Contents

ABSTRACT................................................................................................................................................ 2
1.0INTRODUCTION .................................................................................................................................. 2
2.0 OBJECTIVES ....................................................................................................................................... 3
3.0 THEORY ............................................................................................................................................. 4
4.0 APPARATUS AND MATERIALS ........................................................................................................... 6
5.0 PROCEDURES..................................................................................................................................... 7
6.0 RESULT ............................................................................................................................................ 11
7.0SAMPLE CALCULATION .................................................................................................................... 17
8.0 DISCUSSION..................................................................................................................................... 20
9.0 CONCLUSIONS ................................................................................................................................. 22
10.0 RECOMMENDATIONS.................................................................................................................... 23
11.0 REFERENCES .................................................................................................................................. 23
11.0 APPENDIX ...................................................................................................................................... 24
ABSTRACT

The experiment is done to determine the effects of temperature, substrate concentrations, and
pH on the enzyme activity. Other than that, the experiment is done to calculate the maximum
velocity based on the result obtained whether it is same as in the theory or not. There are 4
experiments being done simultaneously. The enzyme used in this experiment is alpha
amylase. Amylase were being tested with starch by using different parameters; temperature,
pH and substrate concentration. Based on the results that has been plotted and calculated, it
can be said that the graph is successfully constructed. Each effect and parameters are all been
discuss. The objective of the experiment is achieved and hence the experiment is completed
successfully.

1.0 INTRODUCTION

Enzymes are often described as organic catalysts which increase the rate of reaction.
[1]
One of them is amylase. Amylase generally known as the enzyme which catalyses the
hydrolysis of starch and glycogen is among the most important industrial enzymes. Also, the
enzyme has been long studied and it may be said that the history of study amylase is as long
as that of enzymes. Hydrolysis is a common mechanism used by enzymes to break chemical
bonds. The hydrolysis of starch can be measured through the use of an enzyme test or assay.
An enzyme assay will test for the simple presence of enzyme activity but can also be used to
measure the reaction rate of an enzyme-catalysed reaction. The assay can measure either the
appearance of one of the products or the disappearance of one of the substrates over time.

In order to measure the amylase activity, the disappearance of amylase’s substrate

(starch) need to monitored. Starch will react to iodine to form a blue compound. This reaction
is the basis of a colorimetric assay for amylase activity. In this experiment, the broth culture
supernatant which contains the secreted amylase will be incubated with starch. After the
incubation period, a portion of this mixture is combined with acidic iodine. The acid stops the
enzymatic reaction and the iodine reacts with the starch to produce the blue colour. Any
starch that has not yet been hydrolysed by the amylase will turn blue with the intensity of the
blue color can be quantified spectrophotometrically by measuring its absorbance at 620nm.
The greater the change in absorbance between a sample containing the initial amount of
starch not containing enzyme and the hydrolyzed mixture containing the enzyme, the greater
the amount of starch degraded by the enzyme, therefore the greater the activity of the enzyme
being measured.

The aims for this experiment is to determine the effect of temperature on the
enzymatic activity and changes in enzyme concentration of an enzyme-catalyzed reaction,
describe the relationship between substrate concentration and the maximum velocity of an
enzyme and estimation of Michaelis-Menten parameters, effect of pH and temperature on
enzyme activity and kinetics of inhibition. Enzyme activity is dependent upon the
environmental conditions either in nature or in the laboratory. This is because these
conditions can alter the amino acid side chains in protein, affecting protein structure and
folding and sometimes the enzyme’s active site. The effect of some conditions will be
explored in this experiment.

2.0 OBJECTIVES

1. To determine the effect of temperature on the enzymatic activity and changes in

enzyme concentration of an enzyme – catalyzed reaction.
2. To describe the relationship between substrate concentration and the maximum
velocity of an enzyme.
3. To estimate the Michaelis – Menten parameters, effect of pH and temperature on
enzyme activity and kinetics of inhibition.
3.0 THEORY

Enzymes are proteins produced in living cells that helps to accelerate or catalyze the
metabolic processes of an organism. It is very selective in the molecules that they act upon,
called substrates which often reacting with only a single substrate. An active site located at an
enzyme is a placed for the substrate to bind before the reaction takes place. Enzymes can
speed up the chemical reactions but only function within a narrow temperature and pH range,
otherwise they can lose their structure and become denatured. It also involved in the
processes of breaking down of large protein, starch and fat molecules in food into smaller
molecules during digestion [2].

The rate of an enzyme-catalyzed reaction and how it changes in response to certain

experimental parameters are the most fundamental and central approach in enzyme kinetics
[3]
study . The parameters that affect enzyme activity are pH, temperature and substrate
concentration. Figure 1 shows the effect of substrate concentration on the enzyme activity or
velocity, known as reaction rates. The reaction is very slow at the beginning because at very
low concentrations, substrate molecules collide so infrequently with enzyme molecules. As
the substrate concentration increases, the reaction rate increases as enzyme and substrate
molecules collide vigorously. However, when the enzyme molecules approach the maximum
rate at which they can combine with reactants and release products, increasing substrate
concentration has a smaller and smaller effect, until the rate of reaction eventually levels off
[3]
. The enzyme velocity remain constant although the substrate concentration keep
increasing. The maximum velocity is called Vmax.

Figure 1: Michaelis-Menten Model

Michaelis-Menten Equation

Enzyme reacts with substrates, forms an intermediate enzyme-substrate complex

(ES), and a product (P) and regenerated enzyme. This assumes that when the enzyme
complexes with the substrate, it either dissociates into unchanged substrate and enzyme or
proceeds irreversibly forward to product.

The second step’s rate (k2) is the enzyme’s turnover for enzyme-substrate converting
to product. The Michaelis constant, Km, obtain from the ratio of the reactions affecting the
enzyme-substrate complex: its formation (k1), dissociation (k-1), and progress towards
product (k2). A lower Km value indicates that an enzyme has a higher affinity for a substrate.

Michaelis-Menten enzyme’s velocity depends on its Km, substrate concentration, and

maximum possible velocity (Vm), which occurs when there is much more substrate than
enzyme. This means that when the substrate concentration equals Km, the enzyme’s velocity
is half of its Vm.

Lineweaver – Burke Plot

Figure 2: Lineweaver Burk Plot

 Figure 2 above shows the Lineweaver-Burk plot which also known as the double
reciprocal plot. It can be used to calculate Km and Vmax as well as determine the mechanism
of action of enzyme inhibitors. In this plot we see that 1/v o is plotted versus 1/[S] and a
straight line is obtained. The graph shows that the intercept on the x-axis is equal to -1/Km
and the intercept on the y-axis is equal to 1/Vmax.

4.0 APPARATUS AND MATERIALS

APPARATUS MATERIALS
 Beaker  Alpha amylase enzyme
 Measuring cylinder  Starch
 Cuvette  pH buffer solution (pH 4-9)
 Falcon tube rack  DNSA reagent
 Falcon tube  Water bath
 Micropipette and tips
 Label sticker
 Schott bottle
 Vortex mixer
 Spectrophotometer
 Hot plate
5.0 PROCEDURES

A. PREPARATION OF 2% STARCH SOLUTION

1) 4g of soluble starch was mixed in approximately 50ml of cold water.

2) The slurry is added to approximately 100ml of gently boiling water while stirred in a
large beaker.
3) Then, the final volume of 200ml was added and mixed well.

B. EFFECT OF TEMPERATURE

1) One test tube was labeled with 30°C and placed with 1ml of 2% starch solution and
1ml of pH=7 buffer.

2) 2 ml of amylase solution was put onto an additional clean test tube.

3) Both tubes were placed in the 30°C water bath for 5 minutes until the temperature
equilibrated.

4) The content of amylase test tube poured onto the starch test tube and mixed on vortex
meter.

5) The tubes returned to the 30°C water bath.

6) The hydrolysis reaction was let to proceed for 10 minutes.

7) The amylase activity was determined using the method in Appendix 1.

8) Steps 1-7 repeated at different temperature ranging from 30-70°C.

9) Graph of temperature vs. enzyme activity plotted.

C. EFFECT OF PH

1) Five test tubes were labeled with pH 5, 6, 7, 8 and 9 and placed with 1ml of 2% starch
solution and 1 mL of the appropriate buffer to each tube.

2) 2 ml of amylase solution added to each of five additional clean test tubes.

3) All the ten test tubes placed in the 37°C water bath for 5 minutes until the temperature
equilibrated.

4) The content of each amylase test tube was added onto each starch test tube and were
mixed on vortex meter.

5) The tubes were returned to 37°C water bath.

6) The hydrolysis reaction was let to proceed for 10 minutes.

7) The amylase activity was determined using the method in Appendix 1.

8) Graph of pH vs. enzyme activity plotted.

 D. EFFECT OF SUBSTRATE CONCENTRATION

1) A starch solutions varying concentration (0.5, 1.5, 2.0, 2.5 and 3.0% w/v) was
prepared as the substrate.

2) Each tube with starch concentration labeled and placed each of them with 1ml starch
solution.

3) 1ml of ph=7 buffer was added to the tubes.

4) 2ml of amylase solution was added onto five additional clean test tubes.

5) The content of each amylase test tube poured onto the starch test tube and were mixed
on vortex meter.

6) The tubes were returned to 37°C water bath.

7) The hydrolysis reaction was let to proceed for 10 minutes.

8) The amylase activity was determined using the method in Appendix 1.

9) Graph of starch concentration vs. enzyme activity plotted.

Appendix 1 (Demonstration Of Enzyme Activity)

1) After the 10 minutes hydrolysis reaction, the reaction stopped by adding 4ml of DNS
agent.

2) The tubes boiled for 10 minutes and left cooled to temperature.

3) The absorbance of the samples measured at λ= 540 nm.

4) The absorbance value was compared with the glucose standard curve that prepared to
obtain the glucose concentration.

5) The enzyme activity calculated.

Appendix 2 (Glucose standard curve preparation)

1) A standard solution of glucose was prepared at five different concentrations ranging

from 0-2000g/L by serial dilution.

2) 1 ml of each glucose solution was added in test tubes.

3) 1 ml of DNS reagent was added in each test tube and mixed for few seconds on vortex
meter.

4) The test tubes were placed in water bath (T=100°C) for 10 minutes and left cooled at
room temperature.

5) The absorbance value of the samples were measured at λ= 540 nm.

6) The graph of standard curve of absorbance vs. glucose concentration plotted.

6.0 RESULT

a. Glucose Standard Curve

Table 1 : Glucose Standard Curve

Standard Glucose Concentration Absorbance Optical Density (OD)

0 0.8
400 0.48
600 1.66
800 2.05
1000 3.15
1200 3.49
1400 3.82
1800 4.49
2000 5.34

GLUCOSE STANDARD CURVE

6

5
ABSORBANCE OPTICAL DENSITY (OD)

0
0 500 1000 1500 2000 2500
STANDARD GLUCOSE CONCENTRATION

Figure 3: Glucose Standard Curve

 b. Effect Of Temperature

Table 2: The values of absorbance optical density (OD) at different temperature

Temperature (oC) Absorbance Optical Density (OD)

1 2 3 Average OD Real OD
60 0.06 0.08 0.011 0.07 x 33 2.310
70 0.068 0.079 0.056 0.062 x 33 2.046

OD vs Temperature

2.35
Absorbance Optical Density (OD)

2.3

2.25

2.2

2.15

2.1

2.05

2
58 60 62 64 66 68 70 72
Temperature (oC)

Figure 4: The effect of absorbance optical density (DO) at different temperature

 Table 3: The values of enzyme activity at different temperature

Temperature (oC) Real OD (nm) Glucose concentration, Glucose Enzyme activity,

X (g/L) released (mol) V (mol/min)
60 2.310 820 9.11E-3 9.11E-4
70 2.046 700 7.78E-3 7.78E-4

Temperature vs Enzyme Activity

9.20E-04

9.00E-04
Enzyme activity, V (mol/min)

8.80E-04

8.60E-04

8.40E-04

8.20E-04

8.00E-04

7.80E-04

7.60E-04
58 60 62 64 66 68 70 72
Temperature (oC)

Figure 5: The effect of enzyme activity at different temperature

 c. Effect Of PH
Table 4 : The values of absorbance optical density (OD) at different pH values

pH value Absorbance Optical Density (OD)

1 2 Average DO Real OD
5 0.190 0.207 0.199 x 33 6.567
6 0.094 0.173 0.180 x 33 5.940
7 0.100 0.093 0.147 x 33 4.851
8 0.135 0.119 0.127 x 33 4.191
9 0.108 0.114 0.111 x 33 3.663

OD vs pH value
7
6
5
OD (nm)

4
3
2
1
0
0 2 4 6 8 10
pH

Figure 6: The effect of absorbance optical density (DO) at different pH values

Table 5: The values of enzyme activity at different pH values

pH value Real OD Glucose concentration, X Glucose Enzyme activity,

(nm) (g/mL) released (mol) V (mol/min)
5 6.567 2.736E-03 3.040E-05 3.040E-06
6 5.940 2.475E-03 2.750E-05 2.750E-06
7 4.851 2.021E-03 2.246E-05 2.246E-06
8 4.191 1.746E-03 1.940E-05 1.940E-06
9 3.663 1.526E-03 1.696E-05 1.696E-06
 enzyme activity vs pH values
3.50E-06

enzyme activity (mol/min) 3.00E-06

2.50E-06
2.00E-06
1.50E-06
1.00E-06
5.00E-07
0.00E+00
0 2 4 6 8 10
pH

Figure 7 : The effect of enzyme activity at different pH values

d. Effect Of Substrate Concentration

Table 6: The values of absorbance optical density (OD) at different substrate concentrations

Starch Absorbance Optical Density (OD)

Concentration (%) 1 2 3 Average OD Real OD
1 0.139 0.137 0.12 0.138 x 33 4.55
3 0.122 0.047 0.069 0.122 x 33 4.03

OD vs Substrate Concentration
4.6

4.5

4.4
OD (nm)

4.3

4.2

4.1

4
0 0.5 1 1.5 2 2.5 3 3.5
Substrate concentration (%)

Figure 8: The effect of absorbance optical density (OD) reading on different substrate concentration
Table 7: The values of enzyme activity at different substrate concentrations and the data for michaelis-menten

Substrate Real OD Glucose Glucose Enzyme 1/V 1/S

concentration, (nm) concentration, released activity, V
S (%) X (g/mL) (mol) (mol/min)
1 4.55 1.896E-03 2.107E-05 2.107E-06 474608.4480 1.0000
3 4.03 1.679E-03 1.866E-05 1.866E-06 535905.6806 0.3333

Enzyme activity vs substrate

concentration
2.15E-06
enzyme activity (mol/min)

2.10E-06
2.05E-06
2.00E-06
1.95E-06
1.90E-06
1.85E-06
0 0.5 1 1.5 2 2.5 3 3.5
Substrate concentration (%)

Figure 9: The effect of enzyme activity at different substrate concentrations

1/V vs 1/S
540000
530000
520000
510000
1/V

500000
490000
480000 y = -91941x + 566550
R² = 1
470000
0 0.2 0.4 0.6 0.8 1 1.2
1/S

Figure 10: Graph of 1/V vs 1/S for Michaelis-menten

7.0 SAMPLE CALCULATION

1. Determination of glucose concentration, X (g/mL)

From the standard curve of glucose plotted in figure 3, the linear equation of the curve is presented
as:

y = 0.0024x

Where,

x: glucose concentration

y: absorbance reading

Therefore, to calculate the glucose concentration:

𝑦
𝑥=
0.0024

i) Effect of temperature

When T= 60℃, the OD reading is 2.31 nm:

2.31
𝑥=
0.0024
𝑚𝑔 0.001 1𝑙
x = 962.5 𝑥 𝑥
𝑙 1𝑚𝑚 1000𝑚𝑙
x = 9.625x10-4 g/mL

ii) Effect of substrate

When substrate, S= 1%, the OD reading is 4.55nm:

4.55
𝑥=
0.0024
𝑚𝑔 0.001 1𝐿
x=1895.83 𝑥 𝑥
𝑙 1𝑚𝑚 1000𝑚𝐿
x=1.896x10-3mg/L
 iii) Effect of pH

When pH value= 8, the OD reading is 4.191 nm:

4.191
𝑥=
0.0024
𝑚𝑔 0.001 1𝐿
x= 1746.25 𝑥 𝑥
𝑙 1𝑚𝑚 1000𝑚𝐿

x=1.746x10-3 mg/L

2. Determination of glucose released (mol)

MW of glucose = 180 g/mol, Volume of enzyme (amylase) = 2ml

𝑔
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒( )
𝑚𝐿
Mol of glucose released (mol) = x vol. of amylase (mL)
𝑀𝑊 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒

i) Effect on Temperature
9.625x10−4 g/mL
Mol of glucose released (mol) = x 2mL
180 g/mol

= 1.069x10-5mol

ii) Effect on Substrate

1.896x10−3 mg/L
Mol of glucose released (mol) = x 2mL
180 g/mol

= 2.107x 10-5 mol

iii) Effect on pH

1.746x10−3 mg/L
Mol of glucose released (mol) = x 2 mL
180 g/mol

= 1.94x 10-5 mol

 3. Calculation of enzyme kinetic (mol/min)

Duration of hydrolysis reaction is 10 minutes

𝑚𝑜𝑙 𝑚𝑜𝑙 𝑜𝑓 𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑
Enzyme activity ( )=
𝑚𝑖𝑛 ℎ𝑦𝑑𝑟𝑜𝑙𝑦𝑠𝑖𝑠 𝑟𝑒𝑎𝑐𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒

i) Effect on temperature
𝑚𝑜𝑙 1.069𝑥10−5 𝑚𝑜𝑙
Enzyme activity ( )=
𝑚𝑖𝑛 10 𝑚𝑖𝑛

= 1.069x 10-6 mol/min

ii) Effect on substrate

𝑚𝑜𝑙 2.107x 10−5 mol
Enzyme activity ( )=
𝑚𝑖𝑛 10 𝑚𝑖𝑛
= 2.107x 10-6 mol/min

iii) Effect on pH

𝑚𝑜𝑙 1.94x 10−5 mol

Enzyme activity ( )=
𝑚𝑖𝑛 10 𝑚𝑖𝑛
=1.94x 10-6mol/min
8.0 DISCUSSION

In this experiment, we want to study the effect of pH, temperature and substrate concentration
toward the enzymes kinetic. In order to determine the glucose concentration for every sample
in this experiment, a standard glucose curve have to be plotted by using a glucose
concentration with a range concentration of 0 to 2000 g/L by serial dilution. In order to obtain
the value of the glucose standard curve, a number of glucose solution with different
concentration and after several step of procedure, the absorbance optical density (OD) value
was measured for each of the samples. The value then was use to plotted the standard glucose
curve and this graph was use to obtain the glucose concentration for each of the experiment
by interpolating the value of OD that was obtain in the experiment in this graph.

One of the variable that we use to determine the effect on the enzymes kinetics is
temperature. In the figure shows that that graph of OD versus temperature shows that the
graph become decreasing as the temperature increase. From the OD value that was recorded,
the enzymes activity was calculated and a graph of enzymes activity versus temperature was
plotted. Based from the graph that have been plotted, the graph become deceasing as the
temperature increasing. Based from this observation, the temperature of the solution can
affect the enzymes activity where the increasing of temperature will cause the enzymes
activity become lower and after a certain high temperature the enzymes will denatured. As
we already know, the temperature has a general effect on the chemical reaction where the
increasing of temperature can typically cause the increasing of reaction rate. As for the
enzymes, the increasing of temperature causes the kinetic motion of amino acid chain in the
enzymes increase. From the range of 0oC to 40oC, the rate of reaction in the enzyme
increasing by double for every 10oC increasing in temperature. Above 40oC, the increasing of
temperature will cause the reducing of the increase in enzymes activity. Most enzymes reach
its peak in activity between 40oC to 50oC and then it became steep at 55oC and start to drop at
60oC. Thus, it is recommended that to operate the process in the optimum temperature of the
most enzymes which is 40oC to 50oC, but different enzymes have different optimum
temperature in which it can reach a peak performance of enzymes kinetic and high
temperature can cause the enzyme kinetics become decreasing and eventually become zero.
 For different pH values, the graph of absorbance optical density (OD) against
different pH values was plotted. The graphs decrease gradually as the pH value increases.
The graphs that the highest OD reading is at pH 5 with 6.567 nm and the lowest OD reading
is at pH 9 with only 3.663 nm. Then the results followed with a graph of enzyme activity
(mol/min) against different pH values. The graph shows a same pattern as in graph in figure.

There will be a pH, characteristic of each enzyme, at which the net charge on the
molecule is zero. This is called the isoelectric point (pI), at which the enzyme generally has
minimum solubility in aqueous solutions. In a similar manner to the effect on enzymes, the
charge and charge distribution on the substrate(s), product(s) and coenzymes (where
applicable) will also be affected by pH changes. Increasing hydrogen ion concentration will,
additionally, increase the successful competition of hydrogen ions for any metal cationic
binding sites on the enzyme, reducing the bound metal cation concentration. Decreasing
hydrogen ion concentration, on the other hand, leads to increasing hydroxyl ion concentration
which competes against the enzymes' ligands for divalent and trivalent cations causing their
conversion to hydroxides and, at high hydroxyl concentrations, their complete removal from
the enzyme. [4]

For enzyme-substrate reaction, the most favourable pH value is 7 (natural) where it is

being used as buffer in this experiment. At this value, the enzyme is most active and this
value is known as the optimal pH. At pH value above or below the optimum, the rate of
enzyme activity drops off. At extreme pH values, the rate might even drops to zero. [3]

Based on the graph plotted in figure, the result shows that when the concentration of
substrate (starch) increases, the absorbance optical reading (OD) decreases. The graph started
at 1% starch concentration with 4.55 nm OD reading and then decreases to 4.03 nm OD
reading at 3% starch concentration. Next, a graph of enzyme activity (mol/min) against
substrate concentration (%) is also plotted. The graph shows an identical pattern such in the
graph plotted in figure. The graph shows decreasing value of enzyme activity from 2.107E-06
mol/min at 1% starch concentration to 1.866E-06 mol/min at 3% starch concentration. The
same pattern shows that the OD reading is equal to the enzyme activity against the substrate
where the enzyme activity is defined as the amount of glucose formed in reaction
mixture per unit time.
 The graph results in such state because the reaction is already at its maximum reaction
rate at the beginning of the experiment. The enzyme and substrate molecules collide
vigorously at this stage and continuous addition of substrate concentration only caused the
reaction rate to slow down. This is because the enzyme slowly becomes ineffective as it is
being used to the maximum where there is no more free enzyme to bind with the substrate
and all the active sites of enzyme are already bound to the substrate.[3]

Lastly, a graph of michaelis-menten is plotted which is the graph of 1/V vs 1/S.

Theoretically, values of Vmax and Km can be determine from the graph but because the graph
shows neither competitive, uncompetitive and non-competitive inhibition graph, therefore,
the values of Vmax and Km cannot be obtain. The Km value is the rate constant or how much
of substrate concentration is needed by an enzyme to reach to the half of maximum rate or
velocity of enzyme 𝑉𝑚𝑎𝑥 . Each enzyme will has different Km values.

9.0 CONCLUSIONS

The experiment is conducted to study the effects of temperature on the enzymatic

activity and changes in enzyme concentration of an enzyme-catalysed reaction in order to
describe the relationship between substrate concentration and the maximum velocity of an
enzyme and also to study the effects of pH, estimation of Michaelis-Menten parameter and
temperature on enzyme activity and kinetics of inhibition. Based on the results that has been
plotted and calculated, it can be said that the graph is successfully constructed. Each effect
and parameters are all been discuss. The objective of the experiment is achieved and hence
the experiment is completed successfully.
10.0 RECOMMENDATIONS

There are few recommendations that need to be taken to increase the Km and Vmax value
during conducting the experiment:
1) Increase the temperature because temperature effects enzymatic reactions as an
increase in reaction rate until it reaches a peak where enzyme can function well.
2) Use another enzyme as different enzymes has different characteristics.
3) Increase in enzyme concentration as it increase the reaction rate by allowing the
active sites in which substrate may bind to perform reactions.

11.0 REFERENCES
1. The Amylase Research Society (2014). Handbook of Amylases and Related
Enzymes: Their sources, isolation methods, properties and applications.
Retrieved from:
https://books.google.com.my/books?id=2lKeBQAAQBAJ&dq=amylase&source=gbs_
navlinks_s
2. The American Heritage® Science Dictionary Copyright © 2011. Published by
Houghton Mifflin Harcourt Publishing Company.
3. Peter J. Russell et al, Biology: The Dynamic Science 3rd Edition Volume 1, 2014,
page 81.
4. Enzyme Technology Effect of pH and ionic strength. Martin Chaplin (6 August, 2014 )
Retrieved from, www1.lsbu.ac.uk/water/enztech/ph.html
11.0 APPENDIX