EXPERIMENTAL METHODS IN MODERN BIOTECHNOLOGY
Editors
Ibrahill1 Ali N oorbatcha I'v1ohall1ed Is111ail Abdul Karin1
Hall1zah l\1ohd Salleh
nc\!
PressPublishedby:
IIUv] Press
International Islamic Lniversity \lalaysia
First Edition. 2011
£:1 IUI\l Press. IIU\l
All rights resen ed. No part oftbis publication may be reproduced. swred in a retrieval system. or transmitted. in anyform orb.yany means, electronic. mechanical. phoTOcopying, recording, or otherwise.
\\ithout any prior written permission of the pub] isher.
Perpustakaan l'\egara :t\lalay Cataloguing-ill-Publication Data
Ibrahim Ali '\Ioorbatcha, Mohamed Ismail Abdul Karim and I Iamzah IVlohd Salleh ExptTimenral \lethods in\'Iodern Biorechnology
ISBl'\: 978-967-0225-86-9
fvlember of\lajlis Pcncrbitan lImiah \'lala)sia - \lAPl\J (\Jalaysian Scholarly Publishing Council)
Printed by:
HUM PRL'\TI'iG SD:\'. BHD.
'Jo. I.Jalan lndustri Satu Caws 1:3 Taman Pcrindustrian Batll Caves Batll Caws Centre Point
68100 Datu Caws Selangor Darul Ehsan
Preface
The rapid grc1\\1h of biotechnology as the interdisciplinary field involving scientists and engineers has given rise to new challenges in carrying out expcriments in di\'erse areas ranging fl'om examining the biological systems to the purification and production of products of commercial importance. The idea of writing a book on \'Iodern Experimental
\lethods in Biotechnology in such a diverse area is a very daunting task. Ho\wver, there is a need tor such a reference book for senior undergraduate students or beginning researchers in biotechnology, providing an integrated view of the experimental techniques along with the underlying principles, exemplified with case studies or sample results, and pointing out the advantages and limitations of tIle methods. A modest beginning towards this direction has been attempted in this book by combining the expertise and the experience o!" the researchers at the Depal1ment of Biotechnology' Engineering.
International Islamic University f\'Ialaysia and their collaborators who have been doing or teaching these experiments for the past one decade. For example, a selected set of experiments ranging from straight forward liquid-liquid separation process to the more latest direct nucleation control approach for the control of crystal size distribution in crystallization processes have been described in detail. In addition to extraction and purification of some selected compounds. a set or experiments to screen tor metabolic disorders and anti-cancer activities arc described \vith suitable cxampks. Any biotechnology experimental methods books will be incomplete without the inclusion enzyme assay methods, which has been described in this book as a set of three simple experiments. A flavor tor the use afcomputers in biotechnology is provided in a chapter on homology modeling. Even though the list of experiments included in this book is not exhaustive, the reader will find that the experiments covered arc fuJ1y self contained with good explanation of the theory behind the experiments and details abollt how to carry out
these experiments. A more nhaustive eowrage of a wide \ariety of other experiments in biotechnology \\-iJ1 be included in the later editions.
I. A. J\oorbatcha
\1.
r.
A. Karim I-I. \'1. Sallehv
CONTENTS
Preface \'
Chapter
1.Immobilization of Enzymes
FariclahYlls(~(
.,
I.7.
5.
6.
3 4 4 4 4 5 5 ....J
6 7
)
1 1 1 2 2
.,
~
10 10 10 ...j .
4.
Introduction
Scope of This Chapter
Salient Features of Enzyme Immobilization Advantages of Enzyme Immobilization Disadvantages of Enzyme Immobilization I\fethods of enzy'me immobilization 6.1 Adsorption T'vfethod
6.2 Covalent Bonding I\1ctl1od 6 ..3 Entrapment .t\1elhod
6.4 Copoly:merizationorCross-Linking Method 6.5 Encapsulation rvlethod
6.6 CalTier-Free Enzyme Immobilization
6.6.1 Cross-Linked Dissolved Enzymes (ClE) 6.6.2Cross-linkcd Enzyme Crystals (CLEU 6.6 ..3 Cross-linked Enzyme Aggregates (ClEA) I\laterials and I'vlethods
7.1 Immobilization ofu-amylase by Entrapment
7.2 Carrier-fi'ce immobilized enzymes - Cross-linked Enzyme Aggregates (ClEA)
8. Notes and Tips 9. References 10. Further Readings
Chapter 2. Protein Extraction and Purification
Faridah Yusof.,
1..J....
Introduction
Scope of This Chapter
Approaches to Protein Purification
3.1 Development of .Assay tor the Protein 3.2 Extraction of Protein from Sources 3.3 Fractionation ofProtelns
3.3.1 Types of Column C11romatography'Techniques
II I 1 12 12 12 13 [5
VI
3.3.1.1 Ion-Exchange Chromatography 3.3.].2 Gel filtration Chromatography
3.3.1.3 Hydrophobic Interaction Chromatography 3.3.],4 Chromatotocussing
3.3.1.5 Aninity Chromatography 4. Determination of Protein Purity
5. Purification or an 'Inhibitor' Protein from He\'ea brasiliensis Latex
.5.] Materials and ~vIethods
.5.1.I I'vlateria Is .5.1.2 l\lethods
5.1.2.1 Development of Assay for the' Inhibitor' Protein 5.1.2.2 Extraction of Crude Protein fi:om Fresh Latex 5.] .2.3 Fractionation ofC -serum Protein by Column .5.1.2,4 Determination of Purity of' Inhibitor' Protein 5.2 Results and Discussion
6. :Notes and Tips
7 Summary
8. Relerenccs 9. further Readings
Chapter 3. IHethods for Screening the Potential :\'atural Compound for Treating 'letabolic Disorder Diseases: Anti-InflammatOl·y and Griess Assay
r~itricOxide (:'\0) i\IeasurementJ
A::ura Amid. SulmFatie Semoil. {l'(711 Dalila TYOll Chik(ll1dHammed A dcmola
.\1011.5111"
16 16 17 17 18 18 19 19 19 19 19 19 19 21
JI
} )
23 24 24
1.
..J.
4.
6.
Introduction Objective :vlateria Is
\'lethods
4.1 Preparation of I L cell culture media
4.2 Preparation of media with 10% fetal bovine serum (FBS) 4.3 Preparation ofIL PBS-EDTA
4.4 Resuscitation of Frozen Cell Lines 4.5 Subculture of Adherent Cell Lines 4.6 Cells Quantification
4."7 Procedureto treat RA\V264.7 macrophage cells with identified extract 4.8 Preparation0
r
nitrite standards curw4.9 Griess Reaction
4.10 1\itrite Concentration Determination Example 0l'Results Obtained
References Further Reading
vii
25 25
26
17~I
27 28 28 28 28
29
29 30 30 30 31 34
Chapter 4. Factors Affecting Enzyme Assays
Hom::ah Alohd Solleh 1.
4.
6.
8.
1ntroduction to Enzyme Assay
1.1 Factors That Affect Enzyme Activity Objective of The Experiments
rvlateriaIs Enzyme Lab 1
4.1 Ertect of pH on Enzyme Activity 4.1.1 Procedure
Enzyme Lab2
5.1 Efteet of Temperature on Enzyme Activity and Enzyme Stability 5.1.1 Procedure"A".
5.1.2 Procedure "B".
Enzyme lab 3
6.1 Effect of Substrate Concentration on Enzy'me Rates 6.1.1 Procedure
For Further Readings Appendix
35 35 36 36 37 37 38 39 39 39 39 40 40 42 45 46
Chapter 5. Techniques of Extraction and Purification of Fucoxanthin from Brown Sea\veeds
Jnvondi JasHir, Dedi Novielldri, Ham:::oh A/ohdSolidI. Jll1hammad Taher und Ka::uo Miv([shiro
1.
4.
6.
'7I .
Introduction Materials T'vlethods
3.1 Extraction of Fucoxanthin
3.2 Purification ofFucoxanthin (with SiO~ Open Column Chromatography) 3.3 Further PurificationofFucoxanthin (\\-ith ODS Double Column)
3.4 Ilustration or Extraction and Purification of Fucoxanthin fi'om Brown Sca\\eeds
Notes
Acknowledgments References
Appendix
viii
50 51 51 51 52
- ' )
)""
55
57 59 59 62
Chapter 6. Fundamentals of Proximate Analysis in Food Products
Inrondi Ja.nvir and As ZHmb i-HOIn111ed TmvakaliT Tope
1. Introduction
') Determination of Moisture and Total Solids 2.1 Sample Preparation
' ) ! Evaporation \kthods
2.3 Distillation \lethods (Dean and Stark !\'lethod) 2.4 Chemical Reaction f\'kthods
2.4. ] Karl-Fisher method 2.4.2 (ias production methods 3. Analysis Of Ash Content
3.1 Sample Preparation 3.2 Dry Ashing
3.3 Wct Ashing 4. Analysis of Fat::;
4.1 Introduction
4.2 Sample Selection and Presenation 4.3 Detel111ination of Total Fats Content
4.3. 1 So ]vent Extraction
4.3.1.1 Continuous Solvent Extraction rv1ethod:Goldfish \kthod 4.3.1.2 Semicontinuous Solvent Extraction \-1ethod: Soxhlet rvlethod 4.3.2 0Jon solvent Liquid Extraction !\Iethods
4.3.2.1 BabcockJ\kthod 4.3.2.2 Gerber \lethod
4.3.2.3 DetergenT ivIethod 4.3.3 Instrumental methods.
5. Analysis of Proteins
5.1 Determination of Ovcrall Protein Concentration 5. 1.1 Kjeldahl method
5.1.1.1 DigesTion 5.1.1.2 NelfTrali:::afiol1 5.1.1.3 TifrariOJl
5.1.2Enhanced Dumas method
5.1.3 Nlethods using UV-visibIe spectroscopy
5.1.3.1 Direct measurement at 28011111 (AbsOlTJtion \·Icthodl 5.1.3.2 BiureT Method
5.1.3.3 [0\\"1')' fv1cthod 5.1.3.4PlEbinding methods 5. ] .3.5 TurbimeTric method 6. Analysis of Carbohydrates
7. Analysis ol'Fibcr
7.1 Common Procedures of Sample Preparation
7 ') Fiber Determination \1ethod
IX
65 66 66
66 67 67 67 68 69 69 69 70 71 71 71 71
72 72 72
'"i" ....
i.:J
73
7',.:J
73
73
74 74 74 74
75 75 75 76
77 77
78
78 78
79
79 79
7.2.1 Gravimetric I'vlethods
7.2.1.1 Crude Fiber Method
7.2.1.2 Total, Insoluble and Soluble Fiber :"'lethod 7.2.1.3 Chemical }\iIethods
8. References
79 79 7 80 80
Chapter 7. Fish Gelatin Production: Extraction lVlethod and Quality Analysis
Invondi Jaslvir. Hammed A. Mansur andHWTlzuhAi. Saltch
1. Introduction
! Extraction of Gelatin 2.1 rvlaterials
2.2 fvlethods
2.2.1 Skin preparation 2.2.2 Pretreatment 2.2.3 Extraction
2.2.4 Dehydration 3. Quantitati\e Analysis
3.1 Gr3\'imetric method
3.2 Soluble protein content method 4. Quality Analysis
4.1 Preparation of matured gelatin gel
4.2 Gel Strength: detinition and determination 4.3 I'vlcasurcment of Rheo logical Parameters
4.4 Sodium Dodeeyl Sulfate Polyacrylamide Gel Electrophoresis (SDS- PAGE) 4.5 The Foam test (Foam capacity and stability)
4.6 Amino acid proliling
4.7 Analysis of Color, Turbidity and clarity .5. References
81 82 82
82
82 83 84 84
87
87
87
88 89 89 90 91 9J 92 92 93
Chapter 8. Application of Fourier Transform Infrared Spectroscopy Edible Fats and Oils Analysis
Mohamed EhvalhigSCiced Alirghuni 1.
2.
Introduction
1.2 Analytical and Quality Control
1.3 Fourier Transform Infrared (FTIR) Spectroscopy' 1.4 Fourier Transformation
1.5 Transmission technique
1.6 Attenuated total rdlectance (ATR) rvlethodology
2.1 Sample preparations 2.1.1 Gases Samples
x
96 97 97 98 98 99
99
100 100
2.1.2 Liquid samples 2.1 ..3 Solid samples
2.2 fTIR Data Handling Techniques 2.2.1 Detection of overlapped bands 2.2.2 Smoothing and interpolation 2.2.3 Baseline correction
2.2.4 Peak intensity measurements 2.2.5 Spectn:ll stripping (Subtraction) 2.2.6 Ratio method
3. Quantitative Analysis 3.1 Beer-Lambert law
3.2 Classical least squares (K - \1atrixl .3.3 Inverse least squares (p - IVlatrix) 3.4 Partial least square (PLS)
3.5 Principal component regression (PCR)
4. FTlR Spectroscopy Applications On Food And Lipids 4.1 Examples
4.1.1 Slip melting point 4.1.2 Residual soap in oil
4.1.3 Hexane in solvent extracted 011 4.1.4 Palm carotene
4.1.5 Sesamol in sesame seed oil 4.1.6 Gossypol in cottonseed oil 4.t.7 Aflatoxins
4.1.8 Animal fats
4.1.9 Adulteration of'edible fats and oils 5. References
100
100
100 100 100 101 101 101 101 102 102 103 103 103 103 104 106 106 106 106 107 107 107
107 108 108 109
Chapter 9. In J7t1'o Assay for Investigating Potential Anti-Cancer Agents Targeting at
~VletastaticLevel
YlIIl1i ZuhanisHaS-YUH Hashim alldChris J.R. Gill
I.
..,
.:l.
-L
6.
7.
Introduction Scope fvlaterial
3.1 EquipmenL'Apparatus/Softwares Preparation
4.IIvlcthods Notes Reference
List of Abbreviation
xi
115 115 116 116 117 117 118 120 122
Chapter
10.Homology }lodelling Of Pyranose-2-0xidase from Phanerochaete Chrysosporium
Ibrahim Ali Noorbmcho, A::rarul Ashimah Nllr Malld Dom, Ahmad Sidqi Harit!llIddin and Ham::ah Mohd Sol/ch
1, Introduction 2. Literature Revic\\
2.1 Biofucl and enzymatic biofuel cells
} ! Glucose oxidase 2.3 PjTanose-2-oxidase 2.4 Homology \lodel1ing 3, \Taterials and rvlethods
3.1 Homology project design
],2 Homology modeling 4. Resu[t and Discussion
4.1 Description of the results pairwise sequence 4,2 Description of result pairwise sequence alignment 4.3 Ramachandran plot statistics
" Structure Validation 6. References
123 124 124 124 [25 125 126 126 126 131 133 162 139 140 141
Chapter 11. Liquid-Liquid Extraction and its Application for Separation of Organic Acids
Parveel1 Jamal
1. Introduction 143
1.1 Liquid-Liquid Extraction 143
1.2 Principle of ExtractionfiTnn Liquids 143
2. Notes and Tips 144
2.1 Solvent Selection 144
2,2 Propel1ies ofa good solvent 145
2,] Precautions 145
2.4 Class of Organic Compounds and Solubility' factor [45
2.5 Principle of Extracting Different Compounds 146
2.6 Salting Out 148
3. Experimental Part: Separation of acid tl'Olll a mixture containing acid, base and 149
3.1 Purpose 149
3.2 Learning Objectives 149
3.3 Techniques 149
4. \'[aterials 149
). Procedure 150
), 1Flow chart of Experimental Procedure 150
),2 Suggested Timetable 1.50
.5,3 Section A: Extraction of the Acid 151
xii
5.4 Section B: Isolation of the Acid
5.5 Section C: Recrystallization of the Acid 6. Results
6.1 Percent Yield Calculation 6.2 Example of Data calculation 6.3 Calculation
7. References
Chapter 12. Response Surface '\Icthodology (RS"\'1) Design for Bioreactor Operation
Alaizinvan AIel and Najiah Nadir
151 152 152 152 153 153 154
1. Introduction 155
1.1 Design of Experiment J55
1.2 Response Surface l ' v 1 e t h o d o l o g y l 5 5
1.3 Central Composite Design 155
l.4 Box-Behnken Design 156
1.5 Analysis of Variance (AKOVA) 156
1.6 Graphical Analysis 157
l.7 Bioreactor Operation 157
2. r..kthodology 158
2.1 Preparation of Bioreactor 158
2.1.1 Optimization of Fermentation Parameters for Maximum Ethanol 158 2.1.2 Optimization of Dyestuff Adsorption From Aqueous Solution Using 159
2.2 Experimental Design 159
2.2.1 Central composite design 159
2.2.2 Box-Behnken Design 161
3. Results and Discussion 161
3.1 Regression Equation 161
3.1.1 Central composite design 161
3.1.2 Box-behnken design 162
3.2 .A..nalysis 163
3.2.1 Central composite design 163
3.2.2 Box-Behnken Design 164
3.3 Response Surface Curves 164
3.3.1 Central composite design 164
3.3.2 Box-Behnken Design 166
4. Conclusion 168
5. References 168
Chapter 13. Screening Natural Compounds for Antibacterial Activity by Disc Diffusion:Vlethod
Raha AhmadRallS
xiii
1. Jntroduction 170
2. Materials 1"""1)I~
, T\lethods 172
~1.
3.1 Prepare bacterial inoculums 172
.... '1
Adjust inoculums turbidity 172
j.",- '> '>
Plating bacterial inoculums on plate 173
.J .•1
3.4 Place disc on plate 173
....j . )
-
Measure inhibition zone and interpretation 1734. \Jotes 175
5. References 175
Chapter 14. Indicator l\licroorganisms: Detection of
Col~f'orl1land Escherichia coli
Mohamed Ismail Abdul Karim 1.
!
...
4.
5.
6.
7.
Introduction
l.l Enumeration ?vlethods 1.2 Solid l\'ledia
Equipment and materials that are needed are as fo11O\'l/s Media and Reagents
Conventional Method for testing growth of coliforms, fecal coliforms and 4.1 IvlP\J - Presumptive test for colitorms, fecal coliforms and E.coli 4.2 MP\J - Confirmed test for coljforms
4.3MP\J - Confirmed test for fecal coliforms andE. coli 4.4 IvlP)J - Completed test forE coli.
4.5 The IIvIViC Tests
4.6 Solid medium method using Red Bile Agar - coliforms 4.7 Iv1cmbrane Filtration (\·JF)\.Jethod - coliforms
LST -MUG rv1ethod Used for DetectingE coliin Chilled or Frozen Foods References
Appendix
176 177 177 177 178
E. l78
178 179 180 180 181 182 183 183 184 186
Chapter 15. Direct l\ucleation Control: A l\ovel Approach for the Control of Crystal Size Distribution in Crystallization Processes
.i.\1ohdRushdi Abu Bakar, Zoltan Karman A'agv, Ali Nauman Salcf'lniClnd Christopher David Rielly
I. Introduction
! I'vlaterials 3. i\'1ethods
3.1 Uncontro1led Anti-so lvent Addition 3.2 D"\JC by Anti-solvent/Solvent Addition 4. ~otes
xiv
190 192 193 193 194 194
5.
6.
/.
4. I Uncontrolled Anti-so ]vent Addition 4.2 D:-JC by Anti-soh ent'So!vcntAddition References
Further Reading List ofAbbreviation
xv
194 194 197 198 198
190
CHAPTER 15
Direct Nucleation Control: A Novel Approach for the Control of Crystal Size Distribution in
Crystallization Processes
Mohd Rushdi Abu Bakar, Zoltan Kannan Nagy, Ali Nawnan Saleemi and Christopher David Rielly
1. Introduction
Crystallisation from solution is an important unit operation used in various industries as a technique for separating solid materials in purified forms. It is a technique of choice for solid-liquid separation due to its capability of producing high purity materials. In biophannaceutical industry, the crystallisation operation is often critical because it determines the product properties, such as the crystal size distribution (CSD), morphology and polymorphic form. The CSD is of primary importance since it influences subsequent downstream operations such as filtration and drying, and the product therapeutic performance such as dissolution rate, bioavailability and stability (Abu Bakar, 2010). Driven by the United States Food and Drug Administration's (FDA) Process Analytical Technology (PAT) initiative and the Quality-by-Design (QbD) concept, the development of control approaches, which can improve the manufacturing of products with desired properties, has become of significant interest.
PAT-based control approaches involve the use of in situ analysers that are able to measure and monitor product quality and critical process properties in real-time. This real-time process information is integrated into a control framework to provide an optimal control strategy with the aim of producing products with consistent desired quality. In addition to the provision of kinetic data that are required in the development of a robust process, real-time monitoring also allows appropriate remedial actions to be taken during the process if undesirable product quality or process property are identified (Yu et at., 2003).
Lasentec focused beam reflectance measurement (FBRM) is one of the in situ analysers that has been used extensively in the crystallization processes to provide qualitative as well as quantitative information about crystallisation process, \vhich comprises of nucleation and crystal grov.1h (Abu Bakar et al., 2009a; Abu Bakar et at., 2009b; Barrett & Glennon, 2002;
Chew et al., 2007a; Doki et al., 2004; How"ard et al., 2009; Nagy et a1., 2008; Simon et al., 2009). FBRM uses a laser beam sent through fibre optics to an immersion probe tip whereit is finely focused by a rotating lens, which causes the beam to scan in a circular path through a sapphire \vindo\v at a fLxed high speed. The beam then passes into the solution under study and \vhen it hits a solid particle suspended in the solution, light is scattered in many directions, but only light scattered back towards the probe is collected. The solid particle continues to back-scatter the light until the beam reaches the opposite edge of the crystal.
The time period of the backscattering (M) is recorded and multiplied by the scan speed of the beam (vh ) to give the distance between one edge of the solid particle to the other (s).
This distance measured by FBR,.\1 is caI1ed a chord length. Fig. I shows a schematic diagram of backscattered light pulses detection and chord length measurement.
From: Experimental Methods In Modern Biotechnology
Edited by. I. A. Noorbatch, H. M. Salleh andM I. A. Karim. /fUM Press, KL, Malaysia 190
