EXPERIMENTAL METHODS INMODERN BIOTECHNOLOGY

Editors

Ibrahill1 Ali NoorbatchaI'v1ohall1ed Is111ail Abdul Karin1

Hall1zah l\1ohd Salleh

nc\! Press

Published by:IIUv] Press

International Islamic Lniversity \lalaysia

First Edition. 2011£:1 IU I\l Press. IIU\l

All rights resen ed. No part oftbis publication may be reproduced. swred in a retrieval system. ortransmitted. in any form or b.y any 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 IIamzah IVlohd SallehExptTimenral \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 1:3Taman Pcrindustrian Batll CavesBatll Caws Centre Point68100 Datu CawsSelangor Darul Ehsan

Preface

The rapid grc1\\1h of biotechnology as the interdisciplinary field involving scientists andengineers has given rise to new challenges in carrying out expcriments in di\'erse areasranging fl'om examining the biological systems to the purification and production ofproducts 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 isa need tor such a reference book for senior undergraduate students or beginningresearchers in biotechnology, providing an integrated view of the experimental techniquesalong with the underlying principles, exemplified with case studies or sample results, andpointing out the advantages and limitations of tIle methods. A modest beginning towardsthis direction has been attempted in this book by combining the expertise and theexperience o!" the researchers at the Depal1ment of Biotechnology' Engineering.International Islamic University f\'Ialaysia and their collaborators who have been doing orteaching these experiments for the past one decade. For example, a selected set ofexperiments ranging from straight forward liquid-liquid separation process to the morelatest direct nucleation control approach for the control of crystal size distribution incrystallization processes have been described in detail. In addition to extraction andpurification of some selected compounds. a set or experiments to screen tor metabolicdisorders and anti-cancer activities arc described \vith suitable cxampks. Anybiotechnology experimental methods books will be incomplete without the inclusionenzyme assay methods, which has been described in this book as a set of three simpleexperiments. A flavor tor the use a f computers in biotechnology is provided in a chapteron homology modeling. Even though the list of experiments included in this book is notexhaustive, the reader will find that the experiments covered arc fuJ1y self contained withgood 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 inbiotechnology \\-iJ1 be included in the later editions.

I. A. J\oorbatcha\1. r. A. Karim

I-I. \'1. Salleh

v

CONTENTS

Preface \'

Chapter 1. Immobilization of EnzymesFariclahYlls(~(

.,I.

7.

5.6.

3444455

....J

67

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11122.,~

101010

...j.

4.

IntroductionScope of This ChapterSalient Features of Enzyme ImmobilizationAdvantages of Enzyme ImmobilizationDisadvantages of Enzyme ImmobilizationI\fethods of enzy'me immobilization6.1 Adsorption T'vfethod6.2 Covalent Bonding I\1ctl1od6..3 Entrapment .t\1elhod6.4 Copoly:merization or Cross-Linking Method6.5 Encapsulation rvlethod6.6 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'vlethods7.1 Immobilization ofu-amylase by Entrapment7.2 Carrier-fi'ce immobilized enzymes - Cross-linked Enzyme Aggregates(ClEA)

8. Notes and Tips9. References10. Further Readings

Chapter 2. Protein Extraction and PurificationFaridah Yusof

1..,....J.

IntroductionScope of This ChapterApproaches to Protein Purification3.1 Development of .Assay tor the Protein3.2 Extraction of Protein from Sources3.3 Fractionation ofProtelns

3.3.1 Types of Column C11romatography' Techniques

III 112121213[5

VI

3.3.1.1 Ion-Exchange Chromatography3.3.].2 Gel filtration Chromatography3.3.1.3 Hydrophobic Interaction Chromatography3.3.],4 Chromatotocussing3.3.1.5 Aninity Chromatography

4. Determination of Protein Purity5. Purification or an 'Inhibitor' Protein from He\'ea brasiliensis Latex

.5.] Materials and ~vIethods

.5. 1. I I'vlateria Is

.5. 1.2 l\lethods5.1.2.1 Development of Assay for the' Inhibitor' Protein5.1.2.2 Extraction of Crude Protein fi:om Fresh Latex5.] .2.3 Fractionation ofC-serum Protein by Column.5 .1.2,4 Determination of Purity of' Inhibitor' Protein

5.2 Results and Discussion6. :Notes and Tips7 Summary8. Relerenccs9. further Readings

Chapter 3. IHethods for Screening the Potential :\'atural Compound forTreating 'letabolic Disorder Diseases: Anti-InflammatOl·y andGriess Assay r~itric Oxide (:'\0) i\IeasurementJA::ura Amid. SulmFatie Semoil. {l'(711 Dalila TYOll Chik (ll1d Hammed Adcmola.\1011.5111"

1616171718181919191919191921JI

})

232424

1.

..J.

4.

6.

IntroductionObjective:vlateriaIs\'lethods4.1 Preparation of IL cell culture media4.2 Preparation of media with 10% fetal bovine serum (FBS)4.3 Preparation of IL PBS-EDTA4.4 Resuscitation of Frozen Cell Lines4.5 Subculture of Adherent Cell Lines4.6 Cells Quantification4."7 Procedure to treat RA \V 264.7 macrophage cells with identified extract4.8 Preparation 0 r nitrite standards curw4.9 Griess Reaction4.10 1\itrite Concentration DeterminationExample 0 l' Results ObtainedReferencesFurther Reading

vii

25252617~I

2728282828292930303031

34

Chapter 4. Factors Affecting Enzyme AssaysHom::ah Alohd Solleh

1.

4.

6.

8.

1ntroduction to Enzyme Assay1.1 Factors That Affect Enzyme ActivityObjective of The Experimentsrvlateria IsEnzyme Lab 14.1 Ertect of pH on Enzyme Activity

4.1.1 ProcedureEnzyme Lab 25.1 Efteet of Temperature on Enzyme Activity and Enzyme Stability

5.1.1 Procedure "A".5.1.2 Procedure "B".

Enzyme lab 36.1 Effect of Substrate Concentration on Enzy'me Rates

6.1.1 ProcedureFor Further ReadingsAppendix

35353636373738393939394040424546

Chapter 5. Techniques of Extraction and Purificationof Fucoxanthin from Brown Sea\veedsJnvondi JasHir, Dedi Novielldri, Ham:::oh A/ohd SolidI. Jll1hammad Taher undKa::uo Miv([shiro

1.

4.

6.'7I.

IntroductionMaterialsT'vlethods3.1 Extraction of Fucoxanthin3.2 Purification ofFucoxanthin (with SiO~ Open Column Chromatography)3.3 Further Purification of Fucoxanthin (\\-ith ODS Double Column)3.4 Ilustration or Extraction and Purification of Fucoxanthin fi'om Brown

Sca\\eeds

NotesAcknowledgmentsReferencesAppendix

viii

5051515152-'))""

55

57595962

Chapter 6. Fundamentals of Proximate Analysis in Food ProductsInrondi Ja.nvir and AsZHmb i-HOIn 111 ed TmvakaliT Tope

1. Introduction') Determination of Moisture and Total Solids

2.1 Sample Preparation')! Evaporation \kthods2.3 Distillation \lethods (Dean and Stark !\'lethod)2.4 Chemical Reaction f\'kthods

2.4. ] Karl-Fisher method2.4.2 (ias production methods

3. Analysis Of Ash Content3.1 Sample Preparation

3.2 Dry Ashing3.3 Wct Ashing

4. Analysis of Fat::;4.1 Introduction4.2 Sample Selection and Presenation

4.3 Detel111ination of Total Fats Content4.3. 1 So]vent Extraction

4.3.1.1 Continuous Solvent Extraction rv1ethod:Goldfish \kthod4.3.1.2 Semicontinuous Solvent Extraction \-1ethod: Soxhlet rvlethod

4.3.2 0Jon solvent Liquid Extraction !\Iethods4.3.2.1 Babcock J\kthod

4.3.2.2 Gerber \lethod4.3.2.3 DetergenT ivIethod

4.3.3 Instrumental methods.5. Analysis of Proteins

5.1 Determination of Ovcrall Protein Concentration5. 1.1 Kjeldahl method

5.1.1.1 DigesTion5.1.1.2 NelfTrali:::afiol1

5.1.1.3 TifrariOJl

5.1.2 Enhanced Dumas method5.1.3 Nlethods using UV-visibIe spectroscopy

5.1.3.1 Direct measurement at 28011111 (AbsOlTJtion \·Icthodl5.1.3.2 BiureT Method5.1.3.3 [0\\"1')' fv1cthod5.1.3.4 PlE binding methods5. ] .3.5 TurbimeTric method

6. Analysis of Carbohydrates7. Analysis ol'Fibcr

7.1 Common Procedures of Sample Preparation7 ') Fiber Determination \1ethod

IX

65

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737',.:J

7373747474

74757575767777

787878797979

7.2.1 Gravimetric I'vlethods7.2.1.1 Crude Fiber Method7.2.1.2 Total, Insoluble and Soluble Fiber :"'lethod7.2.1.3 Chemical }\iIethods

8. References

79797

8080

Chapter 7. Fish Gelatin Production: Extraction lVlethod and Quality AnalysisInvondi Jaslvir. Hammed A. Mansur and HWTlzuh Ai. Saltch

1. Introduction! Extraction of Gelatin

2.1 rvlaterials2.2 fvlethods

2.2.1 Skin preparation2.2.2 Pretreatment2.2.3 Extraction2.2.4 Dehydration

3. Quantitati\e Analysis3.1 Gr3\'imetric method3.2 Soluble protein content method

4. Quality Analysis4.1 Preparation of matured gelatin gel4.2 Gel Strength: detinition and determination4.3 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 proliling4. 7 Analysis of Color, Turbidity and clarity

.5. References

818282828283848487878788898990919J929293

Chapter 8. Application of Fourier Transform Infrared Spectroscopy EdibleFats and Oils AnalysisMohamed Ehvalhig SCiced Alirghuni

1.

2.

Introduction1.2 Analytical and Quality Control1.3 Fourier Transform Infrared (FTIR) Spectroscopy'1.4 Fourier Transformation1.5 Transmission technique1.6 Attenuated total rdlectance (ATR)rvlethodology2.1 Sample preparations

2.1.1 Gases Samples

x

96979798989999100100

2.1.2 Liquid samples2.1 ..3 Solid samples

2.2 fTIR Data Handling Techniques2.2.1 Detection of overlapped bands2.2.2 Smoothing and interpolation2.2.3 Baseline correction2.2.4 Peak intensity measurements2.2.5 Spectn:ll stripping (Subtraction)2.2.6 Ratio method

3. Quantitative Analysis3.1 Beer-Lambert law3.2 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 Lipids4.1 Examples

4.1.1 Slip melting point4.1.2 Residual soap in oil4.1.3 Hexane in solvent extracted 0114.1.4 Palm carotene4.1.5 Sesamol in sesame seed oil4.1.6 Gossypol in cottonseed oil4. t. 7 Aflatoxins4.1.8 Animal fats4.1.9 Adulteration of'edible fats and oils

5. References

100100100100100101101101101102102103103103103104106106106106107107107107

108108109

Chapter 9. In J7t1'o Assay for Investigating Potential Anti-Cancer AgentsTargeting at ~Vletastatic LevelYlIIl1i Zuhanis HaS-YUH Hashim alld Chris J.R. Gill

I.

..,

.:l.

-L

6.7.

IntroductionScopefvlaterial3.1 EquipmenL'Apparatus/SoftwaresPreparation4.IIvlcthodsNotesReferenceList of Abbreviation

xi

115115116116117117118120122

Chapter 10. Homology }lodelling Of Pyranose-2-0xidase fromPhanerochaete ChrysosporiumIbrahim Ali Noorbmcho, A::rarul Ashimah Nllr Malld Dom, Ahmad SidqiHarit!llIddin and Ham::ah Mohd Sol/ch

1, Introduction2. Literature Revic\\

2.1 Biofucl and enzymatic biofuel cells}! Glucose oxidase2.3 PjTanose-2-oxidase2.4 Homology \lodel1ing

3, \Taterials and rvlethods3.1 Homology project design],2 Homology modeling

4. Resu[t and Discussion4.1 Description of the results pairwise sequence4,2 Description of result pairwise sequence alignment4.3 Ramachandran plot statistics

" Structure Validation6. References

123124124124[25125126126126131133162139140141

Chapter 11. Liquid-Liquid Extraction and its Application for Separation ofOrganic AcidsParveel1 Jamal

1. Introduction 1431. 1 Liquid-Liquid Extraction 1431.2 Principle ofExtraction fiTnn Liquids 143

2. Notes and Tips 1442.1 Solvent Selection 1442,2 Propel1ies ofa good solvent 1452,] Precautions 1452.4 Class ofOrganic Compounds and Solubility' factor [452.5 Principle of Extracting Different Compounds 1462.6 Salting Out 148

3. Experimental Part: Separation of acid tl'Olll a mixture containing acid, base and 1493.1 Purpose 1493.2 Learning Objectives 1493.3 Techniques 149

4. \'[aterials 149). Procedure 150

), 1 Flow 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 Acid5.5 Section C: Recrystallization of the Acid

6. Results6.1 Percent Yield Calculation6.2 Example of Data calculation6.3 Calculation

7. References

Chapter 12. Response Surface '\Icthodology (RS"\'1) Design for BioreactorOperationAlaizinvan AIel and Najiah Nadir

151152152152153153154

1. Introduction 1551.1 Design of Experiment J551.2 Response Surface l'v1ethodologyl551.3 Central Composite Design 155l.4 Box-Behnken Design 1561.5 Analysis of Variance (AKOVA) 1561.6 Graphical Analysis 157l.7 Bioreactor Operation 157

2. r..kthodology 1582.1 Preparation of Bioreactor 158

2.1.1 Optimization of Fermentation Parameters for Maximum Ethanol 1582.1.2 Optimization of Dyestuff Adsorption From Aqueous Solution Using 159

2.2 Experimental Design 1592.2.1 Central composite design 1592.2.2 Box-Behnken Design 161

3. Results and Discussion 1613.1 Regression Equation 161

3.1.1 Central composite design 1613.1.2 Box-behnken design 162

3.2 .A..nalysis 1633.2.1 Central composite design 1633.2.2 Box-Behnken Design 164

3.3 Response Surface Curves 1643.3.1 Central composite design 1643.3.2 Box-Behnken Design 166

4. Conclusion 1685. References 168

Chapter 13. Screening Natural Compounds for Antibacterial Activity by DiscDiffusion:VlethodRaha Ahmad RallS

xiii

1. Jntroduction 1702. Materials 1"""1)

I~

,T\lethods 172~1.

3.1 Prepare bacterial inoculums 172.... '1 Adjust inoculums turbidity 172j.",-

'> '> Plating bacterial inoculums on plate 173.J .•1

3.4 Place disc on plate 173.... - Measure inhibition zone and interpretation 173j.)

4. \Jotes 1755. References 175

Chapter 14. Indicator l\licroorganisms: Detection of Col~f'orl1l andEscherichia coliMohamed Ismail Abdul Karim

1.

!...4.

5.6.7.

Introductionl.l Enumeration ?vlethods1.2 Solid l\'lediaEquipment and materials that are needed are as fo11O\'l/sMedia and ReagentsConventional Method for testing growth of coliforms, fecal coliforms and4.1 IvlP\J - Presumptive test for colitorms, fecal coliforms and E.coli4.2 MP\J - Confirmed test for coljforms4.3MP\J - Confirmed test for fecal coliforms andE. coli4.4 IvlP)J - Completed test for E coli.4.5 The IIvIViC Tests4.6 Solid medium method using Red Bile Agar - coliforms4.7 Iv1cmbrane Filtration (\·JF) \.Jethod - coliformsLST-MUG rv1ethod Used for Detecting E coli in Chilled or Frozen FoodsReferencesAppendix

176177177

177178

E. l78178179180180181182183183184186

Chapter 15. Direct l\ucleation Control: A l\ovel Approach for the Control ofCrystal Size Distribution in Crystallization Processes.i.\1ohdRushdi Abu Bakar, Zoltan Karman A'agv, Ali Nauman Salcf'lni ClndChristopher David Rielly

I. Introduction! I'vlaterials3. i\'1ethods

3.1 U ncontro1led Anti-so lvent Addition3.2 D"\JC by Anti-solvent/Solvent Addition

4. ~otes

xiv

190192193193194194

5.6./.

4. I Uncontrolled Anti-so ]vent Addition4.2 D:-JC by Anti-soh ent'So!vcntAdditionReferencesFurther ReadingList of Abbreviation

xv

194194197198198

190

CHAPTER 15

Direct Nucleation Control: A Novel Approach forthe Control of Crystal Size Distribution inCrystallization ProcessesMohd Rushdi Abu Bakar, Zoltan Kannan Nagy, Ali Nawnan Saleemi and Christopher David Rielly

1. IntroductionCrystallisation 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 forsolid-liquid separation due to its capability of producing high purity materials. Inbiophannaceutical industry, the crystallisation operation is often critical because itdetermines the product properties, such as the crystal size distribution (CSD), morphologyand polymorphic form. The CSD is of primary importance since it influences subsequentdownstream operations such as filtration and drying, and the product therapeutic performancesuch as dissolution rate, bioavailability and stability (Abu Bakar, 2010). Driven by theUnited 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 ofsignificant interest.

PAT-based control approaches involve the use of in situ analysers that are able to measureand monitor product quality and critical process properties in real-time. This real-timeprocess information is integrated into a control framework to provide an optimal controlstrategy with the aim ofproducing products with consistent desired quality. In addition to theprovision of kinetic data that are required in the development of a robust process, real-timemonitoring also allows appropriate remedial actions to be taken during the process ifundesirable product quality or process property are identified (Yu et at., 2003).

Lasentec focused beam reflectance measurement (FBRM) is one of the in situ analysersthat has been used extensively in the crystallization processes to provide qualitative as well asquantitative information about crystallisation process, \vhich comprises of nucleation andcrystal 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 where itis finely focused by a rotating lens, which causes the beam to scan in a circular path through asapphire \vindo\v at a fLxed high speed. The beam then passes into the solution under studyand \vhen it hits a solid particle suspended in the solution, light is scattered in manydirections, but only light scattered back towards the probe is collected. The solid particlecontinues 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 diagramofbackscattered light pulses detection and chord length measurement.

From: Experimental Methods In Modern BiotechnologyEdited by. I. A. Noorbatch, H. M. Salleh and M I. A. Karim. /fUM Press, KL, Malaysia

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