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Protocols and
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Protocols and
Applications in
Enzymology
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Seema Anil Belorkar
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Assistant Professor,
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Microbiology and Bioinformatics Department,

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Atal Bihari Vajpayee University,

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Bilaspur (C.G), India

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Sudisha Jogaiah
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Assistant Professor,
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Laboratory of Plant Healthcare and Diagnostics,

PG Department of Studies in Biotechnology and Microbiology,
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Karnatak University, Pavate Nagar, Dharwad, Karnataka, India

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Academic Press is an imprint of Elsevier
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Notices
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Practitioners and researchers must always rely on their own experience and knowledge in
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To the fullest extent of the law, neither the Publisher nor the authors, contributors, or
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methods, products, instructions, or ideas contained in the material herein.

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A catalog record for this book is available from the Library of Congress
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British Library Cataloguing-in-Publication Data

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A catalogue record for this book is available from the British Library
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ISBN: 978-0-323-91268-6
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For information on all Academic Press publications visit our website at

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Contents
Preface ............................................................................................... xvii
CHAPTER 1 Enzymesdpast, present, and future......................... 1

1.1 General introduction ...........................................................1
1.2 Characteristics of enzymes...................................................1
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1.2.1 Enzyme as a molecule................................................ 4
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1.2.2 Catalytical property ................................................... 5
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1.2.3 Specificity................................................................ 5
1.2.4 Holoenzyme ............................................................. 6
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1.2.5 Turnover number....................................................... 7
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1.2.6 Reversibility............................................................. 7
1.2.7 Weight of enzyme ..................................................... 7
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1.2.8 Sensitivity................................................................ 7
1.2.9 Active site................................................................ 8
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1.3 The concept of cell-free fermentation ....................................8
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1.4 Nomenclature of enzymes....................................................8
1.5 Enzymesdthe present scenario.............................................9
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1.5.1 Substrate binding .....................................................10

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1.5.2 Mechanism of enzyme catalyzed reaction.....................10

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1.5.3 Catalysis.................................................................12
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1.6 Recent developments ........................................................ 12

1.6.1 Immobilization ........................................................12
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1.6.2 Biosensors...............................................................12
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1.6.3 Diagnostic tools .......................................................13

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1.6.4 Metagenomics .........................................................13

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1.7 The future....................................................................... 13

References............................................................................. 14
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CHAPTER 2 Distribution and diversity in microbial

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enzymes ................................................................17
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2.1 Origin of enzyme diversity................................................. 17

2.2 Natural niche as a diversity source ...................................... 17
2.3 Evolution and function of enzymes...................................... 20
2.4 Acidophilic enzymes......................................................... 20
2.4.1 Adaptation at cellular level ........................................29
2.5 Alkaliphiles..................................................................... 34
2.6 Halophilic organisms ........................................................ 36
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2.7 The protistda novel enzyme source .................................... 38

2.8 Future directions .............................................................. 39
References............................................................................. 39
CHAPTER 3 Screening of potential microbes for enzymes
of industrial significance........................................47
3.1 History ........................................................................... 47
3.2 High throughput screening methodsdneed of present day....... 47
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3.2.1 Enrichment culture ...................................................48
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3.2.2 Uncultivables as enzyme sourceda
metagenomic approach..............................................49
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3.2.3 Directed enzyme evolution.........................................50
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3.2.4 Computational biology (in silico studies)......................52
3.3 Throughput methods for screening of enzyme variants............ 53
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3.3.1 Selection screening .................................................53
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3.3.2 Agar plate method ..................................................54
3.3.3 Microtiter plate screening method..............................54
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3.3.4 Fluorescent activated cell sorting...............................55
3.3.5 In vitro compartmentalization ...................................57
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3.3.6 Droplets................................................................58
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3.3.7 Plasmid display ......................................................59

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3.3.8 Phage display.........................................................59

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3.3.9 Ribosome display m-RNA........................................61

3.3.10 m-RNA display ......................................................62
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3.3.11 The c-DNA display.................................................62

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3.3.12 Reporter-based screening .........................................63

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3.3.13 Fluorescence-activated droplet sorting........................64

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3.3.14 Digital imaging ......................................................65

3.4 Conclusion and future perspective ....................................... 66
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References............................................................................. 66
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CHAPTER 4 Solid-state fermentation and submerged

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fermentation for enzyme production ........................71

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4.1 History of fermentation for social benefits ............................ 71

4.2 Louis Pasteur and fermentation........................................... 72
4.3 Fermentation ................................................................... 73
4.3.1 SMF fermentation ....................................................74
4.3.2 Solid-state fermentation.............................................79
4.4 Conclusion and future directions......................................... 85
References............................................................................. 85
Contents vii
CHAPTER 5 Laboratory methods for enzyme assays ...................91

Subchapter 5.1 Assay of enzymedfructosyltransferase ............ 91
Before you begin .................................................................... 91
Reagent preparation ........................................................... 91
Key resources table................................................................. 92
Materials and equipment.......................................................... 92
Day 1dproduction of fructosyltransferase enzyme.................. 92
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Preparation of standard curve for reducing sugars ................... 93
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Fructosyltransferase assay ................................................... 94
Quantification of the enzyme ............................................... 94
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Expected outcomes ................................................................. 95
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Quantification and statistical analysis ......................................... 95
Advantages............................................................................ 95
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Limitations ............................................................................ 95
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Optimization and troubleshooting .............................................. 96
Safety considerations and standards ........................................... 96
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Alternative methods/procedures................................................. 96
References............................................................................. 96
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Subchapter 5.2 Assay of enzymedLipase ............................. 98

Before you begin .................................................................... 98
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Reagent preparation ........................................................... 98

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Lipase assay ..................................................................... 98

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Key resources table................................................................. 99

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Materials and equipment.......................................................... 99

Day 1dproduction of lipase enzyme .................................... 99
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Expected outcomes ............................................................... 100

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Quantification and statistical analysis ....................................... 101

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Advantages.......................................................................... 101
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Limitations .......................................................................... 101

Optimization and troubleshooting ............................................ 101
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Safety considerations and standards ......................................... 102

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Alternative methods/procedures............................................... 102

References........................................................................... 102
Subchapter 5.3 Assay of enzymedprotease ........................ 103
Before you begin .................................................................. 103
Reagent preparation ..........................................................103
Key resources table............................................................... 104
Materials and equipment........................................................ 104
Day 1dproduction of protease............................................104
viii Contents
Preparation of standard curve for L-Tyrosin ...........................105

Quantification of the enzyme ..............................................106
Advantages.......................................................................... 107
Limitations .......................................................................... 107
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Subchapter 5.4 Assay of enzymedxylanases....................... 109
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Before you begin .................................................................. 109
Reagent preparation ..........................................................109
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Day 1dproduction of xylanase enzyme................................110
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Preparation of standard curve for reducing sugars ..................111
Xylanase assay.................................................................111
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Advantages.......................................................................... 112
Limitations .......................................................................... 113
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References........................................................................... 114
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CHAPTER 6 Structure and functions of enzyme kinetics........... 115

6.1 Introduction................................................................. 115
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6.2 Gibb’s free energy and enzyme kinetic............................. 117

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6.2.1 Successful collision .............................................. 117

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6.2.2 The activation energy............................................ 117

6.3 The catalyst................................................................. 117
6.4 Enzyme kinetics and features of ECR .............................. 117
6.5 The transition complex in enzyme-catalyzed reaction ......... 118
6.6 The MichaeliseMenten equation..................................... 119
6.7 Advantages.................................................................. 123
6.8 Disadvantages.............................................................. 123
6.9 The significance of Km and Vmax .................................... 123
Contents ix
6.10 Conclusion .................................................................. 126

References........................................................................... 126
CHAPTER 7 Protocols of important industrial enzymes............. 129
Subchapter 7.1 Large-scale production of
enzymedfructosyltransferase ..............................129
Before you begin .................................................................. 129
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Step-by-step method details.................................................... 130
Preparation of inoculum.....................................................130
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Preparation of production medium.......................................130
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Inoculation of the production medium..................................130
Termination of fermentation ...............................................131
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Optimization and troubleshooting ........................................132

Potential solution to optimize the procedure ..........................132
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Advantages.......................................................................... 135
Limitations .......................................................................... 135
x Contents

References........................................................................... 136
Further reading..................................................................... 137
Subchapter 7.3 Large-scale production of enzymedlipase...... 138
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Advantages.......................................................................... 140
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Subchapter 7.4 Large-scale production of protease ............... 142

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Production of enzyme........................................................143
Termination and enzyme extraction......................................143
Advantages.......................................................................... 144
Limitations .......................................................................... 144
Contents xi

References........................................................................... 145
Subchapter 7.5 Small-scale production of
enzymedfructosyltransferase ..............................146
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Subchapter 7.6 Small-scale production of enzymedlipase ..... 150

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Advantages.......................................................................... 152
Limitations .......................................................................... 152
xii Contents

References........................................................................... 153
Subchapter 7.7 Small-scale production of protease............... 155
Before you begin .................................................................. 155
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Preparation of production medium and inoculation.................156
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Termination and enzyme extraction......................................156
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Limitations .......................................................................... 157
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Advantages.......................................................................... 160
Limitations .......................................................................... 160
Contents xiii

References........................................................................... 161
CHAPTER 8 Strategies to improve enzyme activity for
industrial processes............................................. 163
8.1 Introduction................................................................... 163
8.1.1 Improvement of enzyme-based process ...................... 163
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8.1.2 Strategies to achieve efficient biocatalysis .................. 166
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8.1.3 Heterogeneous biocatalysis ...................................... 170
8.1.4 Substrate engineering.............................................. 171
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8.1.5 Advanced strategies for enzyme improvement............. 171
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8.1.6 Altering the signal peptide ....................................... 172
8.1.7 Metagenomics ....................................................... 172
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8.1.8 Genetic engineering................................................ 173
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8.2 Conclusion.................................................................... 174
References........................................................................... 174
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CHAPTER 9 Scope and relevance of industrial applications ..... 179
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9.2 Enzymes in industries ..................................................... 180

9.3 Common strategies for enzyme immobilization ................... 181
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9.3.1 Use of carriers ....................................................... 181

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9.3.2 Covalent linkages ................................................... 181

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9.3.3 Entrapment............................................................ 182

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9.4 Some important industrial enzymes ................................... 182

9.4.1 Fructosyltransferase ................................................ 182
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9.4.2 Lipases................................................................. 184

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9.5 Biocatalytic membranes................................................... 189

9.6 Conclusion.................................................................... 190
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References........................................................................... 190
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CHAPTER 10 Agroindustrial wastes for enzyme production ...... 197

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10.1 Introduction................................................................. 197

10.2 Agroindustrial wastes: excellent raw material for
production................................................................... 197
10.3 Impact of agroindustrial wastes on environment:
a global concern........................................................... 198
10.4 Waste to value ............................................................. 200
xiv Contents
10.4.1 Cellulose........................................................... 200

10.4.2 Hemicellulose .................................................... 200
10.4.3 Lignin............................................................... 200
10.4.4 Starch............................................................... 200
10.5 Fructosyltransferase ...................................................... 202
10.5.1 Cellulases.......................................................... 202
10.5.2 Proteases........................................................... 203
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10.6 Xylanase..................................................................... 205
10.7 Conclusion .................................................................. 207
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References........................................................................... 207
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CHAPTER 11 Significance of enzymes and their agricultural
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applications....................................................... 213
11.1 Introduction................................................................. 213
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11.2 Role of enzymes in soil ................................................. 215
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11.2.1 Alkaline phosphatase........................................... 215
11.2.2 Enzymes as biopesticides..................................... 216
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11.2.3 Enzymes as antimicrobial agents........................... 217
11.3 Conclusion .................................................................. 218
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Subchapter 11.1 Production of enzyme

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biofertilizerdphosphatase ..................................221
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Before you begin .................................................................. 221

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Preparation of screening medium.........................................222

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Inoculation of the screening medium....................................222

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Advantages.......................................................................... 223
Limitations .......................................................................... 223
Contents xv

References........................................................................... 224
CHAPTER 12 Biotechnological applications of enzymes and
future prospective .............................................. 225
12.1 Introduction................................................................. 225
12.2 Enzymes in medical field............................................... 225
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12.2.1 Enzymes and diagnosis........................................ 225
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12.2.2 Desirable features of diagnostic enzyme ................. 227
12.2.3 In disease diagnosis ............................................ 227
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12.2.4 Coupled enzyme assay......................................... 229
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12.2.5 Immunological reactions...................................... 229
12.2.6 DNA-based diagnostic......................................... 229
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12.2.7 Therapeutic enzymes........................................... 230
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12.3 The biological detergents: a revolution in laundry
industry ...................................................................... 232
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12.3.1 Proteases........................................................... 234
12.3.2 Lipase............................................................... 235
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12.3.3 Amylases .......................................................... 235
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12.3.4 Cellulases.......................................................... 235

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12.4 Conclusion .................................................................. 236

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References........................................................................... 237
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CHAPTER 13 Conclusion ........................................................ 239

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Index...................................................................................................243
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Preface
Protocols and applications in enzymology is a book presenting intensive, coordi-

nated, and synchronized information with the perfect blend of historical informa-
tion, thrust areas in research, and the resultant protocols much desired in the
academics and research labs.
This book is a one-stop solution for students, academicians, research beginners,
50
and entrepreneurs. The initial two chapters reveal the glorious history of enzy-
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mology with the pioneering research and its deep-rooted connection with the discov-
eries by eminent chemists. It explains the diversity an enzyme molecule exhibits and
48
their implications on industrial processes.
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The book describes various stages of screening technologies, conventional Vs
modern, fermentations, and their types and provides exclusive research-based pro-
25
tocols for industrially important enzymes which are desperately required in research
labs and industrial research units. Thus, this book is a link to protocols desired by the
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industries as the intensive outcome of the research community. It also elucidates the
“Waste to value” term by discussing conversion of the trapped energy in wastes into
bioactive molecules. 的
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The book will hopefully update the readers from agricultural to industrial sector
with recent advances in usage of enzymes as frontier tools and finally focusing on
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applications of enzymes in different walks of life and future applications.

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(Dr. Seema Anil Belorkar and Dr. Sudisha Jogaiah)

Authors
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CHAPTER
Enzymes—past, present,
and future
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1.1 General introduction
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Enzymes today need no introduction. They are well recognized and acknowledged
25
in every walk of life from a layman to a research person entangled in molecular
biology protocols. These magical molecules have proved to be an efficient tool in
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almost all industries directly or indirectly.
The term “Enzyme” was proposed by Wilhelm Kuhne, which in Greek means
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“leavened” or “in yeast” in 1877. Enzymes have gained recognition as a Biological
catalyst, which is generally proteins with the exceptions of ribozymes. The nature
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and activity of any enzyme depend upon the sequence of amino acids of the polymer.
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The enzymes can be active as a single chain (monomeric protein) or can have a num-
ber of polymeric chains in form of a functional complex (oligomeric protein). The
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function of an enzyme is dependent on its structure and conformation that in turn

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depends on the amino acid sequence (Berg et al., 2002). The structure of any enzyme
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is a very sensitive and adaptive feature in context to its changing environment. It

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cannot be the sole feature governing its function (Somero, 1978).

The basic development of enzymology is accredited to pioneering observations
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in the field that are enlisted in Table 1.1.

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1.2 Characteristics of enzymes

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Enzymes are biological catalysts. They catalyze biological reactions in a similar

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manner as Chemical catalysts do. The enzymes transform their substrates (reactants)
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to products under normal conditions of temperature and pressure in contrast to

chemical catalyst. Fig. 1.1 elucidates the general path of enzyme action. It is a
two-step reaction. The first step is reversible and forms an unstable ES complex.
If the substrate is specific, the reaction progresses for the second step; otherwise,
the breakdown causes the release of the substrate and a backward reaction takes
place. The second step is irreversible and progresses only in the forward direction
when the enzyme finds its specific substrates.
Protocols and Applications in Enzymology. https://doi.org/10.1016/B978-0-323-91268-6.00007-7 1

Copyright © 2022 Elsevier Inc. All rights reserved.
2 CHAPTER 1 Enzymes—past, present, and future
Table 1.1 Pioneering contributions in enzymology.

Year Noble laureates Contributions
2018 Frances H. Arnold The directed evolution of enzymes
George P. Smith The phage display of peptides and antibodies
Sir Gregory P. Winter
2017 Jacques Dubochet Developing cryo-electron microscopy for the high-
Joachim Frank resolution structure determination of biomolecules
Richard Henderson in solution
50
2016 Jean-Pierre Sauvage The design and synthesis of molecular
05
Sir J. Fraser Stoddart machines
Bernard L. Feringa
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2015 Tomas Lindahl Mechanistic studies of DNA repair
Paul Modrich
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Aziz Sancar
2012 Robert J. Lefkowitz Studies of G-protein-coupled receptors
25
Brian K. Kobilka
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2009 Venkatraman Studies of the structure and function of the
Ramakrishnan ribosome
Thomas A. Steitz
2008
Ada E. Yonath
Osamu Shimomura
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The discovery and development of the green
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Martin Chalfie fluorescent protein, GFP
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Roger Y. Tsien
2006 Roger D. Kornberg The molecular basis of eukaryotic transcription
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2002 John B. Fenn Development of soft desorption ionization methods

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Koichi Tanaka for mass spectrometric analyses of biological

macromolecules
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Kurt Wüthrich Development of nuclear magnetic resonance

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spectroscopy for determining the three-dimensional

structure of biological macromolecules in solution
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1997 Paul D. Boyer Elucidation of the enzymatic mechanism

John E. Walker underlying the synthesis of adenosine triphosphate
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(ATP)
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Jens C. Skou For the first discovery of an ion-transporting

enzyme, Naþ, Kþ-ATPase
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1993 Kary B. Mullis Invention of the polymerase chain reaction (PCR)

method
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Michael Smith Oligonucleotide-based, site-directed mutagenesis

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and its development for protein studies

1990 Elias James Corey The theory and methodology of organic synthesis
1989 Sidney Altman Discovery of catalytic properties of RNA
Thomas R. Cech
1975 John Warcup Cornforth The stereochemistry of enzyme-catalyzed reactions
Vladimir Prelog The stereochemistry of organic molecules and
reactions
1974 Paul J. Flory The physical chemistry of the macromolecules
1.2 Characteristics of enzymes 3
Table 1.1 Pioneering contributions in enzymology.dcont’d

Year Noble laureates Contributions
1972 Christian B. Anfinsen Ribonuclease, especially concerning the
connection between the amino acid sequence and
the biologically active
Stanford Moore conformation
William H. Stein The connection between chemical structure and
catalytic activity of the active center of the
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ribonuclease molecule
1970 Luis F. Leloir Discovery of sugar nucleotides and their role in the
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biosynthesis of carbohydrates
1969 Derek H. R. Barton The development of the concept of conformation
48
Odd Hassel and its application in chemistry
72
1965 Robert Burns Woodward Achievements in the art of organic synthesis
1964 Dorothy Crowfoot X-ray techniques of the structures of important
25
Hodgkin biochemical substances
1963 Karl Ziegler The chemistry and technology of high
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Giulio Natta polymers
1962 Max Ferdinand Perutz The structures of globular proteins
1961
John Cowdery Kendrew
Melvin Calvin
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The carbon dioxide assimilation in plants
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1958 Frederick Sanger Structure of proteins, especially that of insulin
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1957 Lord (Alexander R.) Todd Nucleotides and nucleotide coenzymes

1956 Sir Cyril Norman Mechanism of chemical reactions
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Hinshelwood
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Nikolay Nikolaevich
Semenov
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1955 Vincent du Vigneaud The first synthesis of a polypeptide hormone

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1953 Hermann Staudinger Macromolecular chemistry

1950 Otto Paul Hermann Diels Discovery and development of the diene
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Kurt Alder synthesis

逊
1946 James Batcheller Sumner Enzymes can be crystallized

John Howard Northrop Preparation of enzymes and virus proteins in a pure
马
Wendell Meredith Stanley form

亚
1929 Arthur Harden The fermentation of sugar and fermentative

Hans Karl August Simon enzymes
找
von Euler-Chelpin
代
1926 The (Theodor) Svedberg Disperse systems

1909 Wilhelm Ostwald Catalysis and for his investigations into the
fundamental principles governing chemical
equilibria and rates of reaction
1907 Eduard Buchner Biochemical researches and his discovery of cell-
free fermentation
1902 Hermann Emil Fischer Recognition of the extraordinary services he has
rendered by his work on sugar and purine syntheses
FIGURE 1.1
General mechanism of enzyme-catalyzed reaction.
50
1.2.1 Enzyme as a molecule
05
Enzymes are macromolecules having a high molecular weight (MW) and are dispro-
portionately bigger when compared to their substrate. Generally, the MW of en-
48
zymes covers a broad range. The general properties in relation to its functions are
72
explained in Fig. 1.2.
Dixon and Web (1958) tabulated enzymes in three series or classes (2n 12,000,
25
2n 16,000, and 2n 19,000), where n is integral 0e4. A hypothesis was proposed
qq
by Wright (1962) using experimental data but as the groups of enzymes were
formed, there remained a question of Biasedness. Johnston et al. (1945) proposed
a better approach of the correlation function for validation of the Svedberg hypoth-
的
esis (Svedberg and Pedersen, 1940). The MW of proteins is calculated by Svedberg’s
有
equation depending on the force applied and the mass of the sedimenting molecule.
没
ib
zl
书
子
电
逊
马
亚
找
代
FIGURE 1.2
Properties and functions of enzymes.
The sedimentation velocity was fundamentally used for the determination of molec-
ular weights of proteins. Presently, an approach using Gel filtration Chromatography
and SDS-PAGE are the advanced methods for the MW determination.
As we all know that most enzymes are proteins, their general behavior is
colloidal as an attribute of their high molecular weight. Enzymes that are proteina-
ceous are thermolabile in nature. Generally, enzymes are active at room temperature,
at low temperatures they are reversibly inhibited, and at higher temperatures they are
irreversibly inactivated.
50
Although, this temperature sensitivity can be handled, if enzymes are stored in
the form of dried extracts. The stability of the enzymes increases along with
05
increased shelf life. Every enzyme requires amicable pH and temperature conditions
48
to explicit its highest activity referred as optimum pH and temperature. In zones
before and after the optimum zone, the enzyme activity decreases. Fig. 1.3 explains
72
the maximum enzyme activity in response to optimum pH and temperature
25
conditions.
qq
1.2.2 Catalytical property
的
A most striking feature of the enzyme is its catalytic power that makes it a robust
synthetic tool. In comparison to chemical catalysts, enzymes are wonderful because
有
they catalyze the transformation of substrates at normal temperature and pressure
没
available inside the biological system. Enzyme caters high potential of substrate
conversion as compared to reactions that run by themselves. It remains unaltered af-
ib
ter the reaction is complete, providing the alternative of its recycling for better yield.
zl
书
1.2.3 Specificity
子
The uniqueness of the enzyme is its structural conformation that renders the quality
电
of being an effective catalyst. The catalysis conferred by the enzyme is very focused
逊
马
亚
找
代
FIGURE 1.3
Sensitivity of the enzymes toward pH and temperature.
Table 1.2 Different types of specificities exhibited by enzymes.

Types of enzyme specificities
Bond specificity Enzyme Action on
(relative specificity) Peptidase Peptide bond
Lipase Ester bond
Group specificity Enzyme Action on
(structural Pepsin Peptide bond linked to aromatic amino
specificity) acids like phenylalanine, tyrosine, and
50
tryptophan
05
Substrate Enzyme Action on
specificity Sucrase Sucrose
48
(absolute Arginase Arginine
specificity) Carbonic anhydrase o carbonic acid.
72
Optical specificity Enzyme Action on
(stereo-specificity)
25
L-amino acid oxidase L-amino acids
a-amylase a-1-4 glycosidic linkage of starch and
qq
glycogen
(Cofactor Enzyme Cofactor
specificity) Glucose-6-phosphate NADH
dehydrogenase 的
有
(Geometric Enzyme Substrates
specificity) Alcohol dehydrogenase Methanol and n-propanol to aldehydes.
没
can oxidize
ib
Chymotrypsin Peptide bond and ester bond

zl
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子
and confined to either one or a group of structurally related molecules. The speci-
ficity can be classified into absolute specificity and relative specificity. Nowadays,
电
enzymes are finding immense applications on grounds of Regioselective and Stereo-

逊
selective (Mu et al., 2020). Table 1.2 explains the specificities of the different
马
enzymes along with examples falling in each category.

亚
1.2.4 Holoenzyme
找
Many enzymes have a nonprotein part along with them. Such conjugated enzymes
代
found associated with another kind of molecules are called the holoenzyme. The
protein component is called as the apoenzyme, and the nonprotein component is
called the cofactor. If the cofactor is inorganic in nature, it is referred as prosthetic
group and if it is organic in nature, it is called as coenzyme. The prosthetic groups
are intensely associated with the apoenzyme and are not easily available. Contrarily,
the coenzymes are very loosely bound resulting in easy separation from the protein
component.
The cofactor is a helper micromolecule that confers enzyme, its functionality.
The nonprotein nature makes it thermostable. The main role of cofactor is it
becomes a significant part in the reaction center of the enzyme for catalysis
involving the removal of functional groups. Cofactors have the ability to get associ-
ated with other enzymes and help their functionality after association.
1.2.5 Turnover number

As we all know, enzymes are biological catalysts that remain unaltered after the re-
action is complete. The rate of enzyme catalyst reactions depends upon the number
50
of substrate molecules converted into the product in a given specified time. Turnover
number is therefore defined as the amount of substrate converted into the product in
05
1 s, in the given specified condition (where the concentration of substrate is in satu-
ration). Enzymes exhibit a wide variety of turnover numbers ranging from 0.5 to 105.
48
72
1.2.6 Reversibility
25
When we focus on catalyzed reactions, the enzyme catalyzed reactions differ from
qq
the chemical catalyst in a way that it functions according to the need of the biolog-
ical system. The nature of the enzyme varies in accordance with the role it is
executing in the metabolism. Therefore, enzyme-catalyzed reactions include unidi-
的
rectional, bidirectional types, and even eegulatory reactions.
有
没
1.2.7 Weight of enzyme

ib
Most of the enzymes range between 20 kD and 60,000 kD molecular weight. The 3-
zl
D structure is visualized in terms of a macromolecule with 327 nm in size and even-

tually appears to be very large in context to the cellular environment. The review of
书
the enzyme revealed the large size of the enzyme offers a large surface area of the
子
molecule to circumvent the binding sites of substrates with appropriate orientation.

There are reports of two esterases very small molecular weight enzymes from
电
Candida lipolytica and Bacillus stearothermophilus with 5.7 kDa (56 amino acid
逊
residue) and the Bacillus enzyme is 1.57 kDa (17 amino acids) (Mattey et al.,
马
1998). The highest MW enzymes reported are from Y. lipolytica with a molecular
weight of 120 kDa, the 90 kDa leucine aminopeptidase II from Aspergillus oryzae
亚
(Nicaud et al., 2002), and glucoamylase from Arxula adeninivorans (Swennen

找
et al., 2002).
代
1.2.8 Sensitivity
Yet another feature of enzymes is that they are very sensitive to the environment in
which they are present. The environmental conditions impose tremendous influence
on the three-dimensional structure of the enzyme molecule and hence affect its ac-
tivity and catalytic power to the extent whether it will remain in active state or be
inactive.
The pH, temperature, and concentration of ions are few factors that impede the
biological functioning of the molecule. These consequences are due to the
disturbance of some vital bonds required for the architectural makeup of the cata-
lytic site or the active site of the enzyme, rendering it inactive or denatured. Every
enzyme has a unique sense of tolerance toward a particular limit of pH and temper-
ature or concentration of ions. The tolerance capacity of enzymes is naturally
inherited by the enzyme from the environment it belongs.
1.2.9 Active site
50
Most of the enzymes are high molecular weight molecules, and the majority of their
monomeric amino acids are for maintaining their three-dimensional conformation.
05
They play a structural role. The functional role is played by few amino acids which
48
participate in the catalytically active center.
The three-dimensional conformation creates events or crevices that are narrow
72
and stereospecific for the substrate. The dimensional congruence of the active site
25
and the substrate structure is the foundation for the wonderful specificity phenomena
observed between the enzyme and the substrate. Although the substrate-binding site
qq
and the catalytic centers are different, they reside close to one another inside the
microcatalytic environment of the enzyme molecule. The regulatory enzymes
的
possess an additional site for binding of the modulator molecule to execute positive
or negative regulatory response as the case may be.
有
没
ib
1.3 The concept of cell-free fermentation

zl
Enzyme received acknowledgment due to Edward Buchner (1897) with the Founder
书
enzyme for which he received Nobel Prize. He was the first person to use the prefix
of the substrate to name the enzymedfor example, protease acts upon proteins as
子
substrate, lipase on lipids as substrate, and so forth. Alternative nomenclature de-

电
pends on the type of reaction catalyzed such as polymerase, dehydrogenase, and

逊
oxidoreductase. Thus, the initial pattern of nomenclature depended upon either

the type of the substrate or the type of the reaction used as a prefix for ase.
马
亚
找
1.4 Nomenclature of enzymes

代
A system for the nomenclature of enzymes was developed by the International

Union of Biochemistry and Molecular Biology. The enzymes are assigned a Com-
mission number called as the EC number. The enzymes are divided into six classes
(Florkin and Stotz, 1965), but in 2018, Class 7 was introduced for Translocases
(Tipton, 2018). Every enzyme is designated a four number sequence, which is called
as the EC number for the enzyme. In the Commission number, every digit signifies
information regarding the enzyme classification. The current classes of enzymes are
categorized in Table 1.3.
1.5 Enzymesdthe present scenario 9
Table 1.3 Enzyme nomenclature with recent advances.

Class of
enzyme Name of class Reaction catalyzed
EC 1 Oxidoreductases Oxidation/reduction reaction
EC 2 Transferases Transfer or exchange of certain groups between
substrates
EC 3 Hydrolases Substrate hydrolysis
EC 4 Lyases Removal of a group or its reverse reaction
50
EC 5 Isomerases Interconversion of isomers
05
EC 6 Ligases Synthesis of one product by combining two molecules
of substrate (exergonic reactions)
48
EC 7 Translocases Transmembrane movement of ions or ionic separation
(Tipton, 2018)
72
25
qq
Table 1.4 Enzyme classes with reaction catalyzed.
Class of enzyme Name of class Reaction catalyzed
EC 1 Oxidoreductases 的
Ared þ Box # Aox þ Bred
有
EC 2 Transferases A B þ C/A þ B C
EC 3 Hydrolases A B þ H2 O/A H þ B OH
没
EC 4 Lyases A B#A þ B
ib
ðreverse reaction : synthaseÞ

zl
EC 5 Isomerases A B C#A C B
A þ B þ ATP/A B þ ADP þ Pi
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EC 6 Ligases
EC 7 Translocases
子
电
逊
The first number signifies to which class the enzyme belongs to, for example, EC
1, it denotes the enzyme is oxido-reductases. EC 1 denotes that all the enzymes clas-
马
sified in this class will catalyze the reactions of either oxidation or reduction. Ta-
亚
ble 1.4 helps us to understand the major classes and their subclasses under the
IUB system of nomenclature. The classes are further divided into subclasses that
找
have a differentiating feature like the type of substrate or the type of product or
代
the mechanism of a chemical reaction. On a suitable basis, the enzyme is assigned

the second digit known as the subclass name. Table 1.4 provides an overview of the
reactions catalyzed by each class of enzyme.
1.5 Enzymesdthe present scenario

In the light of the knowledge of the past, we now look at enzymes as an efficient
bioconversion tool (Toone, 2006). The role of enzymes in biological processes
and their support system for cellular metabolism has been fully revealed. Presently,
an upcoming field of enzymology is the intense analysis of pseudo enzymes. This
branch recognizes enzymes that were catalytic in the past, but, in the process of evo-
lution, these proteins have lost their catalytic ability. Active sites and their amino
acid sequence signify their past involvement in catalysis.
Ribozymes are another type of enzyme molecule that has an immense potential
to be explored currently. The enzymes are commercially used for the synthesis of
industrially important products proven to have economic value due to their extreme
50
application in normal human life. Already, enzymes are in the market proving as an
excellent aid for the synthesis of commercial products like antibiotics, medicines, in
05
bakery for hydrolysis, tenderization of meat.
48
1.5.1 Substrate binding
72
The substrate-binding site is a site that is near the active center of the enzyme where
25
the actual catalysis takes place. The common step for initiation of catalysis reaction
qq
is that the substrate must bind to the enzyme. The specificity of binding to a partic-
ular substrate is exerted by the enzyme as an attribute of the complementary shape,
the charge, the hydrophobic or hydrophilic interactions between the substrate, and
的
the enzyme amino acids present in the catalytic in its catalytic center.
有
Specificity is finely demonstrated by the replicating enzymes as well as the
没
proofreading enzymes inside the cell as they cannot afford to cause a single mistake
during the process that they catalyze. The minimal error that is registered by these
ib
enzymes comes to a rate of less than 1 error in 100 million reaction polymerase. The
zl
polymerases are examples of such wonderful specificity.

书
The proofreading mechanism is also found in RNA polymerase, aminoacyl-t-

RNA synthetases, and ribosomes but when compared, the RNA polymerases have
子
a lesser degree of specificity as compared to other DNA polymerases.

电
Alternatively, some enzymes show a broad sense of specificity toward a group of

substrates that are structurally related. They are active upon all the substrates that
逊
have a similar bond or similar functional group and generally are a part of a cascade
马
of reactions.
亚
1.5.2 Mechanism of enzyme catalyzed reaction

找
A normal enzyme catalyzed reaction occurs in two steps. In the initial step, the sub-
代
strate binds to the enzyme and forms an intermediate that is stable and is called as an
enzyme-substrate complex. The enzyme and substrate are specific, then the second
part of the reaction proceeds in which the product is formed and the enzyme is liber-
ated unaltered.
The first step is a reversible reaction and if the substrate is not specific, the
enzyme and the substrate break off back into free enzyme and substrate. If the spec-
ificity is between the enzyme and the substrate that it proceeds in the forward reac-
tion where the product is formed. The second step is an irreversible step, leading to
product formation.
1.5 Enzymesdthe present scenario 11
50
05
48
72
25
FIGURE 1.4
Models explaining the mechanism of enzyme-catalyzed reaction. (A) Lock and key model
qq
explains only the specificity of enzymes. (B) Induced fit model explains the specificity and
flexibility of enzymes.
的
有
To explain the specificity of the enzyme and the mechanism of how the enzyme
没
works, two models were proposed namely the lock and key model and induced fit
ib
model. Fig. 1.4 explains the two models proposed for explaining the key mechanism
of enzyme-catalyzed reactions.
zl
书
1.5.2.1 The lock and key model

The characteristics of enzymes were well explained through the lock and key model
子
by Miller Fisher (1894). In this model, he proposed that the enzyme is a type of lock
电
and the substrate is a type of key. The specificity that exists between the lock and the
逊
key correlates with the specificity of the enzyme and the substrate. As a particular
key is meant to open for a specific lock, the same relationship exists between the
马
enzyme and the substrate. This model was successful in explaining the specificity
亚
existing between the enzyme and the substrate but it proposed the enzyme as a rigid
molecule and therefore could not have the explanation of the transition state that
找
existed in the enzyme-catalyzed reaction.

代
1.5.2.2 Induced fit model

This was the second model proposed in 1958, by Daniel Koschland. This model
considered enzymes as flexible structures and referred that the active site of the
enzyme had the ability to change its shape due to the presence of its specific sub-
strate or upon interaction with the specific substrate. When specific substrate ap-
proaches the enzyme molecule, it induces a structural change in the conformation
of the enzyme which then favors a proper binding of the substrate with the enzyme
molecule to form the enzyme-substrate complex.
Another random document with
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(3.) Brooklyn.
(4.) Queens, consisting of that portion of Queens County to

be incorporated into the Greater New York.
(5.) Richmond, that is, Staten Island.
Power is given to the Municipal Assembly to subdivide these

Boroughs still further, in case of need. The Greater New York
will start with these five Boroughs for administrative
purposes. Your Committee have reconstructed the Borough
system, as submitted in the tentative draft, upon lines which
we are of one accord in believing to be a better and more
appropriate development of the plan for the Greater New York.
These lines give to each Borough various boards through which
the prosecution of local improvements may be facilitated
within the limits of small districts, but reserve to the
Municipal Assembly the right to incur indebtedness and to
authorize the making of contracts."
The draft thus prepared was subjected to criticism in the

commission and in public hearings, and, after amendment and
revision, was reported to the Legislature in February, 1897,
as the charter recommended by the Commission for the
consolidated city called "The Greater New York." It received
some amendment and was passed. On submission, as required by
the State constitution, to the mayors of New York and Brooklyn
and the mayor and Common Council of Long Island City, it was
approved in Brooklyn and Long Island City, but returned
without approval by the mayor of New York. The Legislature
then re-enacted the bill, and it was made law, by the
governor's signature, on the 4th of May, 1897.
NEW YORK CITY: A. D. 1897 (September-November).

Election of the first Mayor of Greater New York.
The first municipal election in Greater New York excited a
passion of interest that was natural in the city itself, but
extraordinary in the degree and the extent to which it spread,
not only throughout the United States, but widely in the
foreign world. The election was looked upon as the test of a
vastly important experiment in the democratic government of an
enormous city. The charter of the great consolidated
municipality had lodged tremendous, unprecedented power and
responsibility in the office of its mayor. The people were
given an opportunity to determine by a single act of suffrage—
by their choice of a single man—the character of their
government. Would they choose that man, at the beginning of
the new system, in the interest of the corrupting organization
in party politics which had misruled the old city of New York
for years, or would they rise to the grand opportunity
afforded them, and set a strong, free, independently honest
man at the head of their local government. Democracy in
municipal affairs, at least, had never been put on trial so
sharply before. To a great number of the citizens of New York
the duty of the hour was plain, and they promptly set their
hands to it. Many months before the election they began the
organization of a Citizens' Union, in which men of all
political parties, sinking every other difference, should join
for the defeat of Tammany and "Boss" Croker, and for the
election to the mayor's office of the best mayor to be found.
With remarkable unanimity, their thought of the man turned to
Seth Low, President of Columbia University, but one time mayor
of Brooklyn, where his vigor, his firmness and his
independence had been conspicuously proved. An extensive
canvass of the city showed so widely spread a feeling in favor
of Mr. Low that he was named at the beginning of September as
the candidate of the non-partisan Citizens' Union. It was
hoped that the whole opposition to Tammany Hall could be
united in support of Mr. Low, representing as he did no
partisan hostility to any organization in national or state
politics. It was especially hoped and believed that the
Republican party organization would endorse the choice of the
Citizens' Union and make Mr. Low (himself a strong Republican)
its own candidate. By nothing less than a general combination
could the compact forces of Tammany Hall be overcome, and that
fact was well understood.
{324}
It was a fact so plain, indeed, that when the head of the
Republican organization in New York persisted in setting a
party candidate in the field, to divide the opposing voters of
the city, there seemed to be small doubt of the intention with
which it was done. The master politicians of the party were
evidently more willing that the vast powers of the mayoralty,
in the organization of the government of Greater New York,
should be wielded by their prototypes of Tammany than that
they should be given to independent hands. The party was
obedient to them, and General Benjamin F. Tracy was put
forward, by a Republican convention held September 28, in
opposition to Mr. Low. The night previous, another candidate
had appeared, in the person of Mr. Henry George, author of the
economic doctrine of the "single tax," supported ardently by a
large following, especially in the Democratic party. A section
of that party, organized under the name of the United Democracy,
had nominated Mr. George, and his nomination was endorsed a
week later by a great assembly which claimed to represent the
Jeffersonian Democracy of New York. On the 30th of September
the nomination of the Tammany Democracy was given to Judge
Robert A. Van Wyck. Between these four principal candidates,
the result of the election was only put in doubt by some
question as to the strength of the Democratic vote which Mr.
George would draw away from Judge Van Wyck. It was a question
extinguished sadly, three days before the election, by the
sudden death of Mr. George. He had not been in good health,
and the strain of the exciting canvass broke him down. His
followers made a hasty nomination of his son, Henry George,
Jr., in his place; but the personal prestige which might have
carried a large vote with them was lost. Of the triumph of
Tammany there was no longer any doubt, and no surprise was
felt (though abundant grief and anger found expression) when
the returns of the voting on November 2d were announced. Judge
Van Wyck was elected by the ballots of 233,997 citizens, against
151,540 cast for Mr. Low, 102,873 for General Tracy, 21,693
for the younger Mr. George. Tammany would have been beaten if
the Republican vote had gone to Mr. Low. Besides the four
principal candidates here named, there were four other
nominees who received small numbers of votes. Lucien Sanial,
put forward by the Social Democrats, received 14,467; William
T. Wardwell, named by the Prohibitionists, received 1,359;
Patrick J. Gleason and Alfred B. Cruikshank, running with
little more than some personal support, received a few
hundreds of votes each.
NEW YORK CITY: A. D. 1899 (April-December).

The Mazet Investigation.
An investigation of charges against the city government, by a

committee of the Legislature, Mr. Robert Mazet, chairman, was
opened in April, 1899, the examination of witnesses being
conducted by Mr. Frank Moss. The investigation followed lines
much the Same as those pursued by the Lexow committee, in
1894, and revealed much the same foul state of things,
especially in the department of police. But there was
evidently less earnestness in the committee; the probing of
iniquities was fill less thorough, and the whole proceeding
was stopped with suspicious suddenness as soon as it drew near
to prominent members of the party by which it was controlled.
It called fresh attention to the rottenness in municipal
politics, and it led to the creation of a new commission for
the revision of the Greater New York charter; but otherwise it
was most unsatisfactory.
NEW YORK CITY: A. D. 1899-1900.

The Ramapo Water Contract.
In August, 1899, Bird S. Coler, Controller of the City,

exposed a gigantic scheme of plunder involved in a contract
with the Ramapo Water Company, which Tammany officials,
assisted, it was said, by some interested Republicans, were
attempting to crowd through the Board of Public Improvements.
The contract would have bound the city for forty years to pay
to the Ramapo Company $70 per million gallons for 200,000,000
gallons of water daily. In his Message to the State
Legislature, January 2, 1901, Governor Odell thus referred to
the matter: "Under chapter 985 of the laws of 1895, as
amended, the Ramapo Water Company was given the power of
condemnation for the purpose of securing to it the water and
lands necessary for its purposes. During the year 1899 an
attempt was made to enter into a contract with this company by
the municipal board empowered to make such contracts. This
proposition, when presented to the citizens of New York, was
severely criticised by them, and the question of continued
municipal ownership of their water supply was thus brought to
their attention. The Legislature of 1900 enacted a law which
made the consummation of such a contract impossible without
the unanimous consent of those empowered to make such
contract. The ownership of water rights sufficient to provide
the city of New York with an ample supply of pure and
wholesome water should be entirely under the control and
direction of the municipality." Action on the subject was
taken by the Legislature, which, in March, repealed the Act of
1895, thus stripping the Ramapo Company of its extraordinary
powers.
NEW YORK CITY: A. D. 1900 (January-September).

The Rapid Transit Tunnel Contract.
Projected Tunnel to Brooklyn.
"The great project of underground rapid transit is now an

assured thing. A few months ago the prospect seemed very dark.
It is true that the rapid transit commissioners, a very able
and upright body of men, with the invaluable aid of a
distinguished engineer, Mr. Parsons, had a good while ago
decided on the route and the plans; but the way seemed blocked
by a series of semi-political and semi-legal difficulties. …
Suddenly these difficulties began to disappear. … The
financial plan adopted was that the city should provide the
money which a contractor would expend in building the road,
the contractor following the plans furnished by the city,
submitting to municipal inspection, and agreeing upon his part
to pay the interest on the bonds sold by the city to obtain
the money, and also to pay enough into a sinking fund to
provide for the ultimate redemption of the bonds. Bids were
called for on November 15, to be opened on January 15. … It
turned out that two well-known contractors were the only
bidders, and the award was given to Mr. John B. McDonald. His
bid was $35,000,000. The theory of this contract is that the
road is to be the property of the city, leased for fifty years
to the contractor, who is to pay a rental that will be large
enough so that the taxpayers will not have expended a penny. …
{325}
The main trunk line will start at the post-office (City Hall
Square) on the south and proceed northward along the spine of
Manhattan Island, following the general direction of Broadway
to Kingsbridge, a distance from the point of beginning of
twelve or thirteen miles. Near the upper end of Central Park,
at a distance of six or seven miles from the point of
beginning, a branch of the tunnel road will take a
northeasterly direction, terminating at Bronx Park, which is
about the same distance north as Kingsbridge, but several
miles further east. The road will have four tracks for six
miles of main line, two of which will be used for local trains
and two for express trains."
American Review of Reviews,

February, 1900.
Work on the great undertaking was begun promptly, and had made
great progress within the first twelve months.
In September, 1900, preliminary steps were taken toward the

construction of a connecting tunnel, under the East River, to
Brooklyn, and through the congested districts of the latter
borough. "At least three years will be necessary for the
preliminary work and actual construction before trains are
running. … Tentative estimates have been made, and these are
said to be from $8,000,000 to $10,000,000. … The route as
contemplated … starts in connection with the Manhattan
proposed tunnel at a point at or near the intersection of
Broadway and Park Row; thence under Broadway and Bowling Green
to Whitehall Street; under Whitehall Street to South Street;
thence under South Street to the East River, and under the
river, striking the Brooklyn shore at a point in Joralemon
Street between the East River and Furman Street, under
Joralemon Street to Fulton Street, to the Borough Hall, out
Fulton Street to Flatbush Avenue, and under this thoroughfare
to the railroad station. On the New York side the route
includes a loop to be built whose debouching point shall lie
between Bowling Green and Exchange Place in Broadway, running
under Broadway to Bowling Green, and thence under Bowling
Green to State Street, to and under Battery Park to Whitehall
Street, thence returning under Whitehall Street and Battery
Park to State Street and to Broadway. The construction calls
for two tracks, and avoids all grade crossings, each track to
have a separate tubular tunnel."
New York Times,

September 28, 1900.
On the 25th of January, 1901, announcement was published that

the Board of Rapid Transit Commissioners had adopted a
resolution definitely providing for the extension of the Rapid
Transit Railroad to Brooklyn. The original plan of route in
Brooklyn had been chosen. The only change made was in
Manhattan. The trains would be run through State St. instead
of Whitehall, as formerly planned, with a loop at the Battery
for Manhattan trains.
NEW YORK CITY: A. D. 1900 (April-May).
Ecumenical Conference on Missions.
See (in this volume)

MISSIONS.
NEW YORK CITY: A. D. 1900 (June).

Great fire at the Hoboken piers.

HOBOKEN.
NEW YORK CITY: A. D. 1900-1901.

Revision of the charter.
Carefully as the Greater New York charter had been drawn, it

proved unsatisfactory in the working, in various respects, and
a commission to revise it was appointed in 1900. The report of
the commission was submitted to the Governor on the 1st of
December, and transmitted, with his approval, to the
Legislature in the following month. In the hands of the
Legislature, the bill embodying the revised charter underwent
considerable amendment, very much, it would seem, to its
detriment. It was passed by the Senate on the 3d of April and
by the Assembly on the 4th, and went to the Mayor of New York
for the submission to his judgment which the State
Constitution of New York requires. Some of the more important
changes in the charter made by the revision, as passed, are
the shortening of the mayor's term of office from four years
(which the revision commission had advised retaining) to two
years, with eligibility for re-election (which the commission
had advised against); an increase of the administrative powers
of the presidents of boroughs; abolition of the municipal
Council and creation of a Board of Aldermen of 73 members;
reorganization of various departments of the municipal
administration.
NEW YORK CITY: A. D. 1901 (March).
Offered gift of $5,200,000 to the Public Library
by Andrew Carnegie.

LIBRARY, NEW YORK PUBLIC.
NEW YORK STATE: A. D. 1894.

The revised Constitution.

CONSTITUTION OF NEW YORK.
NEW YORK STATE: A. D. 1896-1897.

Passage of the Raines Liquor Law.
An Act for the regulation of the liquor traffic, which was and
is the subject of much controversy, was passed in March, 1896,
by the Legislature of the State of New York. From its author,
Senator John Raines, it has borne the name of the Raines Law.
It heavily increased the tax on the selling of liquor, raising
it to $800 on common "saloons" in the city of New York; to $650
in Brooklyn; to $500 in other cities having more than 50,000
and not more than 500,000 inhabitants; and to rates in lesser
cities and towns which ranged from 8100 to $350. It forbade
the licensing of any liquor shop within 200 feet of a
schoolhouse or a church, and also forbade the opening of any
new shop of that character in a residence district without
consent of two-thirds of the property owners. It prohibited
the sale of liquor on Sundays, except in hotels and clubs; but
this provision furnished a means of evasion which was speedily
brought into use. "Raines hotels" and "Raines Clubs," as they
were called, sprang into existence everywhere, sufficiently
answering the requirements of the law to escape its penalties.
These and other defects were considerably remedied by
amendments of the Act in April, 1897. It survived a powerful
attack in the Legislature at that time, the whole strength of
the leading cities in the State being brought against the law.
The country districts were generally united in supporting it,
partly on principle, and partly because of the extent to which
it lightened the burdens of taxation. By apportioning
two-thirds of the enormous revenue raised under the Act to the
towns, counties and cities in which it is collected, and
one-third to the state treasury, the Raines Law fortified
itself strongly in more than the moral sentiment of the
people. Under the Raines Law all local excise boards are
abolished, and the whole licensing and regulating of the
liquor traffic is placed under the supervision of a State
commissioner.

The Black Civil Service Law.

CIVIL SERVICE REFORM: A. D. 1897-1899.
{326}

Primary Election Law.
An Act which aims to make the political party caucus for

nominating candidates, and for choosing delegates to
nominating conventions, a "primary election," conducted under
strict regulations of law and guarded by registration, was
passed by the New York State Legislature and signed by the
Governor March 23, 1898.

New Civil Service Enactment.

CIVIL SERVICE REFORM: A. D. 1897-1899.
NEW YORK STATE: A. D. 1899 (May).
Taxation of public franchises.
A measure of great importance, introducing a new and eminently

just principle in taxation, was carried through the
Legislature of New York in May, by the energetic influence of
Governor Roosevelt. Recommended by the Governor in a special
message on the 27th of March and passed in an unsatisfactory
form, a bill to provide for the taxing of public franchises
which did not promise successful working was being left on his
hands when the Legislature adjourned. He promptly called a
special session and renewed to it his urgent recommendations.
"I recommend," he said, "the enactment of a law which shall
tax all these franchises as realty, which shall provide for
the assessment of the tax by the Board of State Tax
Commissioners, and which shall further provide that from the
tax thus levied for the benefit of each locality, there shall
be deducted the tax as now paid by the corporation in
question. Furthermore, as the time for assessing the largest
and wealthiest corporations, those of New York and Buffalo,
has passed for this year, and as it will be preferable not to
have the small country corporations taxed before the larger
corporations of the city are taxed, I suggest that the
operations of the law be deferred until October 1, of this
year."
Within a few days, the desired bill was passed by both Houses
of the Legislature, signed by the Governor and became a law.
The public franchises to which it relates are defined in its
first section, as follows:
"The terms 'land,' 'real estate,' and 'real property,' as used

in this chapter, include the land itself above and under
water, all buildings and other articles and structures,
substructures, and superstructures, erected upon, under or
above, or affixed to the same; all wharves and piers,
including the value of the right to collect wharfage, cranage,
or dockage thereon; all bridges, all telegraph lines, wires,
poles, and appurtenances; all supports and inclosures for
electrical conductors and other appurtenances upon, above, and
underground; all surface, underground, or elevated railroads,
including the value of all franchises, rights, or permission
to construct, maintain, or operate the same in, under, above,
on, or through streets, highways, or public places; all
railroad structures, substructures, and superstructures,
tracks, and the iron thereon, branches, switches, and other
fixtures permitted or authorized to be made, laid, or placed
on, upon, above, or under any public or private road, street,
or grounds; all mains, pipes, and tanks laid or placed in,
upon, above, or under any public or private street or place
for conducting steam, heat, water, oil, electricity, or any
property, substance or product capable of transportation or
conveyance therein, or that is protected thereby, including
the value of all franchises, rights, authority, or permission
to construct, maintain, or operate in, under, above, upon, or
through any streets, highways, or public places, any mains,
pipes, tanks, conduits, or wires, with their appurtenances,
for conducting water, steam, heat, light, power, gas, oil, or
other substance, or electricity for telegraphic, telephonic,
or other purposes; all trees and underwood growing upon land,
and all mines, minerals, quarries, and fossils in and under
the same, except mines belonging to the state. A franchise,
right, authority, or permission, specified in this
subdivision, shall, for the purpose of taxation, be known as a
special franchise. A special franchise shall be deemed to
include the value of the tangible property of a person,
co-partnership, association, or corporation, situated in,
upon, under, or above any street, highway, public place, or
public waters in connection with the special franchise. The
tangible property so included shall be taxed as a part of the
special franchise."
NEW YORK UNIVERSITY:

The Hall of Fame for Great Americans.
HALL OF FAME.
----------NEW ZEALAND: Start--------
NEW ZEALAND: A. D. 1891-1900.

Democratic experiments.
Labor laws and the land system.
Compulsory industrial arbitration and its working.
"I have been a studious observer of every phase of social life

and legislative change that has taken place in this colony
during the past seven years," wrote U. S. Consul Connolly, at
Auckland, in July, 1896. "I arrived at the very beginning of
the experimental era—and it is no misnomer to call much of the
legislation of the past few years experimental in the truest
sense [see, also (in this volume), AUSTRALIA: RECENT
EXTENSIONS OF DEMOCRACY]. But while it is so, there is a most
gratifying feature which compensates for the violence done to
the feelings of those whose motto has been 'let us permit
matters to remain as they are, they suit us well enough.' That
the legislative innovations of the immediate past have shocked
the sensibilities of a large number of prominent and
well-to-do colonists is unquestionably true, but, at the same
time, as against any inconvenience they may have experienced
on this account, there is the fact of increased prosperity in
nearly every branch of trade and industrial life throughout
the country, farm products are fetching satisfactory prices,
manufacturing industries are running full time and paying good
wages and fair interest on the capital invested, labor is
remuneratively employed, interest on money has fallen from 6
and 7 per cent to 4 and 5 per cent (this of itself, is
sufficient to prove that money is abundant). Millions of
English capital are flowing in for the development of the gold
fields of the colony, and the credit of the country at no
period of its history stood so high on the English market as
it does to-day.
{327}
I may also mention that, through the genuine encouragement
given by the Government to the small-farmer class, the waste
lands of the country are being rapidly taken up wherever land
is found suitable for farming or grazing purposes.
Notwithstanding the admitted prosperity of the colony and the
fact that the Government have had a substantial surplus over
expenditure now for a number of years, the national debt
continues to increase. But the increased indebtedness is not
of the usual character, for the reason that the country has
security for nearly all the money borrowed in recent years.
Money had to be borrowed under Government guaranty to save the
Bank of New Zealand from closing its doors. This was done to
avert financial disaster. …
"Money has been borrowed to purchase large estates for the

purposes of settlement. Those who take up land under this
system, as already stated, pay an annual rental sufficient to
cover the interest on the purchase money and the cost of
administration. The land is always vested in the Government
and this must be regarded as a good asset. One million and a
Half sterling was borrowed last year in England at 3 per cent
per annum. This £1,500,000 loan is called the ' advances to
settlers loan.' This money is lent out to farmers at 4 per
cent per annum. … I need scarcely add that the large
landholders, the mortgage companies, and the money lenders
generally did not favor this kind of legislation, particularly
the cheap advances to settlers, but their opposition was utterly
futile. With the advent of the one-man-one-vote and the
extension of the franchise to women, the power of corporate
wealth in this country appears to have been irrevocably
destroyed. Whether this be for good or evil, I am not, of
course, in a position to say. I can say, however, that no ill
effects of the change are apparent up to the present; on the
contrary, the country is more prosperous and at least as
honestly and as economically administered as it was under the
old régime.
"To say that this country is, in my opinion, more truly

democratic than any country in the world would be merely
stating a simple truth; and to say that the present Government
is a workingman's Government is equally true. A great deal of
the legislation of recent years, however, is in advance of the
requirements and ideas of the people, with the result that
some of it has proved to be annoying and irksome to many. This
is especially true of some of the labor laws. The Government
are honestly endeavoring to place the masses in possession of
their legitimate rights with as little friction as possible,
and at the same time with due regard to vested interests and
the propriety of things generally. But while struggling thus
with the duties and responsibilities of their official
positions, the members of the Ministry are torn asunder by the
clamorous and impracticable demands of the unreasonable and
irresponsible. The sympathies of the Government are
unmistakably with the people, but the honor, the dignity, and
the welfare of the country will not permit them to depart from
a course too inconsistent with the sense of obligation, fair
dealing, integrity, and responsibility which are the admitted
characteristics and duty of all civilized governments. The
great danger at the present moment is too much legislation in
one direction. This is the one thing wherein the Government
find it really hard to resist the demands of organized labor.
There is, however, a very gratifying disposition manifesting
itself among the more reasonable members of the labor
societies to let well enough alone for the present—a
disposition it is much to be hoped may extend throughout the
whole body of the workers. If not, I have no hesitation in
predicting a serious revulsion of public sentiment and
sympathy within the next few years."
United States Consular Reports,

January, 1897, page 35.
"Australian experience seems in many ways to prove the value
of our system of written constitutions, to be construed and
enforced by the courts. The effect on the minds of
ill-informed legislators of the knowledge that they can do
anything for which they can get a majority, is naturally to
beget extravagance and an overweening sense of power, and lead
to excessive experimentation. … It is in devices for the
protection of labor that most of this experimentation occurs.
New Zealand affords the best example of it. It provides
elaborate legal protection for the eight-hour day. A workman
cannot consent to work overtime without extra pay. The state
sees that he gets the extra pay. It looks closely after the
condition of women and children in the factories. It sees that
servant girls are not overcharged by the registry offices for
getting them places. It prescribes one half-holiday a week for
all persons employed in stores and offices, and sees that they
take it. It will not allow even a shopkeeper who has no
employees to dispense with his half-holiday; because if he
does not take it, his competition will injure those who do.
The 'labor department' of the government has an army of
inspectors, who keep a close watch on stores and factories,
and prosecute violations of the law which they themselves
discover. They do not wait for complaints; they ferret out
infractions, so that the laborer may not have to prejudice
himself by making charges. The department publishes a
'journal' once a month, which gives detailed reports of the
condition of the labor market in all parts of the colony, and
of the prosecutions which have taken place anywhere of
employers who have violated the law. It provides insurance for
old age and early death, and guarantees every policy. It gives
larger policies for lower premiums than any of the private
offices, and depreciates the private offices in its documents.
It distributes the profits of its business as bonuses among the
policy-holders, and keeps a separate account for teetotalers,
so that they may get special advantages from their abstinence.
The 'journal' is, in fact, in a certain sense a labor manual,
in which everything pertaining to the comfort of labor is
freely discussed. The poor accommodation provided for servants
in hotels and restaurants is deplored, and so is the
difficulty which middle-aged men have in finding employment.
More attention to the morals and manners of nursemaids is
recommended. All the little dodges of employers are exposed
and punished. If they keep the factory door fastened, they are
fined. If housekeepers pretend that their servants are
lodgers, and therefore not liable to a compulsory
half-holiday, they are fined. If manufacturers are caught
allowing girls to take their meals in a workshop, they are
fined.
{328}
"As far as I can make out, too, without visiting the country,
there is as yet no sign of reaction against this minute
paternal care of the laborer. The tendency to use the powers
of the government chiefly for the promotion of the comfort of
the working classes, whether in the matter of land settlement,
education, or employment, seems to undergo no diminution. The
only thing which has ceased, or slackened, is the borrowing of
money for improvements. The results of this borrowing have
been so disastrous that the present generation, at least, will
hardly try that experiment again."
E. L. Godkin,
The Australian Democracy
(Atlantic Monthly, March, 1898).
NEW ZEALAND:
Labor Laws.
Compulsory industrial arbitration.
"There is not in any other country in the world a more

valuable or more enlightened body of Labour laws than those
now upon the statute book of this progressive colony. They
cover almost every risk to life, limb, health, and interest of
the industrial classes. They send the law, as it were,
everywhere a worker is employed for daily wages to fling the
shield of the state over him or her in the labour of
livelihood. The bare enumeration of these laws will indicate
the far-reaching ground they cover:—The Coal Mines Act, the
Master and Apprentices Act, the Conspiracy Law Amendment Act,
the Trade Union Act, the Servant's Registry Offices Act (for
the protection of servant girls against the risks of dishonest
offices of that kind), Contractors and Workmen's Lien Act,
three amended Employer's Liability Acts, three amended
Shipping and Seamen's Acts, two Shops and Shop-assistants
Acts, the Factories Act, and the Industrial Conciliation and
Arbitration Act of 1894. … The Industrial Conciliation and
Arbitration Act, passed in 1894 … has attracted much attention
outside New Zealand. An Act with a similar purpose, but
permissive in its operations, was passed … in the New South
Wales Legislature in 1892. It was limited in duration to four
years, and was not a success. The New Zealand bill was more
skilfully drawn, and, possessing the element of a gentle
compulsion, has so far achieved its aim. The Act begins by
inviting all parties to join 'in lawful association for the
purpose of protecting or furthering the interests of employers
or workmen in, or in connection with, any industry in the
colony.' Such parties as accept the legal invitation are
allowed to register themselves as 'an industrial union,' and
this step once taken they are enticed on through a network of
solicitations, provisions, and safeguards, until they find
themselves, almost without knowing it, agreeing to everything
that follows. Trades Unions, or any other labour organization,
or any combination of employers, can register as individual
bodies without a mixed association of workers and employers.
Once registered, they are in the network of arbitration:—'The
effect of registration shall be to render the industrial
union, and all persons who may be members of any society or
trade union, so registered as an industrial union at the time
of registration, or who after such registration may become
members of any society or trade union so registered, subject
to the jurisdiction by this Act given to a Board and the Court
respectively, and liable to all the provisions of this Act,
and all such persons shall be bound by the rules of the
industrial union during the continuance of the membership.' …
'Every industrial agreement duly made and executed shall be
binding on the parties thereto, and on every person who at any
time during the term of such agreement is a member of any
industrial union, trade union, or association party thereto,
and on every employer who shall in the prescribed manner
signify to the Registrar of the Supreme Court where such
agreement is filed concurrence therein, and every such
employer shall be entitled to the benefit thereof, and be
deemed to be a party thereto.' … 'In and for every district
there shall be established a Board of Conciliation, to have
jurisdiction for the settlement of industrial disputes
occurring in such district, which may be referred to it by one
or more of the parties to an industrial dispute, or by
industrial agreement.' … 'Every Board shall consist of such
equal number of persons as the Governor may determine, being
not more than six nor less than four persons, who shall be
chosen by the industrial unions of employers and of workmen in
the industrial district respectively, such unions voting
separately, and electing an equal number of such members.' …
Should this body itself be unable to come to a satisfactory
decision it may refer the matter in question to a small
committee of its members fairly representing each side. If a
settlement or reconciliation be unattainable in this way,
either party to the dispute can appeal to the Court of
Arbitration, which is constituted as follows:—'There shall be
one Court of Arbitration for the whole colony for the
settlement of industrial disputes pursuant to this Act. … The
Court shall consist of three members to be appointed by the
Governor, one to be so appointed on the recommendation of the
councils or a majority of the councils of the industrial
associations of workmen in the colony, and one to be so
appointed on the recommendation of the councils or a majority
of the councils of the industrial associations of employers of
the colony.' … 'No recommendation shall be made as to the
third member, who shall be a Judge of the Supreme Court, and
shall be appointed from time to time by the Governor, and
shall be President of the Court.'"
M. Davitt,
Life and Progress in Australasia,
chapter 68.
Honorable W. P. Reeves, lately Agent-General of New Zealand in

England, but who was Minister of Education and Labor in New
Zealand from 1891 to 1896, and who is looked upon as the
principal author of the industrial arbitration laws in that
colony, wrote, during the summer of 1900, on the working of
those laws, in an article contributed to the "London Express,"
as follows:
"The arbitration law has been in constant use in New Zealand

for about four years and a half. During those years there has
never been a time when there has not been a dispute pending
before one or other of the Conciliation Boards or the Central
Arbitration Court. Writing, as I do, at some distance from
London, I cannot say from memory what the exact number of
disputes finally adjusted has been; but, so far, they cannot
be less than sixty or seventy. Most of these have been
carried, on appeal from some Conciliation Board, to the
Arbitration Court and settled there. In about two cases out of
seven the Conciliation Boards have been able successfully to
arrange the disputes. Even where they have not done so, it by
no means follows that their labors have been useless. Very
often the appeal to the Arbitration Court is merely on one or
two points out of many involved, and the advice of the
Conciliation Board is accepted on the others. Often, too, most
of the parties to a dispute have been ready to accept a board's
suggestions, but it has needed the firm hand of the
Arbitration Court to bring one or two stubborn men to
acquiescence.

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Protocols and Applications in

Enzymology Seema Anil Belorkar &

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Sensors and Protocols for Industry 4.0: Industrial

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Concepts and Experimental Protocols of Modelling and

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Karnatak University, Pavate Nagar, Dharwad, Karnataka, India

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Preface ............................................................................................... xvii

CHAPTER 1 Enzymesdpast, present, and future......................... 1

1.5.1 Substrate binding .....................................................10

1.5.2 Mechanism of enzyme catalyzed reaction.....................10

1.6 Recent developments ........................................................ 12

1.6.3 Diagnostic tools .......................................................13

1.6.4 Metagenomics .........................................................13

1.7 The future....................................................................... 13

CHAPTER 2 Distribution and diversity in microbial

2.1 Origin of enzyme diversity................................................. 17

2.7 The protistda novel enzyme source .................................... 38

3.3.7 Plasmid display ......................................................59

3.3.8 Phage display.........................................................59

3.3.9 Ribosome display m-RNA........................................61

3.3.11 The c-DNA display.................................................62

3.3.12 Reporter-based screening .........................................63

3.3.13 Fluorescence-activated droplet sorting........................64

3.3.14 Digital imaging ......................................................65

CHAPTER 4 Solid-state fermentation and submerged

fermentation for enzyme production ........................71

4.1 History of fermentation for social benefits ............................ 71

CHAPTER 5 Laboratory methods for enzyme assays ...................91

Subchapter 5.2 Assay of enzymedLipase ............................. 98

Reagent preparation ........................................................... 98

Lipase assay ..................................................................... 98

Key resources table................................................................. 99

Materials and equipment.......................................................... 99

Expected outcomes ............................................................... 100

Quantification and statistical analysis ....................................... 101

Limitations .......................................................................... 101

Safety considerations and standards ......................................... 102

Alternative methods/procedures............................................... 102

Preparation of standard curve for L-Tyrosin ...........................105

Expected outcomes ............................................................... 112

Quantification and statistical analysis ....................................... 112

Optimization and troubleshooting ............................................ 113

Safety considerations and standards ......................................... 113

Alternative methods/procedures............................................... 114

CHAPTER 6 Structure and functions of enzyme kinetics........... 115

6.2 Gibb’s free energy and enzyme kinetic............................. 117

6.2.1 Successful collision .............................................. 117

6.2.2 The activation energy............................................ 117

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