Experiment D.

Kinetics of Free and

Immobilized Enzyme Systems

이름: 윤초록

학번: 20180422
 1. Introduction

Enzymes are biomolecules that catalyze numerous chemical reactions that are including

biological reactions. They are designed to be highly substrate-specific so they can gain

maximal efficiency in the environment. Also, enzyme involving reaction usually have higher

reaction rates than inorganic catalyst case under same conditions. With enzyme-catalyzed

reaction, we want to know enzyme kinetics model. With enzyme kinetics, we can identify

how fast the reaction proceeds and how long activity maintains. Also, we can determine

which enzyme will be used, how many enzymes will be used and how long the reaction will

take based on enzyme kinetics. Our purpose is to express enzyme-catalyzed reaction rate

mathematically. In this experiment, we will compare catalytic activity of free enzyme and

immobilized enzyme by using the hydrolysis reaction of 4-nitrophenyl butyrate catalyzed by

lipase. 4-nitrophenolate, product, adsorbs at 400 nm in maximum while starting substrate is

transferred for such wavelength. Therefore, using spectrophotometer we can know how far

the reaction has progressed. First, we will make immobilized enzyme beads by using Ca-

alginate gel beads. Then, we will measure the absorbance of both free and immobilized

enzyme systems using spectrophotometer. After that, we will make a plot about the double-

reciprocal graphs and compare the obtained data. Lastly, we can calculate and analyze

enzyme kinetics (Vmax and Km) based on the plot.

In this experiment, our purpose is comprehensive understanding on enzyme/enzyme kinetics

and immobilization of enzymes using sol-gel entrapment with alginate. We can learn why and

how to immobilize enzymes. Also, we can learn how to calculate kinetic constants of

free/immobilized enzymes based on enzyme kinetics model.

 2. Theoretical Background

Enzymes are biomolecules that catalyze numerous chemical reactions that are including

biological reactions. Most of enzymes are included in proteins. In reactions concerned with

enzymes, the molecules which exist at the beginning are called substrates, and different

molecules which enzyme converts into different formation are called products. Substrates

such as starch, protein, lipid, and cellulose for enzymatic reaction, mostly derive from living

organisms. Enzymes perform various functions at interior living organisms. For example,

enzymes synthesize/decompose substances. Then, reaction velocity concerned with each

enzyme with proper substrates is different. There are some factors that can affect the enzyme

reaction velocity Intrinsic properties between substrates and enzymes and diverse inhibition

factors like substrate inhibition can affect the velocity. Also, enzymes are designed to be

highly substrate-specific so they can gain maximal efficiency in the environment.

2-1. Enzymes / biological reaction system related with enzymes

Enzyme is bio-macromolecule that can act as specific, able to be adapted to many different

functions, and very effective biological catalyst. Generally, enzymes are polymer-protein. It

means that most enzymes are protein of tertiary / quaternary structures with one or more

subunits. Enzymes have organic part as well as non-protein part such as cofactors or

coenzymes that greatly contribute to activation of enzymes. Enzymes are named as like ‘their

own substrate/the reaction which they catalyze’ + ‘ase’. For example, lipase is an enzyme that

hydrolyzes a lipid, ‘Lip’ (substrate) + ‘ase’. Alcohol dehydrogenase catalyzes

dehydrogenation (reaction) of alcohol (substrate). There are also enzymes that are not

proteins. These substances can act as enzymes. For instance, RNA can act as enzyme, then it
is called ribozyme (RNA+-zyme). It is important that enzyme is a catalyst so it can lower the

activation energy of corresponding reactions. With Figure 1, we can compare two graphs (a),

and (b). We can clearly figure out the energy hurdle have lowered when enzyme is used. We

utilize enzymes in order to make reaction rate faster.

Figure 1. Energy graph of reaction; (a) with no enzyme (b) with enzyme

Technically speaking, enzyme is a subset of catalyst in principle. However, we use term

‘catalyst’ for inorganic catalyst case (non-enzymatic catalyst) in general. Also, enzyme

typically have higher molecular weight than inorganic catalyst, and have specific binding to

substrate in contrast with inorganic catalyst.

Most industrial catalysts are metals. Most metals have many electrons which are a little

cavalier about exactly how close to the central atom they need to be. Metals and their

compounds can function as catalysts. Because they have either ability to change oxidation

state of substrate (transition metal) or ability to adsorb other substances on surface and

activate them in the process of reaction. Biological catalysts work on a very different

principle in contrast with metals with fast-and-loose electrons. Biological catalysts like
enzymes contain specific pockets for their substrates to fit into. Once substrates are trapped

inside the enzyme, enzyme promotes the reaction either by forming temporary bonds with

substrates to help them fit together, or by simply holding substrates close enough to interact

with each other for actual reaction and converting into the product. Most enzymes are

discovered inside organisms so enzymes generally do not need high temperatures to function

as catalyst while metal catalysts tend to need a bit of an energy kick to function. Also,

enzymes are incredibly specific unlike catalysts. As the substrates fit into active sites inside

the enzyme, the enzyme can only fit these such molecules when catalyzing.

Therefore, enzyme involving reaction usually have higher reaction rates than catalyst case

under same conditions.

We can summarize these contents in table (Table 1).

Catalyst Enzyme

Inorganic catalyst Organic/bio catalyst

Non-specific binding Specific binding to substrate

Low molecular weight High molecular weight

Table 1. Major Difference Between Catalyst and Enzyme

In Figure 2, an enzyme, E is specifically binding to a substrate S so that they form an ES

complex as a reaction intermediate. After such an enzyme catalysis, the reaction makes

product, P and P is released.

 Figure 2. Steric Image of enzyme-catalyzed reaction

There are various types of enzyme. The Table 2 shows 6 representative classes of enzyme.

Class work Example Reaction catalyzed

Alcohol dehydrogenase removes hydrogen from alcohols
Catalyzing
Oxidoreductases oxidoreduction
reaction

Catalyzing Hexokinase (kinase gets involved in phosphorylation)

transfer of
phosphates glucose.
functional groups
such as methyl
Transferases
group or glycosyl
group from one
compound to
another compound
Catalyzing Carboxypeptidase A breaks peptide bond
hydrolytic
Hydrolases cleavage of
amide bond or
other bonds
Catalyzing breaking
of various chemical
Pyruvate decarboxylase removes carboxylic group from
bonds by means
other than pyruvate
Lyases hydrolysis and
oxidation
Often forming a new
double bond or a
new ring structure.
Maleate isomerase involves in cis to trans isomerization of
Catalyzing Maleate.
geometric or
Isomerases
structural change
of molecule

Catalyzing Can be coupled with hydrolysis of diphosphate or

joining of two triphosphate form of ATP.
Ligases molecules by Pyruvate carboxylase
forming a new
chemical bond
Table 2. Classification of Enzyme
 Biological reaction system related with enzymes

There are several steps in modeling biological systems (Table 3).

Step Factors Modeling Example

1 Single substate, enzyme Enzyme reaction Michaelis-Menten

Complex enzyme
2 Complex substrates, enzymes Single cell model
reactions in a cell

3 Single substrate, cell Fermentation Monod equation

Complex Anaerobic digestion

4 Complex substrates, cells
fermentation reaction of organic substrates

Production of useful
5 Substrates, cells, reactor Biological reactor compounds,
wastewater treatment

Process
Substrates, fermentation,
optimization, Design
6 separation and purification, energy Biological plant
of new biological
consumption, economic evaluation
plant

Table 3. Interaction among substrate, enzyme, and cell following several modeling steps

2-2 Michaelis-Menten kinetics

The objective of enzyme kinetics is identifying how fast the reaction proceeds and how long

activity maintains. We can know which enzyme is used, how many enzymes are used and

how long the reaction will take based on enzyme kinetics. Our purpose is to express enzyme-
catalyzed reaction rate mathematically. There was a first mathematical modeling enzyme

kinetics of single-substrate-enzyme-catalyzed reactions. It was developed by V.C.R. Henri in

1902, and by L. Michaelis and M.L. Menten in 1913. We can call this simple enzyme kinetics

modeling as Michaelis-Menten kinetics or saturation kinetics.

Michaelis-Menten kinetics is most famous one of models of enzyme kinetics. Michaelis-

Menten equation describes the rate of enzymatic reaction with [S] ([S] means the

concentration of a substrate S). This model comes from an idea that if the formation of ES

complex quickly reaches equilibrium, then enzymatic reaction can be described in terms of

[S] using equilibrium constant.

We need to clarify reaction rate. If the reaction is

S→P

the reaction rate v with quasi-steady state approximation, is defined by

−dS dP
v= =
dt dt

As a result, the unit of v is mole ∙ volume−1 ∙time −1 . The reaction rate is an intensive quantity,

which means that reaction rate is independent of the system size, defined at each point in the

process. The reaction rate v can be affected by substrate, product, enzyme, temperature, pH,

etc.

There is a typical experiment for enzyme kinetics measurement. It proceeds as follows; At

the beginning, solutions containing a type of substrates and its appropriate purified enzymes

are mixed in a closed isothermal, well-stirred vessel containing a buffer solution that is for

controlling pH. The concentrations of substrates and/or product are recorded periodically.
There are some techniques for following the time progress of the reaction including

spectrophotometric, polarimetric, electrode, and sampling-analysis methods. In general,

initial-rate data are only used. Since the reaction conditions taking in enzyme and substrate

concentrations are known as the best at time zero (t = 0), the initial gradient of (concentration

of substrate or product vs. time) curve is estimated from the data. In the enzyme-catalyzed

reaction, it is essential to express an enzyme unit. Units or units of activity refer the amount

of enzyme which shows a certain amount of catalytic activity under standard conditions for

such particular enzyme.

This Michaelis-Menten kinetics can be taken from a simple reaction scheme (Figure 3) that

includes a reversible step ES formation and a separation step of the ES complex.

Figure 3. Enzymatic Reaction Formula

In Figure 3, an enzyme E binds to a substrate S and forms a complex, ES followed by

product releasing and recovery of the original enzyme. k denotes a reaction rate constant.

Since we assume that complex formation is much faster than product formation and the rate

of reverse reaction of the step 2 is ignorable, we can set the letter 1 as rate-determining step.

When accumulation of product is ignorable at beginning of reaction, we can assume that

second reaction irreversibility often fits.

dP
Then reaction rate v can be described like this; v= =k 2 [ ES]
dt

Then, we do not know the concentration of ES. Thus, we need to substitute by some
measurable factors.

K eq =exp ( −∆RTG )= [[PS ]]

eq
eq

If S → P is irreversible, ΔG should be smaller than 0, so that K eq would have very huge

value. ([P]eq >> [S]eq). As a result, the that S → P is irreversible means that the forward

velocity is much higher than reverse velocity.

d [ P]
v= =k 2 [ ES ]
dt
d [ ES ]
=k 1 [ E ] [ S ] −k−1 [ ES ] −k 2 [ ES ]
dt
[ E ] =[ E o ] −[ ES ]

d [ P]
[P] is proportional to [ES] (v= =k 2 [ ES ]) and the [E] has not connected to substrate (
dt
[ E ] =[ E o ] −[ ES ] ).

Now, we should substitute concentration of ES by some measurable factors.

There are two major approaches used in developing a rate expression; (a) quasi-steady-state

approach (b) rapid equilibrium approach.

a. Quasi-steady state assumption

Under quasi-steady state assumption, the concentration of ES complex does not change on

the time scale of product formation. In most experimental systems, a closed system is used in

d [ ES ]
which [S]0 greatly exceeds the [E]0. Since [E]0 was small, [S]0 >> [E]0, =0 and then,
dt
k 1 [ E ] [ S ]−k−1 [ ES ] −k 2 [ ES ] ≅ 0

[ E ][ S ]
This makes [ ES ] =k 1
k −1 + k 2

Now, we do not know [E] so we need to rewrite [E] with measurable values.

[ E ][ S ] [ E0 ] [ S ]
When we balance [ ES ] =k 1 with [ E ] =[ E ]0− [ ES ], we can get [ ES ] = .
k −1 + k 2 Km+ S

Then, Km (Michaelis-Menten constant) = [k-1+k2]/k1.

b. Rapid equilibrium assumption

Under the assumption that a rapid equilibrium between the E and S to form an ES complex,

we can use the equilibrium coefficient to express [ES] in terms of [S]. The equilibrium

' k−1 [ E ][ S ]
constant is K m = = . We can balance that equilibrium constant with [ E ] =[ E o ] −[ ES ] .
k1 [ ES ]

[ E0 ] [ S ]
Then, [ ES ] = . Then, Km’ (dissociation constant of the ES complex) = k-1/k1.
K m +[S]

If the value of Km' is high, it means that [E] >>[ES]. If the value of Km' is low, it means [ES]

>> [E]. In conclusion, the lower value of Km' is, higher affinity of the enzyme to the

substrate is. When we deal with complex enzyme kinetics, this (b) rapid equilibrium

assumption is preferred to (a) quasi-steady-state assumption so Km' will be used in these

cases.

c. Enzyme kinetics
 d [ P]
The rate of product formation (v = ) with the two assumptions (a), and (b);
dt

d [ P] [ E0] [ S]
v a= =k 2
dt K m+[S ]

d [ P] [E0] [ S ]
v b= =k 2
dt K m '+[S ]

d [ P] [ E 0] [ S] [ E ][ S ] [ E][S]
We focus on v= =k 2 . In previous a. content, [ ES ] =k 1 , Km= .
dt K m+[S ] k −1 + k 2 [ES ]

Then, higher Km means that lower concentration of [ES]. Therefore, if value of Km is high, it

means that affinity between enzyme and substrate is lower.

When we express Vmax = k2[E]0, Vmax reflects how fast enzyme can catalyze the reaction.

There can be also another constant k2/Km (catalytic efficiency) is a measure of how efficiently

an enzyme converts a substrate into product.

d [ P] [ E 0 ] [ S ] V max [ S ]
In summary, we can v= =k 2 = .
dt K m+[S ] K m +[ S]
 Figure 4. Enzyme reaction by Michaelis-Menten kinetics

In Figure 4, we can observe features referred to Michaelis-Menten kinetics (Mathematical

V max [ S ]
equation is v= ). According to Figure 4, the maximum reaction velocity is Vm. Vm is
K m +[S ]

dependent on addition of enzyme, but the addition of substrate does not affect Vm (Vmax =

k2[E]0).

Also, reaction order is dependent on the relative size of the two terms, Km and [S].

V max [ S ]
v=
K m +[S ]

If Km is significantly lower than [S], we can neglect the Km of denominator. The reaction

velocity will be maximal value regardless of the amount of substrate.

If [ S] ≫ K m ,then v=V max [ zero−order reaction ]

If Km corresponds to the S, it results in the half-maximal reaction velocity.

V max [ S ] 1
If [ S ] =K m , then v= = V
[ S ] + [ S ] 2 max
If Km is significantly larger than [S], we can neglect the [S] of denominator. Below equation
is the result

V max
If [ S]≪ K m ,then v= [S ] [ 1 st order reaction ]
Km

M-M Kinetic constants Vmax and Km

Since the reaction order of [S] varies, it is difficult to find reaction constants. However there

is a simple trick for finding constants by taking reciprocal formation on both sides.

V max [ S ] 1 1 Km
v= → = +
K m +[S ] v V m V m [ S ]

1 1
Then, and has a linear relationship, which makes analysis more convenience. This
v [S ]

double-reciprocal plot is also known as Lineweaver-Burk plot.

Figure 5. Double-Reciprocal Plot

 1 1 1 1 Km
A plot of versus shows a linear line (Figure 5). With equation = + , the
v [S ] v Vm Vm[S]

Km 1 −1
value of slope is , y-axis intercept is , and x-axis intercept is . Such a double
Vm Vm Km

reciprocal plot gives good estimates on V m, however it is not necessarily on K m. Because the

error about reciprocal form of a data point is not symmetrical, the reader should take a

treatment carefully in applying regression analysis for such plots. Data points at low [S]

influence the value of slope and intercepts more than those ones at high [S].

Other analyzing methods to get kinetic constants can be utilized. For example, we can use

Km V
Eadie-Hofstee plot (V =V m− ), Hanes-Woolf plot ¿). This experiment focuses on
[S]

calculating kinetic constants, Km and Vm, using the Double-Reciprocal Plot in

free/immobilized enzyme systems and will get such reaction activities

2-3. Immobilized-enzyme technology

Enzyme immobilization is to restrict the enzyme mobility to a confined space. Compared to

free enzyme system, immobilized enzyme system has a several advantages. By applying the

immobilized system, it becomes much easier to reutilize enzymes. It does not require

additional process for recovery and purification of enzyme. Because enzymes are generally

expensive, we can have economic advantage with catalyst reuse for many processes. Also,

this system offers improved stability to heat, pH, organic solvent, protein degeneration,

protein break-down enzyme. This system may supply a better environment for enzyme

activity. In addition, a model system to imitate and understand the action of membrane-bound
enzymes can be described through immobilized enzymes because some of the intracellular

enzymes are membrane-bound. Product purity is usually improved, and effluent treatment

problems are minimized through immobilization.

However, enzyme molecules would be changed through immobilization. 3D structure of

enzyme is very important for substrate-specificity. Therefore, change of enzyme can lower

the activity of enzyme. Therefore, it is important to understand the physical/chemical

property variations which an enzyme would be anticipated to get upon immobilization. There

are several factors that influence the intrinsic enzyme-catalytic activities. Majorly because of

their mass transfer limitation, functional features can also be changed independently on their

intrinsic features.

2-4. Methods of immobilization

There are two types of immobilization method

(a) Physical methods

Adsorption is one of the physical methods. Here, enzymes are fixed through bonding to

either external or internal surface of supporting materials by dispersion force of Vander Waals

interaction. We can use alumina, silica, clay, ceramics, cellulose, starch, activated carbon, and

ion exchange resins as supporting materials. Adsorption is that enzymes are attached on the

surface of support particles, so it is simple method. There is no pore diffusion limit because

enzymes are immobilized on surface not in the pores. Also, enzyme activity maintains well.

The active site of the adsorbed enzyme is usually unaffected, and nearly whole activity is

kept upon adsorption. Such surface immobilization is reversible

However, dispersion force is not strong enough. Especially with the presence of strong
hydrodynamic forces, binding forces are relatively weak. Therefore, if the flow rate of

substrate solution is high, then enzymes are easily detached. Such desorption of enzymes is a

typical problem. Enzymes are also very sensitive to pH, ionic strength, and temperature. Also,

adsorption method has a relatively lower loading capacity. Enzyme activity is likely to be

lower because the active site may be fixed toward surface of supporting materials.

The other physical method is an entrapment. In this experiment, we use entrapment method.

In this method, enzymes can be physically entrapped inside a matrix of a water-soluble

polymer such as polyacrylamide type gels and naturally derived gels. Cellulose triacetate,

agar, gelatin, carrageenan and alginate are representative of polymer matrix of entrapment.

Activated carbon, porous ceramic, and diatomaceous earth are instances of solid matrix.

When the enzyme is immobilized, loss of enzyme activity is unavoidable. However,

entrapment can minimize this. There is little loss of enzyme activity. Also, it has higher

loading capacity than other methods as the enzymes are in matrix or membranes. However,

enzyme leakage from matrix and diffusion resistance is possible.

Figure 6 Schematic Illustration of Examples in Physical Enzyme Immobilization Methods

 (b) chemical method

One is use of ionic interaction. This is method of forming ionic bonds between the enzyme

and ion-exchange resin/matrix. There can be positive ion-exchanger and negative ion-

exchanger. Such amino-cellulose and carboxy-cellulose are largely used.

Figure 7. positive ion-exchanger and negative ion-exchanger

Using the ionic interaction is one type of adsorption, so it is simple to immobilize. Also, it

has higher loading capacity. However, it can be used only for ionic enzyme and enzyme

leakage is available.

Another chemical method is use of covalent bonds. The covalent bonds are formed between

the functional groups of enzyme and corresponding groups on the surface of supporting

materials. In general, peptide bond, diazotization, alkylation, schiff base, disulfide bond are

applied. Because covalent bond is strong enough, the enzymes can be immobilized well.

Also, this method can be utilized under the condition which is a broad range of pH, ionic

strength, and other variables. However, similar to physical adsorption, it can decrease the

functional group of active sites by bounding to surface of supporting materials. It can lead to

loss of enzyme activity and enzyme inactivation.

 The last one is cross-linking. It is characterized by covalent bonding among enzymes

through a polyfunctional reagent. Polyfunctional reagent can be glutaraldehyde, diazonium

salt, hexamethylene diisocyanate, and N-N’ ethylene bismaleimide. This method is cheap and

easy to immobilize because only the enzyme and such reagent are used. However, it has

many drawbacks. The use of cross-linking may reduce active site of enzymes which leads to

enzyme inactivation. This method requires a lot of amount of enzyme and product is not in

fixed state.

Figure 8. Schematic illustration of chemical enzyme immobilization methods

3. Apparatus and materials

Apparatus

1) Beaker (100 ml, 50 ml)

2) Stirrer

3) Magnetic bar

4) Pipet
 5) Glass vial (10 ml, 20 - 30 ml)

6) pH meter

7) burette

8) Filter unit

9) Clock (stopwatch)

Reagent

1) Lipase from C.rugosa (enzyme)

2) Sodium alginate

3) CaCl2

4) 4-nitrophenyl butyrate (substrate)

5) Dimethyl formamide

6) Tris-HCl

7) HCl

4. Experimental Method

Overall scheme for experiment.

In this system, lipase (a subclass of the esterase) is used as enzyme and 4-nitrophenyl

butyrate is used as substrate.

 Figure 9. Enzyme System for Experiment

We will compare catalytic activity of free enzyme and immobilized enzyme by using the

hydrolysis reaction of 4-nitrophenyl butyrate catalyzed by lipase.

4-nitrophenolate adsorbs at 400 nm in maximum while starting substrate is transferred for

such wavelength. Therefore, using spectrophotometer we can know how far the reaction has

progressed.

▷ Lipase immobilization in Ca-alginate gel beads

1) Dissolve sodium alginate at about 1-2% (w/v) in 100mL of distilled water. Use a

microwave to heat the solution for 5 minutes.

2) Cool the sodium alginate solution at room temperature.

3) Prepare 100 mM CaCl2 aqueous solution (about 100 mL)

4) Prepare Tris-HCl (50 mM, pH 7.2) *buffer

*Buffer should not be contaminated by other enzymes. pH can be adjusted by adding small
amount of high-concentrated HCl.

5) Dissolve lipase powder in the Tris-HCl buffer (over 5 mL) to be 10 mg/mL

6) *Mix 4 mL of 10 mg/mL lipase solution and 16 mL of sodium alginate solution

homogeneously

*Mix with pipet, not vortexing

7) Add 20mL of [Lipase + sodium alginate] mixture very slowly into 100 mM CaCl2 (20

mL) solution (burette or syringe usage preferred, add [Lipase + sodium alginate] drop by

drop). At this time, CaCl2 solution should be stirred in the beaker. As soon as the [Lipase +

sodium alginate] entered in CaCl2 solution, spherical type Ca-alginate bead entrapping

Lipase will form. It is finished in about 20 minutes. This gel-formation takes place by ion-

exchange between Na+ alginate and CaCl2, resulting in cross-linking of Ca2+ and alginate to

form gel beads.

8) Wash Ca-alginate bead entrapping lipase once with 100 mM CaCl2 and twice with 50

mM Tris-HCl. (stirring in a beaker for 5-10 minute and filtering the immobilized enzyme)

9) *Dry immobilized enzyme at RT for about 1 hour.

* Freeze-drying is the most effective method to dry enzymes. This experiment temporarily

uses drying at RT

10) Store at 4℃

▷ Free lipase activity measurement

*Use pipettes separately for enzyme and substrate. Be careful not to mix substrate and

enzyme.
 1) To measure the hydrolytic activity of lipase, dissolve the appropriate amount of lipase in

Tris-HCl buffer (50 mM, pH 7.2) (1-5 mg/mL)

2) *Prepare the substrate solution at 6 different concentrations by diluting 4-nitrophenyl

butyrate in Dimethylformamide. [ex: 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, 1.5625

mM]

* a) The substrate should be stored at -20℃. Before using the substrate, wait a while until

the vial temperature is close to room temperature.

b) Be careful not to contaminate substrate stock vial.

c) Six different concentrations of the substrate can be prepared by serial dilution.

d) After the use of substrate vial, store at -20℃.

e) Because Dimethylformamide is an organic solvent, do an experiment in hood.

3) To make a control solution, mix 1.98 mL of 50 mM Tris-HCl (pH 7.2) buffer and 20 μL

of substrate (4-nitrophenyl butyrate).

4) Set baseline of the absorbance with controls.

5) Measure the absorbance of each sample at 400 nm using spectrophotometer. The detailed

measurement condition is as *follows:

*Mix 890 : 10 : 100 = Tris-HCl buffer (50 mM, pH 7.2) : Substrate solution : Lipase

solution in volume ratio

Mix well using a pipette

6) Measure the absorbance increase at 400 nm time-dependently. It can increase abruptly, so

that use a stopwatch. Wash the cuvette before each measurement.

 7) Measure the A400/min with different substrate concentrations.

▷ Immobilized lipase activity measurement

1) Prepare the substrate solution at 6 different concentrations by diluting 4-nitrophenyl

butyrate in dimethylformamide [ex: 50 mM, 25 mM, 12.5 mM, 6.25 mM, 3.125 mM, 1.5625

mM]

2) Add 100 μL of the substrate solution in 9.9 mL of Tris-HCl buffer (50 mM, pH 7.2) in a

50 mL tube.

3) Add 5 mg immobilized enzyme, vortex the tube, and gather the supernatant at pre-

determined time point (ex: 0 min, 5 min, 10 min, 15 min, 20 min)

4) *Measure the absorbance at 400 nm of gathered supernatant

*Be careful not to add enzyme particle.

5) Measure the A400/min with different substrate concentrations.

5. Meaningful Questions

1) In this enzyme-catalyzed kinetic equation, there is no term of substrate inhibition. How

mechanism does substrate inhibition work and what equation can I use based on substrate

inhibition?

2) I think that the degree of specificity of an enzyme is different. Is there a method for

measure substrate-specificity? Can a tiny-volumed molecule (so this molecule can penetrate

inside of enzyme) with proper functional group be catalyzed by enzyme or just only
structurally similar molecule be catalyzed by enzyme?

3) In contrast with free enzyme system, I think that bulk concentration of product/substrate

and concentration nearby immobilized enzyme of product/substrate are a little different. Can

do this affect significantly the kinetic equation?

4) With entrapment, the loss of enzyme activity is minimized (Assume that Km is almost

d [ P ] V max [ S ]
same). Then, in just mathematical calculation, v= = , there may not be constant
dt K m+[S ]

difference between immobilized and free enzyme system. (Because same [S], [E]0, (same E,

S ->) k). Then if v of immobilized and free enzyme system are meaningfully different, what

factor can affect them?

5) Why the double-trip plot provides a good estimate for Vm; however, it is not necessarily

in Km?

6. References

1) Chemical & Biomolecular Engineering Laboratory, CBE301, Daejeon, KAIST, p 37-48.

2) [CBE 301] Chemical and Biomolecular Engineering Laboratory Experiment Part D,

박상혁, 김한을, 이창현, Dept. of Chemical & Biomolecular Engineering, 2021.

3) Speeding up reactions: biological vs. chemical catalysts, S.E. Gould, Scientific American,

2014.