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Transcript of MODERN BIOTECHNOLOGY - Hörbücher · Modern biotechnology : ... Growth of the New Biotechnology...
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MODERN BIOTECHNOLOGYConnecting Innovations in Microbiology and Biochemistry to Engineering Fundamentals
Nathan S. MosierMichael R. Ladisch
A JOHN WILEY & SONS, INC., PUBLICATION
InnodataFile Attachment9780470473405.jpg
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MODERN BIOTECHNOLOGY
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MODERN BIOTECHNOLOGYConnecting Innovations in Microbiology and Biochemistry to Engineering Fundamentals
Nathan S. MosierMichael R. Ladisch
A JOHN WILEY & SONS, INC., PUBLICATION
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Copyright 2009 by John Wiley & Sons, Inc. All rights reserved
Published by John Wiley & Sons, Inc., Hoboken, New JerseyPublished simultaneously in Canada
No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning, or otherwise, except as permitted under Section 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923 (978) 750-8400, fax (978) 750-4470, or on the web at www.copyright.com. Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030, (201) 748-6011, fax (201) 748-6008, or online at http://www.wiley.com/go/permission.
Limit of Liability/Disclaimer of Warranty: While the publisher and author have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifi cally disclaim any implied warranties of merchantability or fi tness for a particular purpose. No warranty may be created or extended by sales representatives or written sales materials. The advice and strategies contained herein may not be suitable for your situation. You should consult with a professional where appropriate. Neither the publisher nor author shall be liable for any loss of profi t or any other commercial damages, including but not limited to special, incidental, consequential, or other damages.
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Library of Congress Cataloging-in-Publication Data:
Mosier, Nathan S., 1974- Modern biotechnology : connecting innovations in microbiology and biochemistry to engineering fundamentals / Nathan S. Mosier, Michael R. Ladisch. p. cm. Includes index. ISBN 978-0-470-11485-8 (cloth) 1. Biotechnology. I. Ladisch, Michael R., 1950- II. Title. TP248.2.M675 2009 660.6dc22 2009001779
Printed in the United States of America
10 9 8 7 6 5 4 3 2 1
http://www.copyright.comhttp://www.wiley.com/go/permissionhttp://www.wiley.com
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CONTENTS
Preface xvAcknowledgments xviiList of Illustrations xix
1 Biotechnology 1
Introduction 1The Directed Manipulation of Genes Distinguishes the New
Biotechnology from Prior Biotechnology 2Growth of the New Biotechnology Industry Depends on
Venture Capital 3Submerged Fermentations Are the Industrys Bioprocessing
Cornerstone 10Oil Prices Affect Parts of the Fermentation Industry 10
Growth of the Antibiotic/Pharmaceutical Industry 11The Existence of Antibiotics Was Recognized in 1877 11Penicillin Was the First Antibiotic Suitable for Human
Systemic Use 12Genesis of the Antibiotic Industry 12Other Antibiotics Were Quickly Discovered after the
Introduction of Penicillin 13Discovery and Scaleup Are Synergistic in the Development of
Pharmaceutical Products 15Success of the Pharmaceutical Industry in Research, Development,
and Engineering Contributed to Rapid Growth but Also Resulted in Challenges 15
Growth of the Amino Acid/Acidulant Fermentation Industry 16Production of Monosodium Glutamate (MSG) via Fermentation 17The Impact of Glutamic Acid Bacteria on Monosodium
Glutamate Cost Was Dramatic 17Auxotrophic and Regulatory Mutants Enabled Production of
Other Amino Acids 17Prices and Volumes Are Inversely Related 19Biochemical Engineers Have a Key Function in All Aspects of
the Development Process for Microbial Fermentation 21
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vi CONTENTS
References 22
Homework Problems 24
2 New Biotechnology 27
Introduction 27
Growth of the Biopharmaceutical Industry 28The Biopharmaceutical Industry Is in the Early Part of Its
Life Cycle 31Discovery of Type II Restriction Endonucleases Opened a New
Era in Biotechnology 33The Polymerase Chain Reaction (PCR) Is an Enzyme-Mediated,
In Vitro Amplifi cation of DNA 33
Impacts of the New Biotechnology on Biopharmaceuticals, Genomics, Plant Biotechnology, and Bioproducts 34
Biotechnology Developments Have Accelerated Biological Research 35
Drug Discovery Has Benefi ted from Biotechnology Research Tools 36
The Fusing of Mouse Spleen Cells with T Cells Facilitated Production of Antibodies 36
Regulatory Issues Add to the Time Required to Bring a New Product to Market 36
New Biotechnology Methods Enable Rapid Identifi cation of Genes and Their Protein Products 39
Genomics Is the Scientifi c Discipline of Mapping, Sequencing, and Analyzing Genomes 39
Products from the New Plant Biotechnology Are Changing the Structure of Large Companies that Sell Agricultural Chemicals 42
Bioproducts from Genetically Engineered Microorganisms Will Become Economically Important to the Fermentation Industry 43
References 45
Homework Problems 47
3 Bioproducts and Biofuels 49
Introduction 49
Biocatalysis and the Growth of Industrial Enzymes 49Glucose Isomerase Catalyzed the Birth of a New Process for
Sugar Production from Corn 51Identifi cation of a Thermally Stable Glucose Isomerase and an
Inexpensive Inducer Was Needed for an Industrial Process 53The Demand for High-Fructose Corn Syrup (HFCS) Resulted
in Large-Scale Use of Immobilized Enzymes and Liquid Chromatography 53
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CONTENTS vii
Rapid Growth of HFCS Market Share Was Enabled by Large-Scale Liquid Chromatography and Propelled by Record-High Sugar Prices 55
Biocatalysts Are Used in Fine-Chemical Manufacture 56
Growth of Renewable Resources as a Source of Specialty Products and Industrial Chemicals 58
A Wide Range of Technologies Are Needed to Reduce Costs for Converting Cellulosic Substrates to Value-Added Bioproducts and Biofuels 59
Renewable Resources Are a Source of Natural Plant Chemicals 63Bioseparations Are Important to the Extraction, Recovery, and
Purifi cation of Plant-Derived Products 64
Bioprocess Engineering and Economics 65
Bioseparations and Bioprocess Engineering 66
References 67
Homework Problems 71
4 Microbial Fermentations 73
Introduction 73
Fermentation Methods 75Fermentations Are Carried Out in Flasks, Glass Vessels,
and Specially Designed Stainless-Steel Tanks 75
Microbial Culture Composition and Classifi cation 78Microbial Cells: Prokaryotes versus Eukaryotes 78
Classifi cation of Microorganisms Are Based on Kingdoms 81Prokaryotes Are Important Industrial Microorganisms 81Eukaryotes Are Used Industrially to Produce Ethanol,
Antibiotics, and Biotherapeutic Proteins 82Wild-Type Organisms and Growth Requirements in
Microbial Culture 83Wild-Type Organisms Find Broad Industrial Use 83Microbial Culture Requires that Energy and All Components
Needed for Cell Growth Be Provided 86
Media Components and Their Functions (Complex and Defi ned Media) 86
Carbon Sources Provide Energy, and Sometimes Provide Oxygen 86
Complex Media Have a Known Basic Composition but a Chemical Composition that Is Not Completely Defi ned 89
Industrial Fermentation Broths May Have a High Initial Carbon (Sugar) Content (Ethanol Fermentation Example) 91
The Accumulation of Fermentation Products Is Proportional to Cell Mass in the Bioreactor 92
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viii CONTENTS
A Microbial Fermentation Is Characterized by Distinct Phases of Growth 93
Expressions for Cell Growth Rate Are Based on Doubling Time 94
Products of Microbial Culture Are Classifi ed According to Their Energy Metabolism (Types I, II, and III Fermentations) 96
Product Yields Are Calculated from the Stoichiometry of Biological Reactions (Yield Coeffi cients) 102
The EmbdenMeyerhof Glycolysis and Citric Acid Cycles Are Regulated by the Relative Balance of ATP, ADP, and AMP in the Cell 104
References 105
Homework Problems 108
5 Modeling and Simulation 111
Introduction 111
The RungeKutta Method 112Simpsons Rule 112Fourth-Order RungeKutta Method 113Ordinary Differential Equations (ODEs) 115
RungeKutta Technique Requires that Higher-Order Equations Be Reduced to First-Order ODEs to Obtain Their Solution 115
Systems of First-Order ODEs Are Represented in Vector Form 116
Kinetics of Cell Growth 117Ks Represents Substrate Concentration at Which the Specifi c
Growth Rate Is Half Its Maximum 120
Simulation of a Batch Ethanol Fermentation 122Ethanol Case Study 123
LuedekingPiret Model 127
Continuous Stirred-Tank Bioreactor 128
Batch Fermentor versus Chemostat 132
References 133
Homework Problems 135
6 Aerobic Bioreactors 141
Introduction 141
Fermentation Process 144Fermentation of Xylose to 2,3-Butanediol by Klebsiella
oxytoca Is Aerated but Oxygen-Limited 144Oxygen Transfer from Air Bubble to Liquid Is Controlled by
Liquid-Side Mass Transfer 153
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CONTENTS ix
Chapter 6 Appendix: Excel Program for Integration of Simultaneous Differential Equations 159
References 161
Homework Problems 162
7 Enzymes 165
Introduction 165
Enzymes and Systems Biology 165
Industrial Enzymes 166
Enzymes: In Vivo and In Vitro 167
Fundamental Properties of Enzymes 169
Classifi cation of Enzymes 170
Sales and Applications of Immobilized Enzymes 172
Assaying Enzymatic Activity 173Enzyme Assays 181
Batch Reactions 187
Thermal Enzyme Deactivation 187
References 192
Homework Problems 195
8 Enzyme Kinetics 199
Introduction 199
Initial Rate versus Integrated Rate Equations 200Obtaining Constants from Initial Rate Data Is an Iterative
Process 204
Batch Enzyme Reactions: Irreversible Product Formation (No Inhibition) 207
Rapid Equilibrium Approach Enables Rapid Formulation of an Enzyme Kinetic Equation 207
The Pseudo-Steady-State Method Requires More Effort to Obtain the Hart Equation but Is Necessary for Reversible Reactions 209
Irreversible Product Formation in the Presence of Inhibitors and Activators 210
Inhibition 212Competitive Inhibition 213Uncompetitive Inhibition 214(Classical) Noncompetitive Inhibition 216Substrate Inhibition 217
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Example of Reversible Reactions 220Coenzymes and Cofactors Interact in a Reversible Manner 223
KingAltman Method 225
Immobilized Enzyme 234Online Databases of Enzyme Kinetic Constants 236
References 237
Homework Problems 238
9 Metabolism 243
Introduction 243
Aerobic and Anaerobic Metabolism 245Glycolysis Is the Oxidation of Glucose in the Absence of Oxygen 245Oxidation Is Catalyzed by Oxidases in the Presence of O2,
and by Dehydrogenases in the Absence of O2 246A Membrane Bioreactor Couples Reduction and Oxidation
Reactions (R-Mandelic Acid Example) 247Three Stages of Catabolism Generate Energy, Intermediate
Molecules, and Waste Products 248The Glycolysis Pathway Utilizes Glucose in Both Presence
(Aerobic) and Absence (Anaerobic) of O2 to Produce Pyruvate 249Glycolysis Is Initiated by Transfer of a High-Energy Phosphate
Group to Glucose 250Products of Anaerobic Metabolism Are Secreted or Processed
by Cells to Allow Continuous Metabolism of Glucose by Glycolysis 253
Other Metabolic Pathways Utilize Glucose Under Anaerobic Conditions (Pentose Phosphate, EntnerDoudoroff, and Hexose Monophosphate Shunt Pathways) 255
Knowledge of Anaerobic Metabolism Enables Calculation of Theoretical Yields of Products Derived from Glucose 257
Economics Favor the Glycolytic Pathway for Obtaining Oxygenated Chemicals from Renewable Resources 258
Citric Acid Cycle and Aerobic Metabolism 259Respiration Is the Aerobic Oxidation of Glucose and Other
Carbon-Based Food Sources (Citric Acid Cycle) 260The Availability of Oxygen, under Aerobic Conditions,
Enables Microorganisms to Utilize Pyruvate via the Citric Acid Cycle 260
The Citric Acid Cycle Generates Precursors for Biosynthesis of Amino Acids and Commercially Important Fermentation Products 264
Glucose Is Transformed to Commercially Valuable Products via Fermentation Processes: A Summary 264
Essential Amino Acids Not Synthesized by Microorganisms Must Be Provided as Nutrients (Auxotrophs) 267
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CONTENTS xi
The Utilization of Fats in Animals Occurs by a NonTricarboxylic Acid (TCA) Cycle Mechanism 267
Some Bacteria and Molds Can Grow on Hydrocarbons or Methanol in Aerated Fermentations (Single-Cell Protein Case Study) 269
Extremophiles: Microorganisms that Do Not Require Glucose, Utilize H2, and Grow at 80100 C and 200 atm Have Industrial Uses 270
The Terminology for Microbial Culture Is Inexact: Fermentation Refers to Both Aerobic and Anaerobic Conditions While Respiration Can Denote Anaerobic Metabolism 271
Metabolism and Biological Energetics 272
References 272
Homework Problems 273
10 Biological Energetics 277
Introduction 277
Redox Potential and Gibbs Free Energy in Biochemical Reactions 277
Heat: Byproduct of Metabolism 286
References 292
Homework Problems 293
11 Metabolic Pathways 295
Introduction 295Living Organisms Control Metabolic Pathways at Strategic and
Operational Levels 296Auxotrophs Are Nutritionally Defi cient Microorganisms that
Enhance Product Yields in Controlled Fermentations (Relief of Feedback Inhibition and Depression) 296
Both Branched and Unbranched Pathways Cause Feedback Inhibition and Repression (Purine Nucleotide Example) 299
The Accumulation of an End Metabolite in a Branched Pathway Requires a Strategy Different from that for the Accumulation of an Intermediate Metabolite 301
Amino Acids 305The Formulation of Animal Feed Rations with Exogeneous
Amino Acids Is a Major Market for Amino Acids 306Microbial Strain Discovery, Mutation, Screening, and
Development Facilitated Introduction of Industrial, Aerated Fermentations for Amino Acid Production by Corynbacteriumglutamicum 308
Overproduction of Glutamate by C. glutamicum Depends on an Increase in Bacterial Membrane Permeability (Biotin-Defi cient Mutant) 309
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xii CONTENTS
A Threonine and Methionine Auxotroph of C. glutamicum Avoids Concerted Feedback Inhibition and Enables Industrial Lysine Fermentations 310
Cell (Protoplast) Fusion Is a Method for Breeding Amino Acid Producers that Incorporate Superior Characteristics of Each Parent (Lysine Fermentation) 312
Amino Acid Fermentations Represent Mature Technologies 313
Antibiotics 314Secondary Metabolites Formed During Idiophase Are Subject
to Catabolite Repression and Feedback Regulation (Penicillin and Streptomycin) 314
The Production of Antibiotics Was Viewed as a Mature Field Until Antibiotic-Resistant Bacteria Began to Appear 317
Bacteria Retain Antibiotic Resistance Even When Use of the Antibiotic Has Ceased for Thousands of Generations 318
Antibiotic Resistance Involves Many Genes (Vancomycin Example) 318
References 320
Homework Problems 323
12 Genetic Engineering: DNA, RNA, and Genes 331
Introduction 331
DNA and RNA 332DNA Is a Double-Stranded Polymer of the Nucleotides:
Thymine, Adenine, Cytosine, and Guanine 332The Information Contained in DNA Is Huge 332Genes Are Nucleotide Sequences that Contain the Information
Required for the Cell to Make Proteins 333Transcription Is a Process Whereby Specifi c Regions of the
DNA (Genes) Serve as a Template to Synthesize Another Nucleotide, Ribonucleic Acid (RNA) 333
Chromosomal DNA in a Prokaryote (Bacterium) Is Anchored to the Cells Membrane While Plasmids Are in the Cytoplasm 333
Chromosomal DNA in a Eukaryote (Yeast, Animal or Plant Cells) Is Contained in the Nucleus 334
Microorganisms Carry Genes in Plasmids Consisting of Shorter Lengths of Circular, Extrachromosomal DNA 334
Restriction Enzymes Enable Directed In Vitro Cleavage of DNA 337
Different Type II Restriction Enzymes Give Different Patterns of Cleavage and Different Single-Stranded Terminal Sequences 339
DNA Ligase Covalently Joins the Ends of DNA Fragments 341
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CONTENTS xiii
DNA Fragments and Genes of 150 Nucleotides Can Be Chemically Synthesized if the Nucleotide Sequence Has Been Predetermined 342
Protein Sequences Can Be Deduced and Genes Synthesized on the Basis of Complementary DNA Obtained from Messenger RNA 343
Genes and Proteins 344Selectable Markers Are Genes that Facilitate Identifi cation of
Transformed Cells that Contain Recombinant DNA 344A Second Protein Fused to the Protein Product Is Needed to
Protect the Product from Proteolysis (-Gal-SomatostatinFusion Protein Example) 346
Recovery of Protein Product from Fusion Protein Requires Correct Selection of Amino Acid that Links the Two Proteins (Met Linker) 347
Chemical Modifi cation and Enzyme Hydrolysis Recover an Active Molecule Containing Met Residues from a Fusion Protein (-Endorphin Example) 347
Metabolic Engineering Differs from Genetic Engineering by the Nature of the End Product 348
References 349
Homework Problems 350
13 Metabolic Engineering 355
Introduction 355
Building Blocks 359
l-Threonine-Overproducing Strains of E. coli K-12 359Genetically Altered Brevibacterium lactoferrin Has Yielded
Improved Amino AcidProducing Strains 360Metabolic Engineering May Catalyze Development of New
Processes for Manufacture of Oxygenated Chemicals 362Gene Chips Enable Examination of Glycolytic and Citric
Acid Cycle Pathways in Yeast at a Genomic Level (Yeast Genome Microarray Case Study) 362
The Fermentation of Pentoses to Ethanol Is a Goal of Metabolic Engineering (Recombinant Bacteria and Yeast Examples) 364
Metabolic Engineering for a 1,3-Propanediol-Producing Organism to Obtain Monomer for Polyester Manufacture 370
Redirection of Cellular Metabolism to Overproduce an Enzyme Catalyst Results in an Industrial Process for Acrylamide Production (YamadaNitto Process) 373
References 377
Homework Problems 379