS REACTOR
DESIGN
BPS 4105

ENZYME
 2
Enzyme

The fundamental part of biocatalysis

the BIOCATALYST itself
Biochemically a large complex of protein
ternary/quarternary structure
Undoubtedly the most important group
of substance in all living systems

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Enzyme Classification

Oxidoreductase
Catalyses redox reactions (ex: alcohol dehydrogenase)
Transferase
Catalyses functional group transfer reactions (ex: aminotransferase)
Hydrolase
Catalyses hydrolysis reactions (ex: protease, amylase, lipase)
Lyase
Catalyses removal/formation of a double bond with group transfer (ex: fumarase)
Isomerase
Catalyses isomerisation reactions (ex: glucose isomerase, triose phosphate isomerase)
Ligase
Catalyses single bond formation by eliminating the elements of water(ex: amynoacyl-transfer
RNA synthetase)
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Enzyme as Biocatalyst

Enzyme can dramatically reduce the activation energy of

reactions in the way that chemical catalysts can; enzymatically-
catalysed reactions may have rate 103-108 times faster than
uncatalysed ones
As a biocatalyst enzyme only promotes the reaction rate, not
shifting the equilibrium
Thermodynamically-non-spontaneous reactions will not
become spontaneous even by catalyst/enzyme addition
As a biomolecule, enzyme is more fragile than chemical
catalysts as it can only survive in limited environment conditions

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Factors that Influences Enzyme Ability
to Act as a Biocatalyst:

Answer in 5 minutes!

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Factors that Influences Enzyme Ability
to Act as a Biocatalyst:

Temperature
pH
Cofactors
Substrate suitability

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Enzyme

Enzymatic Reaction Kinetics

Enzymatic Reactor Systems

Immobilised Enzymes

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ENZYMATIC REACTION
KINETICS
 9
Chemical Reaction Kinetics

Governed by physico-chemical aspects

Generally annotated as r
The exact equation of r depends on the type of
reaction:
Elementary (species coefficients equal reaction order)
Complex
Most chemical reactions
Enzymatic
Biochemical

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Simple Enzymatic Reaction Kinetics

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Simple Enzymatic Reaction Kinetics

=
+
Through either fast equilibrium or pseudo steady-state approach, the
simplest form of enzymatic reaction mechanism is available.
This mechanism is known as Michaelis-Menten (fast equilibrium) or Briggs-
Haldane (pseudo steady-state) models
Values of rmax and Km constants are generally known as Michaelis-Menten
constants and can be evaluated using several options of techniques
using experimental data (Lineweaver-Burk, Hanes-Woof, etc.)

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Example 1

The following data is given for an experiment of enzymatic

glycylglycine hydrolysis rate with a certain enzyme loading:

Determine all Michaelis-Menten constants

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Example 1 - Solution

A. First, change the Michaelis-Menten equation

into a linear form easiest: Lineweaver-Burk
1 1 1
= = +
+ []

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Linearisation with Microsoft Excel

6000.00000

5000.00000 y = 4266.3x + 1997.8 Intercept:

R = 0.9928 1/rmax = 1997.8
1/r (min/mmol)

4000.00000 rmax = 5.01 x 10-4 mmol/min

3000.00000
Slope:
2000.00000 Km/rmax = 4266.3
Km = 2.135 mM
1000.00000

0.00000
0.000 0.100 0.200 0.300 0.400 0.500 0.600 0.700
1/S (1/mM)

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Linearisation with MATLAB

Intercept:
1/rmax = 1997
rmax = 5.01 x 10-4 mmol

Slope:
Km/rmax = 4266
Km = 2.135 mM

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More Complex Enzymatic Reactions

Multisubstrate reactions
Compulsory order
Random order
Allosteric enzymes
Inhibited enzymes
Competitive
Non-competitive
Uncompetitive
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Multisubstrate Reactions (A,B)

Compulsory Order
A binds before B
0
=
1 1
+ + +
1
+

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Multisubstrate Reactions (A,B)

Random Order
A and B binding are regarded as
happening simultaneously

0
=
+ +

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Allosteric Enzymes

Forenzymes with multiple substrate-

binding site (e.g. regulatory enzymes)

=
+

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Inhibited Enzymes

Competitive Inhibition

=

1+ +

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Inhibited Enzymes

Non-competitive Inhibition

=

1+ +

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Inhibited Enzymes

Substrate Inhibition

= = =
+ 2
+ + 1+
2 2

Low substrate conc. High substrate conc.

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ENZYMATIC REACTOR
SYSTEMS
 24
Enzymatic Reactor Systems

The enzymatic reaction rates have

been defined in previous slides
Reactor design cases vary according
to reactor type and modes
Scopes in this course: ideal reactors
Batch
CSTR
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The difference between reactor lies on the mass balance

CA
qin V qout
in rA out
CA,in d(VCA)/dT CA,out

, , + =

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BATCH ENZYMATIC
REACTOR
 27
Batch Reactor

No stream enters or exits the reactor during operation

CA
qin V qout
in rA out
CA,in d(VCA)/dT CA,out

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Mass Balance of a Batch Reactor

Previously, it is known in constant-density batch

reactors that:

=

In enzymatic reactions, species A is the

substrate, therefore:

=
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Mass Balance of a Batch Reactor

Mass balance of product depends on the

overall reaction stoichiometry
sS pP +qQ
Therefore, the mass balance of product(s)
become:

=


=
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conservation equations

= = =


=
+
constitutive equation

By solving these equations simultaneously, reactor design

problems can be solved.
Most of the time, it is easier to work on the substrate first and
the product(s) later

=
+ 21/11/2017
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Analytical Solution

Substrate mass balance:

=
+
Solving this differential equation results in:
[]0 0 1 0
ln + = or ln + =
[] 1
While this form is useful for calculations of reaction time for desirable
conversion, it is very inconvenient to use for other cases, e.g. when
conversion is to be evaluated for given reaction time
This can be solved numerically, using Newton-Raphson method or
other iterative methods 21/11/2017
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Numerical Solution

As reaction rate statements become more complicated,

analytical solutions become more inconvenient to use; even if
the solution exists, it is not very useful to use
Solution Numerical Solution why not go all the way?
MATLAB can be useful for solving systems of ordinary differential
equations (ODEs)
Basic numerical methods (Euler, Runge-Kutta)
Built-in MATLAB functions (ode45, ode23, etc., all based from Runge-Kutta
method)
Extremely useful for complex reaction kinetics that are normally impossible to
solve analytically 21/11/2017
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Example 2

A following enzymatic reaction

A 2B
Has the Michaelis-Menten saturation constant (Km) of 37.2 mM
and rmax = 1.78 mM/min with enzyme loading of 1 mM.
A. If this reaction is carried out in a 1 L batch reactor using
initial A concentration of 12 M and enzyme loading of 2
mM, determine how long the reaction must be carried out
to obtain 95% A conversion.
B. If this reaction is carried out with the same condition with
A., for forty hours, determine the A conversion and the
concentration of B.

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Example 1B-Solution

As previously known:
[]0 0 1 0
ln + = or ln + =
[] 1

By putting in all known values, the reaction time can be

calculated. In this case, the second form is easier to use.
Keep in mind that rmax is proportional to enzyme loading in
a linear fashion
Therefore, t = 53.89 hours
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Example 1C-Solution

Similar to 1B, as previously known:

[]0 0 1 0
ln + = or ln + =
[] 1
It is impossible to transform either equation so that [S]
becomes the variable of t
This equation can be solved using iterative method,
which in this case is demonstrated using GoalSeek in
Microsoft Excel, yielding XS=70.8% (guessed with
Xs,0=60%) 21/11/2017
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Calculating B concentration:
Amount of reacted substrate = 1/2 x amount of formed product

[A]0-[A] = 1/2([B]-[B]0)
Assuming no B exists in the beginning:
[B]=2([A]0-[A])= 17.00 mM

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Example 1C-Solution-Using MATLAB

In addition to analytical (plus a bit of numerical)

method, fully-numerical method can be used to
simultaneously finish this problem.
All known equations should be listed in their canonical
form first:

=
+

=2
+ 21/11/2017
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All known equations should be listed in their canonical

form first:

= =2
+ +
Boundary conditions:
t = 2400 min, [S]0 = 12 M, [P]0 = 0 M
In this example, ode45 function will be used in lieu of
manually-written Runge-Kutta method for easiness
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clear
clc

%Define Boundary Conditions%

tspan=[0 2400]; %reaction time = 2400 s 39
C0=[12;0]; %S0 = 12 M, P0 = 0 M

%Define ode45%
[t,C]=ode45(@reactorequations,tspan,C0);

%Define Conversion Statement

X=1-C(:,1)./C0(1,1); %X=1-(S/S0)
%Plot Result
figure
subplot(1,2,1)
plot(t,C(:,1),t,C(:,2)) %concentration profile
subplot(1,2,2)
plot(t,X) %conversion profile

%Define ODE Functions%

function dCdt=reactorequations(t,C) %X(1)=S, X(2)=P

%Define Constants
rmax=3.56e-3; %rmax = 3.56 mM
Km=37.23e-2; %Km = 37.23 mM

%Define Michaelis-Menten Kinetics

r=rmax.*C(1)./(Km+C(1)); %r=rmax*S/(Km+S)

%Define ODEs
dCdt=zeros(2,1); %2x1 zero matrix
dCdt(1)=-r; %mass balance of S
dCdt(2)=2.*r; %mass balance of P 21/11/2017
end
 40

Here, conversion of A equals 67.69% and final concentration of B

equals 16.25 M, which slightly differs from results from analytical
solution.
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CONTINUOUS STIRRED-TANK
ENZYMATIC REACTOR
 42
CSTR

Reactor is perfectly mixed:

All parameters (T, C, etc.) are homogeneous within mixture inside tank
Effluent condition is identical to that inside the tank
Steady state: no accumulation, equal flow rate for feed and effluent
Assume: constant density

CA
qin V qout
in rA out
CA,in d(VCA)/dt=0 CA,out

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Previously, it is known that:

0
= = 0 =

In enzymatic reactors this becomes:
[]0 []
= = []0 =

Finishingthis problem requires the statement of r,
which is already provided by the Michaelis-
Menten rate statement
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Therefore:
[]0 []
= = []0 =


+ +
While not actually mathematically complex, the same
problem from the batch model comes:
There is no problem to calculate residence time and
reactor volume if reactor conversion target is already
defined
However, it is much more complicated if conversion is to
be evaluated for certain residence time
This can be solved using numerical method (again), e.g. 21/11/2017
Newton-Raphson method