IndustrialEnzymeApplications
Editedby AndreasVogelandOliverMay
Editors
Dr.AndreasVogel c-LEctaGmbH R&DEnzymeDevelopment Perlickstr.5 04103Leipzig Germany
Dr.OliverMay DSMNutritionalProductsLtd Wurmisweg576 4303Kaiseraugst Switzerland
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Contents
Preface xiii
PartIOverviewofIndustrialEnzymeApplicationsandKey Technologies 1
1.1IndustrialEnzymeApplications–OverviewandHistoric Perspective 3 OliverMay
1.1.1PrehistoricApplications 3
1.1.2GrowingtheScientificBasis 5
1.1.3TheBeginningofIndustrialApplicationsandtheEmergingEnzyme Industry 12 References 21
1.2EnzymeDevelopmentTechnologies 25 AndreasVogel
1.2.1Introduction 25
1.2.2IdentificationofWild-TypeEnzymes 26
1.2.2.1SelectionParametersforStartingEnzymes 28
1.2.3EnzymeEngineering 30
1.2.3.1TypesofEnzymeModifications 30
1.2.3.2GeneralEngineeringStrategies.LibraryDesignandGeneration 30
1.2.3.3ScreeningforBetterEnzymes 37
1.2.4ImpactofEnzymeDevelopmentTechnologiesTodayand Tomorrow 38 Acknowledgments 41 References 41
1.3EukaryoticExpressionSystemsforIndustrialEnzymes 47 LukasRieder,NicoTeuschler,KatharinaEbner,andAntonGlieder
1.3.1EukaryoticEnzymeProductionSystems 47
1.3.2SpecialConsiderationsforWorkingwithEukaryoticExpression Systems 47
1.3.2.1ChoiceofExpressionHost 47
1.3.2.2ComparisonofCellStructureandTheirInfluenceonMolecular Biology 49
1.3.3DifferencesinVectorDesignforEukaryoticandProkaryoticHosts 51
1.3.4DifferencesinRegulationofGeneExpressioninEukaryotesand Prokaryotes 56
1.3.4.1DifferentTypesofPromoters 58
1.3.5IndustrialEnzymeProduction 58
1.3.6EnzymeProductiononIndustrialScale 61
1.3.6.1HomologousProteinProduction 61
1.3.6.2HeterologousProteinProduction 62 References 63
1.4ProcessConsiderationsfortheApplicationofEnzymes 71
SelinKaraandAndreasLiese
1.4.1BiocatalystTypesUsedinIndustrialProcesses 71
1.4.2EnzymeImmobilizationforBiocatalyticProcesses 74
1.4.3ReactionMediumAppliedinEnzymaticCatalysis 76
1.4.3.1MonophasicSystems–OrganicMedia 77
1.4.3.2MultiphasicSystems–Liquid/LiquidMixtures 80
1.4.3.3MultiphasicSystems–Gas/LiquidMixtures 83
1.4.3.4MultiphasicSystems–Solid/LiquidMixtures 84
1.4.4AppropriateReactorTypesinEnzymeCatalysis 87
1.4.5AssessmentCriteriaforEnzymaticApplications 90 References 92
PartIIEnzymeApplicationsfortheFoodIndustry 95
2.1EnzymesUsedinBaking 97
JokeA.PutseysandMargotE.F.Schooneveld-Bergmans
2.1.1Introduction 97
2.1.2TheBakingProcess–TheBaker’sNeeds 98
2.1.2.1FlourQualityandStandardization 98
2.1.2.2MixingandDoughHandling 100
2.1.2.3FermentationandDoughStability 105
2.1.2.4BakingandOvenSpring 109
2.1.3TheBreadQuality–TheConsumers’Needs 111
2.1.3.1ColorandFlavor 111
2.1.3.2ShelfLife 112
2.1.4TrendsandOpportunitiesforBakingEnzymes 116
2.1.4.1FineBakingandConfectionary 116
2.1.4.2ConsumerPreference:Health,IndividualValues,and Convenience 117
2.1.5Conclusion 118 References 119
2.2ProteinModificationtoMeettheDemandsoftheFood Industry 125 AndrewEllis
2.2.1FoodProteins 125
2.2.2ProcessingofFoodProtein 127
2.2.3EnzymesintheProcessingofFoodProteins 127
2.2.4FoodProteinValueChain 130
2.2.5RecentEnzymeDevelopments 131
2.2.5.1SimpleProteinModification(ValueLevel3) 131
2.2.5.1.1DevelopingMicrobialAlternativestoPlantandAnimalEnzymes 131
2.2.5.2SpecializedEnzymeModification(ValueLevel4) 134
2.2.5.2.1WheyProteinHydrolysates 134
2.2.5.2.2PlantProteinHydrolysates 134
2.2.5.3HighlySpecificProteinModification(ValueLevel5) 135
2.2.5.3.1GlutenModification 135
2.2.5.3.2AcrylamideReduction 135
2.2.5.3.3BioactivePeptides 136
2.2.6EnzymestoMeetFutureNeeds 137 Acknowledgments 139 References 139
2.3DairyEnzymes 143
PeterDekker
2.3.1Introduction 143
2.3.2Coagulants 145
2.3.2.1TraditionalRennets 147
2.3.2.2MicrobialRennets 148
2.3.2.3FermentationProducedChymosin 151
2.3.3RipeningEnzymes 152
2.3.3.1Proteases/Peptidases 153
2.3.3.2Lipases/Esterases 154
2.3.4Lactases 154
2.3.4.1NeutralLactase 156
2.3.4.2AcidLactase 158
2.3.4.3GOSProduction 158
2.3.5MiscellaneousEnzymes 161
2.3.5.1Oxidases/Peroxidases 161
2.3.5.2Phopholipases 162
2.3.5.3Cross-linkingEnzymes 162
2.3.5.4Preservation 163
2.3.6NewDevelopments 163 References 163
2.4EnzymaticProcessfortheSynthesisofCellobiose 167
BirgitBrucherandThomasHäßler
2.4.1EnzymaticSynthesisofCellobiose 167
2.4.2Cellobiose–PropertiesandApplications 168
2.4.3ExistingRoutesforCellobioseSynthesis 170
2.4.4EnzymeDevelopment 171
2.4.5ProcessDevelopment 173
2.4.5.1SynthesisofCellobiose 174
2.4.5.2PurificationofCellobiose 174
2.4.6SummaryandFuturePerspective 176 References 176
2.5EmergingField–SynthesisofComplexCarbohydrates.Case StudyonHMOs 179
DoraMolnar-Gabor,MarkusJ.Hederos,SebastianBartsch,andAndreasVogel
2.5.1IntroductiontoHumanMilkOligosaccharides(HMOs) 179
2.5.1.1DiscoveryandFunctionofHMOs 179
2.5.1.2StructureofHMOs 180
2.5.1.3HMOProduction,RegulatoryAuthorizations,andCommercial Launch–HistoricalOverview 181
2.5.2GlycomA/STechnologiesTowardCommercialHMO Production 184
2.5.2.1WholeCellMicrobialFermentationtoHMOs(InVivo Process) 185
2.5.2.2TheGlycom InVitro ConcepttoDiversifyHMOBlends 187
2.5.2.3ValidationoftheHMODiversificationConceptwithNon-optimized Enzymes 187
2.5.3EnzymeDevelopment 189
2.5.3.1Optimizationofthe α1-3/4Transfucosidase 189
2.5.3.2Optimizationofthe α2-6Transsialidase 192
2.5.4ApplicationsoftheOptimizedEnzymesfortheHMOProfiles 195
2.5.4.1Scale-UpoftheLacto-N -fucopentaoseIII(LNFP-III),Sialyl Lacto-N -neotetraose(LST-c),andSialylLacto-N -tetraose(LST-a) HMOProfiles 195
2.5.5ConclusionandPerspective 197 References 198
PartIIIEnzymeApplicationsforHumanandAnimal Nutrition 203
3.1EnzymesforHumanNutritionandHealth 205 YoshihikoHirose
3.1.1Introduction 205
3.1.2CurrentProblemsofEnzymesinHealthcareBusiness 205
3.1.3EnzymesinExistingHealthcareProducts 206
3.1.3.1DigestiveEnzymes 206
3.1.3.1.1DigestiveEnzymesinUnitedStates 206
3.1.3.1.2TherapeuticDigestiveEnzymes 207
3.1.3.2AcidLactase 207
3.1.3.3 α-Galactosidase(ADG) 208
3.1.3.4Dextranase 208
3.1.3.5GlucoseOxidase 208
3.1.3.6AcetobacterEnzymes 210
3.1.3.7Laccase(PolyphenolOxidase) 210
3.1.4NewEnzymeDevelopmentsinHealthcareProducts 211
3.1.4.1Transglucosidase 211
3.1.4.2Laccase 211
References 215
3.2EnzymeTechnologyforDetoxificationofMycotoxinsinAnimal
Feed 219
DieterMoll
3.2.1IntroductiontoMycotoxins 219
3.2.2MycotoxinMitigationStrategies 220
3.2.3EnzymeApplications 224
3.2.4FUMzyme® 225
3.2.4.1TheSubstrate:Fumonisins 225
3.2.4.2EnzymeDiscovery 227
3.2.4.3EnzymeSelection 230
3.2.4.4EnzymeActivityAssays 232
3.2.4.5EnzymeCharacterizationandEvaluation 233
3.2.4.6EnzymeFeedingTrialsandBiomarkerAnalysis 234
3.2.4.7EnzymeEngineering 237
3.2.4.8EnzymeProduction 238
3.2.4.9EnzymeRegistration 239
3.2.5FutureMycotoxinases 240
3.2.6Conclusions 242 References 243
3.3PhytasesforFeedApplications 255
NikolayOutchkourovandSpasPetkov
3.3.1PhytaseAsaFeedEnzyme:IntroductionandSignificance 255
3.3.2HistoricalOverviewofthePhytaseMarketDevelopment 256
3.3.3FromPhytatetoPhosphorus:StepbyStepActionofthePhytase 259
3.3.3.1PropertiesofPhytate 259
3.3.3.2PhytasesStructuralandFunctionalClassification 260
3.3.3.2.1PhytasesfromtheHistidineAcidPhosphatases(HAP) Superfamily 261
3.3.3.2.2 β-PropellerPhytase(BPP) 261
3.3.3.2.3CysteinePhytase(CPhy) 263
3.3.3.2.4PurpleAcidPhytases(PAPhy) 263
3.3.3.2.5ClassificationofthePhytasesBasedonPhytateDephosphorylation Steps 263
3.3.4NutritionalValuesofPhytaseinAnimalFeed 265
3.3.5PhytaseApplicationAsFeedAdditive 265
3.3.6EffectivePhytateHydrolysisintheUpperDigestiveTractofthe Animal 266
3.3.7KineticDescriptionofIdealPhytases 269
3.3.8ResistancetoLowpHandProteases 271
3.3.9TemperatureStability 271
3.3.10InlieuofConclusion:LessonsfromPhytaseSuperDosingTrials 274 References 275
PartIVEnzymesforBiorefineryApplications 287
4.1EnzymesforPulpandPaperApplications 289 DebayanGhosh,BikasSaha,andBaljeetSingh
4.1.1RefiningandFiberDevelopmentEnzyme 290
4.1.1.1MicroscopicEvaluation 291
4.1.1.2EvaluationofEnzyme-TreatedHandsheets 293
4.1.1.2.1CaseStudy1 293
4.1.1.2.2CaseStudy2 295
4.1.2DrainageImprovementEnzyme 296
4.1.2.1CaseStudy3 299
4.1.2.2CaseStudy4 300
4.1.3StickiesControlEnzyme 301
4.1.3.1CaseStudy5 303
4.1.4DeinkingEnzymes 306
4.1.4.1CaseStudy6 307
4.1.5HardwoodVesselBreakingEnzyme 308
4.1.5.1FiberTesterImageAnalysis 308
4.1.6NativeStarchConversionEnzyme 310
4.1.7BleachBoostingEnzyme 312
4.1.7.1CommonBleachingAgents 312
4.1.7.1.1CaseStudy7 313
4.1.7.2OvercomingChallengesFacedbyBleachingEnzymesinPulpand Paperindustry 315
4.1.8PaperMillEffluentTreatmentEnzymes 315
4.1.8.1CaseStudy8 316
4.1.9SlushingEnzyme 317
4.1.9.1CaseStudy9 317
4.1.9.2RoleofEnzymesinPulpandPaperIndustry–EndNote! 318 References 319
4.2EnzymesinVegetableOilDegummingProcesses 323 ArjenSein,TimHitchman,andChrisL.G.Dayton
4.2.1Introduction 323
4.2.2GeneralSeedOilProcesses 324
4.2.2.1Phospholipids 325
4.2.2.2AMolecularViewoftheDegummingProcess 327
4.2.3EnzymaticDegumming 330
4.2.3.1PhospholipaseC 331
4.2.3.2WaystoCopewithPoorConversion/PoorQualityOilsinPLC-Based Processes 333
4.2.3.3PhospholipaseA 336
4.2.4EnzymaticDegumminginIndustrialPractice 337
4.2.4.1IntroductionHurdles 341
4.2.5OtherApplicationsofEnzymesinOil–Outlook 343
4.2.5.1EnzymaticInteresterificationofTriglycerideOils 343
4.2.5.2Biodiesel 344
4.2.5.3Enzyme-AssistedDecoloring 344
4.2.5.4Enzyme-AssistedOilExtraction 344
4.2.6Conclusion 345 Acknowledgments 345 References 345
PartVEnzymesusedinFineChemicalProduction 351
5.1KREDs:TowardGreen,Cost-Effective,andEfficientChiral AlcoholGeneration 353
ChrisMicklitsch,DaDuan,andMargieBorra-Garske
5.1.1Introduction 353
5.1.2Ketoreductases 355
5.1.3CofactorRecycling 356
5.1.4CodeEvolver® ProteinEngineeringTechnology 358
5.1.5ReductionofaWideRangeofKetones/Aldehydes 358
5.1.6CriticalSelectivityToolsforEnantiopureAsymmetricCarbonyl Reduction 364
5.1.7ExamplesofImprovedKREDsforImprovedManufacturing 369
5.1.8KREDs:GoingGreenandSavingGreen 373 References 377
5.2AnAldolasefortheSynthesisoftheStatinSideChain 385 MartinSchürmann
5.2.1Introduction–Biocatalysis 385
5.2.1.1EnzymesasBiocatalystsinChemicalProcess 385
5.2.1.2BiocatalyticRoutestotheStatinSideChain 387
5.2.2TheAldolaseDERAinApplication 387
5.2.2.1DERA-CatalyzedAldolReactions 387
5.2.2.2FeasibilityPhaseofDERA-EnabledStatinSideChainProcess 390
5.2.3DirectedEvolutionandProteinEngineeringtoImproveDERA 392
5.2.3.1RationalDesign 392
5.2.3.2DirectedEvolutionofDERA 394
5.2.3.3OtherApproachestoSuitableorImprovedDERAs 396
Contents
5.2.3.4OtherApplicationsofProcessIntermediatesandtheDERA Technology 397
5.2.4Conclusions 398 Acknowledgments 400 References 401
Index 405
Preface
Industrialenzymeapplicationsarepartofoureverydaylifesincemankinddiscoveredthebenefitsoftransformingmilk,grapes,andgrainstodurable,palatable, moretastefulproductssuchasyoghurt,wine,beer,andbread.Atthattime,the microorganismsandenzymesthatledtothistransformationwereusedunconsciously.Sincethen,ourknowledgeaboutenzymes,andthedevelopmentoftechnologiesonhowtoadapt,produce,andapplythemhasimproveddramatically, especiallyinthelastfewdecades.Thishasledtoawidespectrumofenzyme applicationsthatwecomeacrossinproductsfromthefoodanddrink,chemical, pharmaceutical,biorefinery,andthehumanandanimalnutritionindustry.
Thisbookshalldirecttheperspectivetoavarietyofenzymetypesandtheir applicationsthatareactuallyusedinindustrialprocesses.Whenweconceived thisbook,weaskedthefollowingquestions:Whichsolutionscanenzymesprovidetoaddresstheneedsofindustriesandconsumers?Whichenzymesprovide whichsolutions,andwhyhavetheseparticularenzymesbeenchosenandare successfullycompetingwithothersolutions?Whatwasthedecisiveadvantage oftheuseofenzymesoverthecompetitiveprocess?Whatenzymefeatureswere requiredandhowweretheseobtainedduringenzymedevelopment?
Thebookiswrittenbyexpertsfromdifferentindustrieswhodevelopandapply enzymesintheirteamstoaddressvariousproblems:fromenabling“simple”cost reductionandprocessinnovationsforexistingproducts(whichoftengoeshand inhandwithecologicbenefits)toproductinnovations,developingnewproductsthatprovideadditionalvalue(e.g.sustainability,purity,nutritional)forconsumers.Weareveryhappythatwecouldattractauthorsfromvariousindustries thatwerewillingtoshareinsightsintotheirwork.Writingpublicationsfrom anindustrialpositionisoftennottheprimepriorityandisconflictedbycompanypolicies.Particularlyforthisreason,thecontributionsinthisbookprovide uniqueandindividualinsightsintoindustrialdriversandstrategiesfortheimplementationofenzymaticprocesses.
ThistextbookonIndustrialEnzymeApplicationwillserveasareferenceguide foracademicandindustrialresearchersandprovidesaunique,industrialperspectiveonthedevelopmentandapplicationofenzymes.Itwillhelpunderstand theindustrialdriversinsearchingforanddevelopinganenzymaticsolution.
xiv Preface
Wealsohopethedescribedexamplesinspirestudentsandpractitionersto developnewapplications,providingourworldwithmoresustainablesolutions thatenzymesareperfectlycapableofproviding.
c-LEcta,Leipzig
DSM,Kaiseraugst
April2019
AndreasVogel OliverMay
PartI
OverviewofIndustrialEnzymeApplicationsand KeyTechnologies
1.1
IndustrialEnzymeApplications–Overviewand HistoricPerspective
OliverMay
DSMNutritionalProducts,Wurmisweg576,4303Kaiseraugst,Switzerland
Thefieldofindustrialenzymeapplicationshasbeenextensivelyreviewed, includingitshistoricbackground[1–4].Thereforethischapterwillnotrepeat theexcellentworkofothersbutprovidehistoriccontextinthefollowingchapters andgiveageneraloverviewofthefield,includingtopicsthatwehavechosennot tocoverindetailinthisbook.Itisalsomeantasatributetothemanypioneering scientiststhatenlighteneduswiththeirbrilliantmindsaboutthemiraclesoflife andhowenzymeswork(seeSection1.1.2)andtothevisionaryentrepreneurs shapingagrowingmultibillionenzymebusiness(seeSection1.1.3).
Whilewehavecomealongwayinunravelinghowenzymeswork,wehave stillnotcapturedallthedetailsonhowtheyachievethehugerateaccelerationof thereactionstheycatalyze,norhaveweexploredexhaustivelytheirfullpotential inexistingaswellasnewapplicationfields.Thisleavesroomforusandfuture generationsstandingontheshouldersofgiantssomeofwhicharementionedin thefollowing,orarehiddenintheprehistoricworldthatappliedenzymes,even withoutknowingtheyexisted.
1.1.1PrehistoricApplications
Enzymesplayedanimportantroleearlyinthehistoryanddevelopmentof humanity.TheNeolithicRevolutionofaround12500yearsagomarkedthe transitionoflifestylesfromhuntingandgatheringtoagricultureandsettlement. Withthistransition,farmingpracticeswereinvented,leadingtodomestication ofplantsandanimals.Whilesafestorageofhuntedorgatheredfoodwas certainlyalreadyimportantduringtheNeolithictimes,theunconscioususe ofmicrobesandtheactionoftheirenzymesallowedtopreservethefoodand supplyittootherswhocouldfocusonotheractivitiesoutsidetheprimary productionoffood.Certainly,peoplealsoenjoyedimprovedpalatabilityora desirabletasteoffoodthatwasincontactwithmicroorganismsthatcontributed tothesedesiredproperties.
Whilenoonecanreallydeterminetheexactdatewhenthefirstproductswere madeinwhichenzymesfrommicrobes,plant,oranimaltissuesplayedsucha
IndustrialEnzymeApplications, FirstEdition.EditedbyAndreasVogelandOliverMay. ©2019Wiley-VCHVerlagGmbH&Co.KGaA.Published2019byWiley-VCHVerlagGmbH&Co.KGaA.
1.1IndustrialEnzymeApplications–OverviewandHistoricPerspective
beneficialrole,thefirstscientificallyprovenevidence,basedonresiduesfound inpotteryvesselsforcheesemaking,datesbackto7500yearsago[5].Whata greatinventiontopreservemilkwithoutafridgeandmakeitpalatableaswellas enjoyable!Today’senzymeapplicationsindairyarecoveredinChapter2.3and theachievementsofChristianHansen,anentrepreneurstartingaverysuccessful enzymeandstarterculturebusinessin1874,aredescribedinSection1.1.3.
Thefirstindicationofenzyme-assistedgrainprocessingtoproduceanalcoholicbeveragewasfoundintheNeolithicvillageofJiahuinChinaanddatesback to7000bce[6].Basedonchemicalanalysesoforganicsabsorbedintoancient potterytheauthorshaveshownthatamixedfermentedbeverageofrice,honey, andfruit(hawthornfruitand/orgrape)wasbeingproduced.Theearliestproof ofwineproductiondatedto5400–5000bce,attheNeolithicsiteofTepein Mesopotamia[7]wheretartaricacidwasfoundinanoldjar,andaround5000bce fromgrapejuiceresiduesfoundinDikiliTashinGreece[8].Forover2500years Aspergillus strainshavebeenextensivelyusedinChinaasstarterculturesingrain (soy,rice)fermentation,atraditionalpracticeforproductionofricewine(sake)or otherdistilledproducts(shochu),whichwereimportedfromJapanbyBuddhist monks[9].TheJapanesewordKojistillusestheChinesecharacter( )thatmeans (wheat)grainsfermentedbyfungi.Today,grainprocessingandmaltproduction areseparateindustrieswhereasintheWesternworldmaltproductioncanbe consideredasthefirstsectorthatindustrializedenzymeproduction(seealso Section1.1.3).
Nexttobeingusedforfermentativeprocesses,Kojihasbeenusedasdigestiveaid,asfirstdescribed2500yearsagoinaChineseclassicbookentitled “Zuo-Zhuan,”intheEnglishChronicleofZuoorCommentaryofZuo[10]. Inthedescription,wheat-basedKojiwasusedtotreatdigestionproblems. Thistraditionwaslaterturnedintothefirstindustrialapplicationofafungal enzymebyJokichiTakamine,oneofthepioneeringentrepreneursdiscussed inSection1.1.3andwhoalsoinspiredtheapplicationofenzymesfornutrition andhealth,reviewedbyYoshihikoHiroseinChapter3.1.Thegoodoldtradition offermentedfoodiscarriedthroughtodateandisestimatedtoprovideabout 20–40%ofourfoodsupplytoday[11].
TheEgyptians,whoalreadyusedyeasttobrewbeer,begantoemploymicroorganismsaswellasmalttomakebreadforwhichsampleswerefoundindifferent archeologicalsitesdatingto2000–1200bce[12].Thisancientmaltapplication inbakingdevelopedintoamajorfieldofenzymeapplication,whichisreviewed byPutseysandSchooneveldinChapter2.1.
However,itisnotonlyfoodthathasbenefitedfromenzymeslongbeforetheir existencewasknown.Oneofthefirsttechnical(materials)applicationscanbe foundinleatherprocessing,whichprovidedancientcivilizationwithleatherfor waterskins,bags,boats,orshoesasearlyas7000to3300bce[13].Inthistraditionalprocess,thebatingstepthatsoftensthematerialwasafermentativeprocess.Itreliedonenzymesproducedbybacteriafoundinpigeonsordogdung, whichwasaddedinthisstep.Thereplacementofdungbyenzymeswasahuge improvementthatwasstartedbyOttoRöhm,whosepioneeringworkandthe foundationofhisenzymecompanyaredescribedinSection1.1.3.
Enzymeapplicationsinorganicsynthesis(seeChapters5.1and5.2),feed(see Chapters3.2and3.3),textiles,detergents,andinbiorefineries(seeChapters 4.1–4.2)obviouslydonotrootbacktoancienttimesandareinventionsofthe twentiethcentury,whicharefurtherdiscussedinSection1.1.3.Morecomprehensivereviewsofdetergent,textile,andbiofuelapplicationscanbefoundhere [14–17].
1.1.2GrowingtheScientificBasis
Whydoessciencematterifpeoplealreadyenjoyedthebenefitsofenzymesfor thousandsofyearsasdescribedabove?Well,whilehumankindindeedenjoyed thedesiredeffectsofenzymessuchasreducingspoilageofvaluablerawmaterials,creatinganappealingtaste,orsupportingbetterdigestionoffoodproducts, thedesiredeffectssometimesjustdidnothappen,orturnedinhighlyundesireddirections.Sciencewasthereforeneededtoreduceorpreventsuchfailures. Applyingtheinsightscienceprovidedwasnotonlymakingexistingapplications morerobust.Sciencealsoenabledthediscovery,development,andefficientproductionofnewenzymeswithdesiredproperties,whichopenedupnewopportunitiestobroadentheirapplicationfield.Thefollowingsectionhighlightskey achievementsofmanybrilliantmindsinhistorythatcreatedthescientificfoundationonwhichtoday’scommercialsuccessisbuilt.
“Seeingisbelieving,”andseeingmicroorganismswasnotpossiblebeforethe pioneeringDutchscientistandgiftedcraftsmanAntonivanLeeuwenhoekdevelopedananalyticalinstrumentwithsofarunprecedentedmagnifyingpower.With hisspeciallypreparedlenseshecreatedsimplebutstillverypowerfulmicroscopesthatcouldreacha300-foldmagnification.Withthesemicroscopes,he couldnotonlyseelivingmicrobes,whichheoriginallyreferredtoasanimalcules (fromLatin animalculum = “tinyanimal”),butalsoalreadymadeobservations in1675thatbroughthimveryclosetorecognizingwhich“magicforces”are behindsomeofthedesiredeffectscreatedbymicroorganismsandtheirenzymes. Figure1.1.1showsacopyofhisfamouslettertotheRoyalSociety,whichwas translatedinEnglishbyHenryOlden,theEditoroftheJournalPhilosophical TransactionsoftheRoyalSociety,wherehenotonlydisclosedthediscoveryof “livingcreatures”butalsocommentedonthe“bubblingwater”comparingitwith fermentingbeer[18].
Leeuwenhoekdecidednottosharethedetailsofpreparingthelenseswiththe scientificcommunityandtookthissecretwithhimwhenhediedin1723.Because hekepthisknowledgesecret,hisdiscoveriesweredoubtedorevendismissed duringthefollowingcenturyasotherscientistscouldnotreproducehisresults.
Afterthediscoveryoflivingmicrobes,anotherimportantachievementwas reportedbyAnselmePayenandJean-FrancoisPersoz.TheFrenchchemistswere workingatasugarfactorytoimprovetheprocessofstarchconversion.In1833, PayenandPersozreportedthatanalcoholprecipitateofmaltextract(probablytheoldestandhighestvolumeindustrialenzymeproduct)containedasubstancethatconvertedstarchintosugar[19].Theynamedthesubstance“diastase” aftertheGreekword δι ́ αστασις (diastasis)(aparting,aseparation)asitcaused
Figure1.1.1 ExcerptofLeeuwenhoekfamouslettertotheRoyalSociety,whichwastranslated inEnglishbyHenryOlden,theEditoroftheJournalPhilosophicalTransactionsoftheRoyal Society.
thestarchinthebarleyseedtotransformquicklyintosolublesugarsandhence allowedseparationofthehuskfromtherestoftheseed.Theywerealsothefirst toproposeanomenclatureforsuchsubstancesusingthesuffix“ase”afterthe rootthatindicateswhichsubstancewasmodifiedbythebiomolecule.Thiswas thebeginningofasystematicenzymenomenclaturethatisusedtodate,with theexceptionofproteolyticenzymesendingwith“in,”suchassubtilisin.Today, “diastase”referstoany α-, β-,or γ-amylase(EC3.2.1.1-3)thatcanbreakdowncarbohydratesandareappliedinmanydifferentindustrialapplications.Anoverview ofseveralenzymesbasedontheECnomenclatureandtheirapplicationfields, someofwhicharefurtherdiscussedbelow,isprovidedinTable1.1.1.Itisperhapsinterestingtonotethatthesefundamentaldiscoveries,whichinspiredmany innovations,weremadeinthecontextofindustrialresearch.
Soonafterthefirstpreparationofdiastasefromaplantsource,additional enzymeclasseswereisolated.Importantexampleswereproteases(EC3.4.X.X), whichhydrolyzeproteinsandpeptides.In1836,TheodorSchwannstudiedthe processofhumandigestionandsucceededwithisolatinganenzymethathe calledpepsin[20].Thisisthefirstenzymepreparedfromanimaltissue,which
1.1.2GrowingtheScientificBasis
Table1.1.1 Overviewofenzymeclassesusedindifferentapplications.
ECnumberEnzymenameApplicationFunction
Oxidoreductases
1.1.3.4GlucoseoxidaseBakingGluten modification/dough strengthening BrewingOxygenreduction/shelf lifeimprovement
DairyMilkpreservation
TextileBleaching
1.1.3.5HexoseoxidaseBakingGluten modification/dough strengthening
1.10.3.2LaccasePulpandpaperBleaching TextileBleachingofdyetoprevent backstaining VariousCorktreatment
VariousPolymerizationoflignin forproductionofwood fiberboards
1.11.1.6CatalaseBrewingShelfliveimprovement
DairyMilkpreservation TextileHydrogenperoxide removal
VariousWastewatertreatment
1.11.1.7PeroxidaseBakingDoughimprovement
1.13.11.12LipoxygenaseBakingWhiteningofbreadcrumb Transferases
2.3.2.13TransglutaminaseDairyTextureimprovement
2.4.1.5DextransucraseVariousProductionofdextrans Hydrolases
3.1.1.3TriacylglycerollipaseBakingBreadimprovement DairyModificationofcheese flavor DetergentGreasystainremoval PulpandpaperPitchremoval
3.1.1.11PectinmethylesteraseBeverageYieldincreaseforfruit (berry,apple)juices,citrus fruitpeeling
3.1.1.26GalactolipaseBakingEmulsification
3.1.3.83-PhytaseBeverageMashinginbrewing FeedPhosphaterelease (Continued)
8 1.1IndustrialEnzymeApplications–OverviewandHistoricPerspective
Table1.1.1 (Continued)
ECnumberEnzymenameApplicationFunction
3.1.3.266-PhytaseBeverageMashinginbrewing
FeedPhosphaterelease
3.2.1.1 α-AmylaseBakingAntistaling BeverageMashing(brewing),apple juiceproduction
DetergentRemovalofstarch containingstains
StarchprocessingStarchhydrolysisforsyrup production
FeedDegradationofstarch
VariousViscosityreductioninoil drilling
TextileDesizing
3.2.1.2 β-AmylaseBeverageMashing(brewing)
3.2.1.3GlucoamylaseBeverageMashing(brewing),apple juiceproduction
StarchprocessingHydrolysisof maltooligosaccharidefor syrupproduction
VariousToothpaste
3.2.1.4Endo-1,4-β-glucanaseDetergentSoftening,color improvement
TextileCottonfinishing,denim ageing
3.2.1.6Endo-1,4(3)-β-glucanaseFeedIncreasedfeedefficiency
3.2.1.8Endo-1,4-β-xylanaseBakingImproveddough stability/handling
FeedIncreasedfeedefficiency
TextileFlaxretting PulpandpaperPulpbleaching
3.2.1.55ArabinosidaseBeverageApplejuiceproduction
3.2.1.60Endo-1,4-β-mannanaseDetergentRemovalofguargum containingstains
TextileFlaxretting PulpandpaperPulpbleaching
VariousViscosityreductioninoil drilling
3.2.1.91Exo-cellobiohydrolaseDetergentSoftening,color improvement
TextileCottonfinishing PulpandpaperMechanicalpulping (Continued)
Table1.1.1 (Continued)
ECnumberEnzymenameApplicationFunction
3.4.21.26ProlyloligopeptidaseBeverageImprovestabilityofbeer SupplementReductionofallergen
3.4.21.62SubtilisinDetergentRemovalofproteinaceous stains FeedImprovedfeedefficiency VariousMembranecleaning
3.4.21.63OryzinFeedImprovedfeedefficiency
3.4.23.4ChymosinDairyCheeseclotting
3.4.23.18AspergillopepsinIFeedImprovedfeedefficiency
3.4.23.22EndothiapepsinDairyCheeseclotting
3.4.23.23MucorpepsinDairyCheeseclotting
3.4.24.28BacillolysinFeedImprovedfeedefficiency
Lyases
4.1.1.5 α-AcetolactatedecarboxylaseBeverageDiacetylremovalforbeer flavorenhancement
4.2.2.2PectatelyaseTextileCottonscouring
4.2.2.10PectinlyaseBeverageFruitjuiceproduction, citruspeeling
Isomerases
5.3.1.5XyloseisomeraseStarchprocessingConversionofglucoseinto fructoseforhighfructose cornsyrupproduction
5.3.4.1ProteindisulfideisomeraseVariousHairwaving Ligases
6.3.2.28DipeptideligaseBioconversionProductionofdipeptides
Source:Aehle2007[1].AdaptedwithpermissionofJohnWiley&Sons.
foundearlyapplicationsintheleatherindustrytoremovehairandresidual tissuefromanimalhidespriortotanning(aprocessthatproducesleatherfrom hides).Itwasalsousedintherecoveryofsilverfromdiscardedphotographic filmsbydigestingthegelatinlayerthatholdsthesilvercompound[21].Another importantcontributionofSchwannwashisrecognitionthat“Alllivingthings arecomposedofcellsandcellproducts,”whichbecamethefoundationofwhat isknowntodayasthecelltheory[22].
Probablyoneofthemostinfluentialscientistsofthenineteenthcenturywas LuisPasteur.Heisbestknowntothepublicforhisinventionoftheheattreatmenttechniqueof,forexample,milkandwinetostopbacterialcontamination, aprocessnowcalledpasteurization.Hewasalsoveryinfluentialinthefieldof medicinebyrecognizingthatpartlyinactivatedmicrobescanresultinimmunity, whichlaidthefoundationforvaccinationthatsavedmillionsofpeople’slives [23].Furthermore,Pasteuralsolaidthefoundationforourunderstandingof
1.1IndustrialEnzymeApplications–OverviewandHistoricPerspective
molecularasymmetry(chirality).Hewasseparatingdifferentcrystalshapes fromeachothertoformtwopilesoftartariccrystals:insolution,oneformof thesecrystalsrotatedlighttotheleftandtheothertotheright,whileanequal mixtureofthetwoformscanceledeachother’seffectanddidnotrotatethe polarizedlight.Hefurtherobservedthatasolutionofthistartaricacidderived fromlivingthingsrotatedtheplaneofpolarizationoflightpassingthroughit, whereastartaricacidderivedbychemicalsynthesishadnosucheffectalthough itselementalcompositionwasthesame.Today,weknowthatthisphenomenon isattributedtothespecificpropertiesofenzymes.Theycaninducechirality ofreactingwithprochiralcompoundsordiscriminatedifferentenantiomers, whichisavaluablepropertyofenzymesforthesynthesisofenantiomerically purecompoundsasreviewedinChapters5.1and5.2.
Pasteuralsostudiedalcoholicfermentationwhereheprovedthatliving microorganisms(yeast)areindeedresponsibleforfermentation:hehasshown thattheskinofgrapeswasthenaturalsourceofyeastsandthatsterilizedgrapes andgrapejuiceneverfermented.In1862,whenstudyingthefermentation ofsugartoalcoholbyyeast,LouisPasteuralongwithFerdinandCohnand RobertKochcametotheconclusionthatthisfermentationwascatalyzedby a“vitalforce”containedwithintheyeastcells,whichwerecalled“ferments” [24].Hethenmovedontoinvestigatetheconversionofalcoholintovinegar andconcludedthatpellicle,whichhecalled“theflowerofvinegar,”servedasa methodoftransportfortheoxygeninairtoamultitudeoforganicsubstances, layingthefoundationforapplyingbiocatalystsinchemicalsyntheses[25]. In1886,BrownconfirmedPasteur’sfindingsandgavethecausativeagentin vinegarproductionthename Bacteriumxylinum.Brownalsofoundthatthis bacteriumcouldoxidizepropanoltopropionicacidandmannitoltofructose [26].Suchoxidativebiotransformationstepswerelaterappliedintheproduction ofl-ascorbicacid(vitaminC)asdescribedinSection1.1.3.
WilhelmFriedrichKühne,aprofessorofphysiologyattheUniversityofHeidelberg,wasthefirsttoproposetheterm“Enzyme”for“ferments”inapublication from1876asshowninFigure1.1.2[27].Despitethefactthathewasworkingon digestiveenzymes(trypsin)frompancreas,thewordenzymeisderivedfroma Greekword“���� ζ��μo�� ”meaning“inyeast.”
Theconnectionbetweenthefunctionoffermentingyeastandenzymeswas finallyprovedbyEduardBuchnerin1897.Hestudiedtheabilityofyeastextracts tofermentsugarintheabsenceoflivingyeastcells.Inaseriesofexperiments attheUniversityofBerlin,hefoundthatsugarwasfermentedevenwhenthere werenolivingyeastcellsinthemixtureandnamedtheenzyme(mix)thatbrought aboutthefermentationofsucrose“zymase”[28].In1907,hereceivedtheNobel PrizeinChemistry“forhisbiochemicalresearchandhisdiscoveryofcell-free fermentation.”
Oncetheexistenceofenzymeswasshown,researchbegantoprovidedetailed insightsintotheiruniquepropertiesandfunctions.Thefactthatenzymesare veryspecificcanbeconcludedfromPasteur’sdiscoveryofchirality.While heobservedtheeffect,Pasteurdidnotyetmakethedirectlinkbetweenthe phenomenonofchiralityinducedbylivingmicroorganismsandthespecificity ofenzymesthatwerediscoveredlater,asjustdescribed.Astartingpointto
Figure1.1.2 ExcerptofKühne’soriginalpublicationfrom1876whereheintroducedtheword enzymesforthefirsttime.
describeenzymespecificitywasprovidedbyEmilFischer,whosuggestedin1894 thatenzymesandtheirsubstratespossessspecificcomplementarygeometric shapesthatfitexactlyintooneanother.Thismodelisreferredtoas“thelockand key”model[29].Whilethismodelcanbeusedtoexplainenzymespecificity,it failstoexplainoneofthecorefunctionsofenzymesasacatalyst,whichisthe rateaccelerationobtainedbystabilizationofatransitionstate.In1958,Daniel Koshlandsuggestedamodificationtothelockandkeymodel,whichisknownas the“inducedfit”model:sinceenzymeshavearatherflexiblestructure,theactive siteiscontinuouslyreshapedbyinteractionswiththesubstrate[30].Thisbrought usclosertoanexplanationforhowenzymesfunctionbutarealbreakthroughin ourunderstandingwasachievedbyenzymestructuredetermination.
LysozymewasthefirstenzymecrystallizedbyEdwardPenleyAbraham andRobinsonin1937[31]enablingmanyyearslatertheelucidationofthe three-dimensionalstructureofheneggwhitelysozymedescribedbyDavid ChiltonPhillipsin1965.Heobtainedthefirst2-ÅresolutionmodelviaX-ray crystallography[32].AsaresultofPhillips’determinationofthestructureof lysozyme,itwasalsothefirstenzymetohaveadetailed,specificmechanism suggested,whichprovidedanexplanationforhowenzymesspeedupachemical reaction.
StartingwiththecrystallographicdataofRosalindFranklin[33];theelucidationoftheDNAstructurein1953[34]by,andtheawardingoftheNobelPrize to,Watson,Crick,andWilkins[35];theunravelingofthegeneticcodeforwhich MarshallWarrenNirenberg,HarGobindKhorana,andRobertWilliamHolley receivedtheNobelPrizein1968[36];andtheintroductionofrecombinantDNA
1.1IndustrialEnzymeApplications–OverviewandHistoricPerspective technologyin1973byCohenetal.[37],thefieldofmolecularbiologyrevolutionizedthecost-efficientproductionofenzymes,whichisdescribedinChapter1.4. Italsoprovidedefficienttoolsforenzymediscoveryandengineering,whichare furtherdiscussedinChapter1.2.
Buildingontheabove-describedworkandmanymorescientificachievements,thefollowingsectionhighlightsthepioneeringworkofentrepreneurs thatmarkedthebeginningoftheindustrialapplicationofenzymesandthe developmentofamultibillionEuroenzymeindustry.
1.1.3TheBeginningofIndustrialApplicationsand theEmergingEnzymeIndustry
Lookingbackattheancientenzymeapplicationsandthescientifichistory describedabove,itisnotsurprisingthatthefirstindustrialapplicationsof enzymesdidnotmakeuseofisolatedenzymesbutcrudeextracts(suchasmalt extracts),orevenwholecellbiocatalysts.
Thisisalsotrueforthesynthesisofchemicalcompoundsintheso-called biotransformationprocesses.Vinegarproductionisperhapstheoldestand bestknownexampleofsuchanindustrialscalebiotransformationprocess inwhichoxidationofethanoliscatalyzedbyaceticacidbacteria.Another pioneeringexampleofanindustrialbiotransformationprocessistheproduction ofl-( )ephedrine,whichwasintroducedbyKnollAG(todayBASF)inthe 1930s[38].TheprocessisbasedonthediscoveryofNeubergandHirschwho showedthatinpresenceofyeast,benzaldehydecancondensewithacetaldehyde toopticallyactive1-hydroxy-1-phenyl-2-propanone,whichisthenchemically convertedtol-( )ephedrine[39].Thiswasthefirstexampleofanenzymatic C–Ccouplingreactionatanindustrialscale.Morerecentexamplesofindustrial applicationsofaldolasescatalyzingC–Ccouplingreactionsarereviewedin Chapter5.2.
KluyveranddeLeeuw,twoDutchscientistsfromDelft(NL),demonstrated in1924that Acetobactersuboxydans canoxidized-sorbitoltol-sorbose[40]. ThisbecameanimportantintermediateintheReichstein–Grüssnersynthesis ofl-ascorbicacid(vitaminC)[41].Thissynthesiswasturnedintoanindustrial processbyRoche(RocheVitamins,nowpartofDSM)inthe1930s[42].
In1953,Petersonetal.reportedthat Rhizopusarrhius canconvertprogesteroneinto11-α-hydroxyprogesterone,whichwasusedasanintermediateinthe synthesisofcortisone[43].Thismicrobialhydroxylationsimplifiedandconsiderablyimprovedtheefficiencyofthemultistepchemicalsynthesisofcorticosteroid hormonesandtheirderivatives.AlthoughthechemicalsynthesisfromdeoxycholicaciddevelopedatMerck(Germany)wasinprinciplepossible,itwastoo complicatedanduneconomical:31stepswerenecessarytoobtain1kgofcortisoneacetatefrom615kgofdeoxycholicacid.Theintroductionofmicrobial 11-α-hydroxylationofprogesteronedramaticallyreducedthepriceofcortisone from$200to$6pergram.Furtherimprovementshaveledtoanestimatedprice oflessthan$1pergram[44].
1.1.3TheBeginningofIndustrialApplicationsandtheEmergingEnzymeIndustry 13
Anotherremarkableexampleofapplyingawholecellbiocatalystistheproductionofacrylamide.Inthemid-1970s,NittoChemicalIndustries(nowMitsubishi Rayon)introduceditsbiocatalyticproductionusinganitrilehydrataseinthe formof Rhodococcus cells.Thisprocessisconsideredamilestoneinindustrial biocatalysisdemonstratingtheimpressiveefficiencyofenzymesystemsoperatingatproductconcentrationsofupto700g/lfortheproductionofalow-cost commoditychemical.Italsodemonstratesthatenzymescanbeappliedinavery crudeformaswholecellbiocatalyst,providingamorecost-competitivesolution. Theenzymeprocessisalsoa“greener”solutionbyconsuminglessenergy andavoidingheavymetalwastewaterproblemsofthealternativechemical process[45].
Attheendofthe1970sToyoJozo(Japan)incollaborationwithAsahiChemical Industry(Japan)pioneeredtheindustrialproductionof7-aminocephalosporanic acid(7-ACA)byachemo-enzymatictwo-stepprocessstartingfrom cephalosporinC.Inthe1990sdevelopmentsfromGist-Brocadesapplied metabolicaswellasproteinengineering,whichenabledfurtherbreakthroughs notonlyfortheproductionofcephalosporinCbutalsoforthechemo-enzymatic synthesisofseveralderivativesasreviewedinChapter5.1.
Intheearly1980sanotherpioneeringprocesswasintroducedbyDegussa (nowEvonikIndustries).IncollaborationwithProf.KulaandProf.Wandreya racemicresolutionprocesswasdevelopedforproductionofenantiomerically pureaminoacids(e.g.l-methionine)applyinganl-aminoacylase(EC3.5.1.14) from Aspergillusoryzae inanenzymemembranereactor(EMR).Sincethen, manyotherracemicresolutionprocesseshavebeendeveloped[46].Amore recentexampleusinganon-animal-derivedpigliveresteraseisreviewedin Chapter5.1.
ThesamegroupfromJülichandDegussathatdevelopedtheabovementioned EMRprocessalsointroducedthefirstreductiveaminationprocessinthe1990s fortheproductionofl-tert -leucineandl-neopentylglycine.Inthisprocess,a leucinedehydrogenase(EC1.4.1.9)isusedforreductiveaminationofaketoacid togetherwithaformatedehydrogenase(EC1.2.1.2)forrecyclingthecofactor NADH.Morerecentexamplesofapplyingdehydrogenasesarepresentedin Chapter5.2.Amorecomprehensiveoverviewofenzymesusedinorganic synthesisandindustrialbiotransformationsisprovidedhere[2,3].Whilethe above-describedapplicationfieldofenzymesdelivershighlyvaluablesolutions forchallengingsynthesisproblemsforthechemical,finechemical,andthe connectedpharmafield,itsimpactongrowingthetotalenzymebusinessisstill verylimited,asshowninFigure1.1.3.
Intheearlyhistoryof brewing,maltproductionwasanintegratedpartofthe brewingprocess.Inthemaltingstep,thewatercontentandtemperatureofgrains frommalt,sorghum,orwheatwereadjustedtoallowthemtogerminate.During thisgerminationprocess,grainsthatalreadycontainenzymes(e.g. β-amylases) produceadditionalenzymessuchas α-amylases.Afterstoppingthegermination processbyaheatingstep,themaltproductisobtainedandcontainsamixof enzymesthatconvertthestarchofthegrainsintoafermentablesugarsolution. Duringthelastcenturies,thismaltingstepwasseparatedfromthebrewingprocess,makingmaltprobablythefirstcommercialandtodatethehighestvolume
Leather/textile
Starch processing
Dairy
Baking
Pulp and paper
Health supplements
Feed
Detergent
Biofuel
Gist-brocades MAXATASE (bacterial protease for detergent)
Novo ALCALASE (bacterial protease for detergent)
Gebrüder Schnyder (bacterial protease for textile)
Novo THERMOZYM (amylase for textile)
Schweizerische Ferment AG (pectinase for fruit proc.)
Novo (trypsin for leather)
Röhm (pectinase for fruit processing)
Boidin & Effront (bacterial amylase for textile)
Schweizerische Ferment AG (malt for textile)
Rohm’s BURNUS (pacreatic trypsin for textile)
Rohm’s OROPON (pacreatic trypsin for leather)
Take-diastase (fungal amylase for starch & health supplement)
Chr. Hansen’s (Rennet)
Figure1.1.3 Historiceventsandestimatedgrowthofenzymebusinessfrom1875until2025,brokendownintodifferentapplicationfields.
1.1.3TheBeginningofIndustrialApplicationsandtheEmergingEnzymeIndustry 15 enzymeproduct.Itssuccesscanbeattributedtothefactthatitenabledaneasier, faster,andmoreconsistentbrewingprocess.However,themaltenzymes,first isolatedbyPayenandPersozin1833asdescribedintheprevioussection,do havesomelimitations.Theycanonlyworkatcertaintemperatures,pHvalues,or containunwantedsideactivitiesgivingrisetomanyenzymeinnovationsforthe beverageindustry,whichiscoveredinChapter2.2.Accesstostandardizedand relativelypureenzymepreparationswasalsoanimportantfactortointroduce amylasesinthebakingindustries,whichstartedtobeappliedinthebeginningof thetwentiethcenturywithmanyfollowinginnovationscoveredinChapter2.1.
ThehistoryofmodernenzymetechnologyreallybeganwhenChristian Hansen,inajointventurewithpharmacistH.P.Madsen,openedthefirst enzymefactoryin1873.TheystartedthefirstDanishproductionofpepsin preparationsfor treatingdigestiveproblems.
ChristianHansenalsobeganworkingwithrennet,theenzyme-richsubstance extractedfromthefourthstomachofruminants.Theseextractswereusedsince thousandsofyearsforthemanufactureofcheesetomakethemilkcoagulate. Asdemandrosesharplyinthenineteenthcentury,ChristianHansenbegana seriesofexperimentsontheproductionofrennet.In1874theChr.Hansen’s Teknisk-KemiskeLaboratoriumopened,whichis,today,knownasthecompany Chr.Hansen.InaconvertedmetalworkshopinCopenhagenhebeganthe commercialproductionofstandardizedquality-assured,liquidanimalrennet for dairyapplications,whicharefurtherdescribedinChapter2.3.Justasin thepreviouslydescribedexampleforthebrewingindustry,simplificationand standardizationtoreduceprocess/batchfailureswithanegativeeconomic impactfortheemergingcheeseindustrywasagainanimportantdriverand successfactorfortheintroductionofacommercialenzymeproduct.
IncontrasttoWesterncountries,whichoriginallyusedmaltintheirbrewing processesasdescribedabove,Asiancountriesusedthetraditional“Kojiprocess.” Inthisprocess Aspergillus strainsareusedasstarterculturestofermentriceor soyforproductionofricewine(sake),soysauce(shoyu),soybeanpaste(miso), anddistilledspirits(shochu)[47].BelievingthatKojimadefrom A.oryzae could revolutionizetheAmericandistilleryindustryandoutcompetemalt,Jokichi TakamineadaptedtheKojiprocessfortheproductionofdiastase(amylase), whichhepatentedin1894.Thisisreportedtobethefirstpatentonamicrobial enzymeprotectingtheproductionofdiastaticenzymebygrowingfungionbran andusingaqueousalcoholtoextracttheenzymeasdescribedinthepatent applicationshowninFigure1.1.4[48].
Whilehisinitialattemptstointroducemicrobialdiastaseintheconservative Americanbrewingindustrywasnotcommerciallysuccessful,healsorealized thatthisenzymepreparationhaspotentialmedicalapplications.Parke,Davis &CompanyofDetroitreceivedthelicenseontheamylaseandmarketedit successfullyunderthebrandnameTaka-diastaseasadigestiveaidtotreat dyspepsia,whichiscausedbyincompletedigestionofstarch.Thisexampleand thepepsin-basedproductdevelopedbyChr.Hansencanbeseenasstarting pointsfortheapplicationfieldof enzymesupplements,whicharefurther describedinChapter3.1byHirose.Takamineexpandedhisbusinessoperations inbothenzymesandpharmaceuticalsandfoundedthreemajorcompanies:
Figure1.1.4 JokichiTakamine’spatentfrom1894,thefirstenzymepatentprotectinga processforproductionofdiastasefromafungalsource.FromUSpatentoffice.
SankyoPharmaceuticalCompanyofTokyo,theInternationalTakamineFerment CompanyofNewYork,andtheTakamineLaboratoryofClifton,NewJersey. Withhisdeath,theInternationalFermentCompanyofNewYorkwasdissolved. Takamine’sson,Eben,continuedrunningTakamineLaboratory.Afterhisdeath in1953,Eben’swidowsoldthecompanytoMilesLaboratoriesendingupin today’senzymebusinessofDowDuPontasdescribedinFigure1.1.5.
Takamine’sworkonfungalamylaseswassucceededbyBoidinandEffront, whopioneeredtheworkonbacterialenzymesusingsurfacefermentation.One oftheirlandmarkpatentswasobtainedin1917claimingtheuseofamylases inprocessesabove80 ∘ Candunderalkalineconditions,whichisadvantageous inindustrial starchprocessing [49].AfewyearslatertheyfoundedthecompanySocietéRapidaseinSeclin(France)commercializingnotonlyenzymes importanttotheemergingstarchindustrybutalsopioneeringenzymesfor textileapplications [50].Priortotheintroductionofenzymes(initiallyin theformofmaltorpancreasextract)textilesweretreatedwithacid,alkali,or oxidizingagents,orsoakedinwaterforseveraldayssothatnaturallyoccurring microorganismscouldbreakdownthestarchthatisusedassizingagent. However,bothmethodsweredifficulttocontrolandsometimesdamagedor discoloredthematerial,whichcouldbepreventedbyusingenzymes.Asshown intheoverviewofcompaniesinFigure1.1.5,SocietéRapidasewaslateracquired byGist-Brocades(nowpartofDSM).
2017 DuPont and Dow merge to DowDupont; Enzyme Business is part of new Specialty Products Company
2013 Novozymes acquires Iogen Enzyme Business
NovozymesDowDuPontAB Enzymes
2011 DuPont acquires Danisco DuPont
2007 Novozymes acquires Biocon’s Enzyme Business
2005 Danisco acquires Genencore International from Eastman
2002 Genencore International acquires Enzyme Biosystems and RhodiaFeed & Brewing Danisco
2001 Röhm Enzyme GmbH renamed in AB Enzymes
2000 Novo splits into Novozymes, Novo Nordisk A/S, and Novo A/S Novozymes
1999 Degussa-Hüls sells Röhm Enzyme GmbH to Associated British Foods; Danisco acquires Cultor
1995 Genencore International acquires non-food enzyme business from Gist-Brocades and Enzyme business from Solvay
1990 Solvay purchases enzyme business from Bayer
1989 Hüls acquires Röhm GmbH
1982 Genentech and Corning create Genencore, which becomes part of Eastman Kodak
1981 Corning acquired fermentation business of Rohm & Haas; Gist-Brocades acquires Société Rapidase
1976 Genentech founded
1968 Nederlandsche Gist-en Spiritusfabriek and Brocades merge to form Gist-Brocades
Enzyme Biosystems Rhodia Feed & Brewing
Genencore International Non-food enzyme business
Solvay Enzyme business
Genencore (Eastman Kodak)
Genentech Corning
Gist-Brocades
Enzymes
Röhm Enzyme (Deguss-Hüls)
1998 DSM acquires Gist-Brocades DSM Brocades
1967 Novo acquires Schweizerische Ferments AG from Sandoz
1963 Sandoz acquires Schweizerische Ferment AG
1953 Miles acquires Takamine Laboratories
1925 Novo Terapeutisk Laboratorium founded Novo Terapeutisk Laboratorium
1922 Societe Rapidase founded Sandoz
1915 Takamine Laboratories founded
1907 Röhm Enzyme founded
1905 Schweizerische Ferment AG founded 1869 Nederlandsche Gist-en Spiritusfabriek founded
Schweizerische Ferment AG
Société Rapidase
Röhm Enzyme DSM
Röhm GmbH (Hüls)
Rohm & Haas
Röhm GmbH
Nederlandsche Gist-en Spiritusfabriek
Figure1.1.5 Overviewofcompanyrootsstartingwithpioneeringentrepreneurstotodays’worldleadingenzymesuppliers.
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CHAPTER XIV THE GIANTS
THEY stood on the dock of a river where great ships leave their burden of iron ore.
“There she comes!” exclaimed Mr. Prescott, pointing to a freighter that was slowly drawing near.
“No giants in sight yet,” said Billy.
“It’s your eyes that are not seeing,” returned Mr. Prescott. “That boat herself is a giantess. Watch.”
Hardly had the great boat been made fast to her moorings before, in some mysterious way, the hold of the ship opened wide from stem to stern.
Then somebody touched a lever somewhere, and over the hold swung a row of buckets that, opening like two hands, grabbed into the ore, and seizing tons of it, swung back to the dock. A touch of another lever unloaded it into huge storage bins.
“Billy Bradford,” said Mr. Prescott, “weren’t those the hands of a giant?”
“Sure, sir,” answered Billy, who stood staring in wonder.
“That ore,” said Mr Prescott, “came from a surface mine up in the pine woods of Lake Superior, a thousand miles away.
“Perhaps, gentlemen, you may like to know that the American supremacy in iron is largely due to those open pit mines up in Minnesota.
“Much of the ore in that region is so near the surface that a steam shovel can easily strip off the ‘overburden’ of the soil and the roots of pine trees.
“When that was done, giant hands seized that ore, lifted it up, and loaded it into bins, high up on the bluffs.
“Then a man, not a giant, touched a treadle, and another kind of giant, named ‘gravity,’ made the ore run from the bottom of the car into a bin.
“Chutes from the ore bin ran into the hold of the steamer, and almost before she had been tied to the dock she was ready to come down here.
“Giants or not, Billy Bradford?”
“Iron giants,” answered Billy.
“Rather different, Mr. Bradford,” said Dr. Crandon, “from fishing ore with tongs.”
“We’ve moved along a great way since that time,” said John Bradford, “and most of our progress has been due to iron.”
“Giants don’t do all the work even now,” said Mr. Prescott. “They make short work of iron mountains and surface deposits, but most of them are too large to work underground; though we mustn’t forget that Giant Electricity works down there with the men.
“Giant Gravity helps too, for, when they work below the deposit, he caves the ore down. Of course some ores are so hard that they can’t be caved, so there is still some mining for the men to do.”
“Was there,” asked Billy, trying to speak in a sort of offhand way, “an iron mountain where this iron came from?”
“There are some,” answered Mr. Prescott, “up in that region.”
Billy had been paying very close attention to what Mr. Prescott had been saying. There was something that he wanted especially to find out. He felt very sure, now, that he was hearing about an iron mountain that he had heard about once before.
He felt very sure, but he wouldn’t ask any more questions, because that was the secret that he had with Thomas Murphy.
The others started for the car But Billy stood a moment longer to look at the giant hands that, having finished their work, were hanging idly in the air. The hold of the ship, emptied of its burden, was already beginning to close.
“Beginning to believe in giants, aren’t you?” said Mr. Prescott, as Billy stepped into the car.
“The next giant will be a hungry fellow, and he is very, very tall; so he eats a great deal.”
“An iron-eater, is he?” queried Dr. Crandon.
“We ourselves will have something to eat before we visit him,” said Mr. Prescott, ordering Joseph to drive back to the hotel.
“Mr. Prescott,” said Dr. Crandon, as they sat at table, “is iron ever found in a pure state, like gold, for instance?”
“It is practically never found in a pure state,” answered Mr. Prescott, “except the meteoric iron, ‘the stone of heaven.’”
Billy looked at him questioningly.
“That was rather technical, wasn’t it, Billy? You see, I was talking to a technical man. Just between you and me, meteoric iron comes down from the sky, from what we call shooting stars. Sometimes large pieces are found. I suppose that much of it falls into the sea. It is the purest iron that has ever been found.”
“What about magnetic iron?” asked Dr. Crandon. “Where does that come from?”
“At the present time,” answered Mr. Prescott, “most of it comes from Sweden and Norway. It makes the best kind of steel.
“Ages ago, the first was found in Magnesia,” said Mr. Prescott casting a quick glance around the table.
“The people there found certain hard, black stones which would attract to themselves bits of iron and steel. So they named them magnets, from Magnesia, the place where the stones were found,” finished Mr. Prescott, with another look around the table.
“It’s of no use, Prescott,” said Dr Crandon, “you needn’t look at us. We don’t any of us know even where to look for Magnesia. Don’t suppose we could find it even if we had a map.”
“I presume you remember, Crandon,” said Mr. Prescott, “the place that boasted that ancient wonder of the world, the Temple of Diana.”
“Ephesus!” said Dr. Crandon, quickly. “I do happen to know that Ephesus is in Asia Minor.”
“Then,” said Mr. Prescott, still keeping his face very grave, “I should strongly advise your finding Ephesus first. That’s in the near neighborhood of Magnesia.”
“Thank you,” said Dr. Crandon gravely. “Though I did not know where magnetic iron came from, I do happen to know that it is sometimes called ‘lode-stone.’
“And I know, too, that Sir Isaac Newton—he’s the one, Billy, who ran down Giant Gravity—had a ring set with a lode-stone that could lift two hundred and fifty times its own weight.”
“And I know,” said Mr. Prescott, “that I am very grateful to Dr. Crandon for telling me about the new electro-magnet that I now have at the mill. I feel very much easier, now, about my workmen’s eyes.”
“Do you mean,” asked Billy, “that thing that you brought home that I thought was a new desk telephone?”
“It does resemble a telephone,” said Dr Crandon, “only it has a tip instead of a mouthpiece. It’s a great thing for taking bits of steel out of eyes.”
“Isn’t there such a thing,” asked John Bradford, “as a magnetic separator?”
“Glad to hear from you once more, Bradford,” said Mr. Prescott, with a smile. “It has been some time since you have said anything.”
“I have been having too good a time,” said John Bradford, “to want to talk. I should like, now, to have you tell us about the separator.”
“It is an electro-magnetic drum. When the finely crushed ore is poured on it in a stream, the drum attracts the iron, while the earthy
matter, which is non-magnetic, falls off by the action of gravity The iron is carried on by the drum, until a brush arrangement sweeps it off into a truck.
“That is a case, Billy, where Giant Gravity and Giant Electromagnet fight over the ore, and each gets away with a part of it.
“Perhaps I ought to explain to you that, when a bar of soft iron is put inside an insulated coil of copper wire and a current of electricity is passed through it, it becomes a powerful magnet. That is what we mean by an electro-magnet. The advantage of that is that it ceases to be a magnet when the current ceases, so it can be controlled. You will see some before I am through showing you giants.
“There is also an electric cleaner that collects the iron that is left in the corners of cars. Those devices save iron. Strange as it may seem, however, not all iron will respond to the magnetic cleaners.”
“Is there,” asked Dr. Crandon, “any danger that the iron in the world will be exhausted?”
“I hardly think so,” answered Mr. Prescott. “The available ores, in the single range that we were talking about this morning, run up into the trillions of metric tons.”
“I read something the other day,” said John Bradford, “about some iron that had been found in Sweden, up beyond the arctic circle.”
“That,” said Mr. Prescott, “is one of the most extensive deposits in the world. The countries of the western part of Europe draw upon that supply.
“It is very likely that we haven’t found all the iron yet, and even more likely that we shall find a way to make use of the poorer ores.
“By the way, Billy, there is one kind of iron called ‘iron pyrites.’ It looks so much like gold that it has deceived many a poor fellow into thinking that he had found gold. It well deserves the name ‘fool’s gold.’ It doesn’t even make good iron. I’ll show you some when we go home. Now we’ll go to see the iron-eater.”
Ten minutes later Billy exclaimed:
“He’s tall!”
“Not quite a hundred feet,” said Mr. Prescott.
“He’s black!” said Dr. Crandon.
“He roars!” added John Bradford.
“And,” said Mr. Prescott, “even if he could be moved, he’s rather too valuable for a circus manager to buy, for he cost a million dollars. I really think he’s the most fearful thing ever made by man. The Germans, though, did a great thing for iron when they evolved the blast furnace.”
“Makes our cupola,” said John Bradford, as they stopped before the tall iron stack, “look very small.”
“Ours,” said Mr. Prescott, “is only a dwarf, but he does something like the same work; only here they put in iron ore instead of pig iron. Blast furnaces make pig iron.”
“THE
MOST FEARFUL THING EVER MADE”
“What diet,” asked Dr. Crandon, “do they give this giant?”
“You’re bound to think professionally, aren’t you, Crandon? He’s restricted to coke, iron ore, and limestone, but they feed him very often. They see, too, that he has plenty of hot air to breathe.
“The old problem used to be how to get heat enough to melt the ore. That was solved by a Scotchman, who originated the use of the hot blast.
“The gas produced by the furnace used to be wasted. Now they utilize it in the hot-blast stoves. That accounts for some of the huge pipes attached to the furnace. Come this way, and I’ll show you a stove.
“Here it is, almost as tall as the furnace itself. This giant, also, is encased in an armor of iron plates. If we could look inside, we should see that it is almost filled with open brick work that resembles a honeycomb.
“They send hot gas over the brick work till the stove is hot, then they shut off the gas and start the engine that blows in cold air. That, heated by the bricks, is forced into the furnace.
“One of those great pipes up there is where they draw off the slag. It is so much lighter than the iron that it rises to the top, like cream on milk.
“Down here they draw off the iron. Sometimes they keep it hot for the next process; sometimes it is made into pig iron.”
“What,” asked Dr. Crandon, “becomes of the slag?”
“That depends somewhat on the chemical composition of the slag. Some kinds are broken up to be used as foundation for roads; others are granulated by being run into water, and so made into cement. Over in Germany, where the ores are rich in phosphorus, they grind up the linings of the furnace to make phosphatic fertilizers for the farmers.”
“Then,” said Dr. Crandon, “the making of iron involves the use of chemistry, doesn’t it?”
“It certainly does,” answered Mr Prescott; “from the chemical composition of ores to the finished product. We are learning a great deal just now from the chemists about steel alloys.
“I didn’t tell you that from the gas they sometimes save ammonia, tar, and oils, before it is fed to the hot-blast stoves.”
“By-products,” said Dr Crandon, “seem to be a feature of modern industry.”
“It is high time,” said Mr Prescott, “that waste should receive attention.”
“Before we leave this giant I must tell you that he already has a dangerous rival—listen, Billy, for it’s almost a David and Goliath story —in a little electric smelter. Some of them can be moved about like a portable sawmill.
“Up in Sweden, where the ores are among the purest in the world, they use electric smelters and make steel direct from the ore.”
“Any more giants?” asked Billy.
“You’ll think so,” answered Mr. Prescott, “before I am through with them; but we’ve seen enough for to-day. Next time I’ll show you giants that have done something more than to make iron, for they have really reduced the size of the world.”
“Whew!” exclaimed Dr. Crandon.
“Before that,” said Mr. Prescott, “I am going to introduce you to some pygmies.”
CHAPTER XV THE PYGMIES
“SHALL we need glasses, Prescott, in order to see your pygmies?” asked Dr. Crandon, the next morning, while they were waiting for the car.
“I will agree to furnish all the glasses needed,” answered Mr. Prescott.
Much as Billy wanted to know what Mr. Prescott was going to show them, he had made up his mind to trust to his eyes to find out.
John Bradford was learning so many things that he had long wanted to know that he was simply enjoying things as they came along, and being thankful.
“To the office of the steel works, Joseph,” said Mr. Prescott.
On past the great yard of the blast furnace they went, then along by some high brick walls until they stopped in front of a two-story cement building.
Then they followed Mr. Prescott till he stopped at the head of the stairs, and knocked at a door.
“Come in,” shouted somebody in a cordial voice.
“Hullo, Harry, old fellow!” said the owner of the voice, still more cordially, as he came forward with outstretched hand.
“This,” said Mr Prescott, “is my classmate, Mr Farnsworth, who is at the head of the laboratory.”
After he had introduced John Bradford and Dr. Crandon, he added, “And this is Billy Bradford.”
Then he said, “I’ve brought these friends of mine to see your show. We’ve been to see some of the giants in the iron industry. Now I
want them to have a look at your pygmies.”
“Pygmies they shall see,” said Mr. Farnsworth, with an appreciative smile. “Hardly a technical term, but a good way, Harry, to get hold of the facts. Pygmies they shall be.
“Sit down, all of you,” he said, pointing to chairs by his low, broad table.
Pushing back the sliding door of a case behind the table, he took out a tray containing small round pieces of iron and steel.
“Shall I tell you about these specimens, or will you ask me?”
“Just give us a general idea, Jack,” answered Mr. Prescott; “we might ask the wrong questions.”
“Then, Billy Bradford,” said Mr. Farnsworth, smiling at Billy, “I’ll explain to you, and the others may listen.
“You see we chemists analyze the ores before they are smelted; so we know something about what kind of pig iron we shall have. But when we want to know what kind of finished iron or steel we have from a given process, we can’t tell much by analyzing it, so we have to depend on our microscopes.
“Metals crystallize, if they have just the right conditions. Each metal has its own form; so, if you could find a single crystal, you would recognize it by its form.
“But when melted iron grows solid, the crystals are crowded so close together that, when it is prepared for the microscope, and polished like this, the surface looks as if it were made up of ‘crystal grains.’
“Sometimes crystallization takes place in steel if it is subjected to long repeated jar. Many accidents in engines are due to that.”
As he took the cover off his microscope, Mr. Farnsworth said:
“I suppose, Harry, that your ‘pygmies’ are the elements that are found in the various kinds of iron?”
“The same,” answered Mr. Prescott.
“Then I shall tell Billy Bradford that some of the pygmies are enemies and others are friends; some need to be driven away, and others should be invited to come in.
“The most numerous enemies are the Carbon pygmies. The blast furnace drives most of them off, but they have to be fought in the pig iron, too.
“Sulphur pygmies are about the worst of all, because they make the iron brittle. They are practically the hardest to drive away.
“Phosphorus pygmies haven’t a good reputation, but they are in much better standing than the Sulphur enemies.
“Now, if you’ll look in here—this is the purest and the softest Swedish bar iron—you’ll see where the edges of the crystals come together. These are friendly Ferrite pygmies, crowding close together Ferrum is the Latin name for iron; you must remember that.”
“If I didn’t know,” said John Bradford, when he took his turn, “I should think I was looking at some sort of wood with a very fine grain.”
“This,” said Mr. Farnsworth, changing the specimen, “has black and white streaks in it; that means that the iron has begun to be steel. When it has light patches like these in it, we know that it has taken up more carbon, and has grown harder.
“So it goes,” he said, showing one after another of the specimens. “You can see for yourself that, if friendly pygmies stand in line, taking hold of hands, that would make a good kind of iron to draw out into a wire. If enemies stand around in groups, they make the iron easy to break.
“When we want steel for chisels, for example, we invite Tungsten to come in; when we want certain parts for automobiles we call in some Vanadium pygmies.”
“So,” said Mr. Prescott, “while we need the giants to make the pig iron, the real value of the iron and steel depends on the pygmies.”
“That’s about the size of it,” said Mr. Farnsworth.
“Anything the trouble with you, young chap?” asked Dr Crandon. “You haven’t spoken for ten minutes. Feel bad anywhere?”
“No,” answered Billy “I was just wishing I could know about all those things.”
“I’m glad it’s nothing worse than that,” said Dr. Crandon.
“Now,” said Mr. Prescott, “we’ll start for some more giants. Coming, Farnsworth?”
“Sorry, not to-day. Call again!”
“The steel mill comes next on my program,” said Mr. Prescott, when they went out. “I want you to see a Bessemer converter, an open hearth, and some crucibles, because that practically covers the different methods of making iron and steel.
“Here is the Bessemer converter. You see it is an iron cylinder made of wrought iron plates, and it tapers off at the top in a conical end. See. It is swinging down to be filled almost as easily as you can turn your hand over. In a moment it will stand up again, twenty-five feet tall.
“Bessemer got hold of the idea that air could be used instead of fuel. They say he risked his life in his experiments. He worked a long time, but he won, and the Bessemer converters started the boom in steel.
“See it come up again, with fifteen tons of hot pig iron in it. Down in the bottom of the converter is a blast chest where the air is forced in under pressure, after it has been blown into a tank by blowing engines.”
“O-o-oh!” exclaimed Billy, as the top of the converter seemed to burst into flame, and a shower of sparks came down.
“That,” said Dr. Crandon, “is surely a fearful sort of thing!”
Then the flame began to drop slowly, and they saw that the converter itself was safe.
“This process burns out all the carbon. Bessemer was trying to make wrought iron when he started out. Now they put back the right
amount of carbon, and make the iron into steel.
“It’s a chemical process. When the air strikes the hot metals the oxygen unites with them, and they burst into flame. The whole process takes between fifteen and twenty minutes.”
“I am very sure,” said Dr. Crandon, “that I shouldn’t like to work here.”
“When we get to the open hearth process, which is the rival of the Bessemer,” said Mr. Prescott, “I expect that none of you will want to work there.”
“For my part,” said John Bradford, slowly, “I prefer Prescott mill.”
“So do I,” said Billy.
“Which reminds me,” said Mr. Prescott, “to tell you that I have been looking at some machines to help in the foundry. They will help about lifting and ramming; but they won’t do away with the work of men.
“Here we are, gentlemen, before a Siemens-Martin open hearth. This is a continuous process. It was evolved by Sir William Siemens, a German-English engineer, and his brother. Then a man named Martin, a Frenchman, I understand, found a way to mix the iron and steel that are put on the hearth, so it bears both the names.
“We’ll just look in. It is a large, shallow basin, made of bricks, partly filled with iron. Both hot air and gas are burned on top of the iron. The process takes seven or eight hours; but it produces larger quantities of steel than the Bessemer converters can do.
“Then, too, it furnishes all kinds of iron and steel, for they sample it as it burns, and draw off the steel at any percentage of carbon that they want.
“Cast iron has a great deal of carbon in it; steel has much less; and wrought iron has almost none.
“Now, we’ll go over to the crucible furnace.”
They walked slowly across the yard.
“There are no giants here,” said Mr Prescott, “with the exception of the furnaces in which they set the crucibles; and they are small, compared with the furnaces that we have seen.”
They found themselves in a long room lined with shelves of clay crucibles, about eighteen inches in height. On the sides of the room, under the shelves, were rows of small furnaces, each large enough for two crucibles.
“The crucible process,” said Mr. Prescott, “gives us our finest steels. It is a simple melting together of iron and charcoal. The carbon of the charcoal passes into the iron. When the crucibles are filled, they are set in the furnace, and left for several days.
“They make a special kind of crucible steel over in Sheffield.”
While he was saying that, Mr. Prescott glanced at Billy, but Billy was looking at the furnace, and did not hear what Mr Prescott said.
Mr. Prescott looked at him hard, as he said:
“The home of the crucible is Sheffield.”
“Sheffield,” said Billy, turning, “is where they make good jackknives.”
“Want to see a genuine Sheffield?” asked Mr. Prescott, putting his hand into his pocket.
That time he didn’t have to attract Billy’s attention, for Billy stood waiting.
“See,” said Mr. Prescott, pulling out a chain that had a knife on it, and opening the blades. “See, it has Sheffield on both blades.”
Billy’s eyes saw the “Sheffield.” Then they saw something else, for on the side of the knife was a little silver plate, and on it—he had to look twice—was “Billy Bradford.”
“That’s a good knife,” said Billy.
The three men smiled, each his very best smile.
“Thank you, Mr. Prescott,” said Billy as he took the knife. Then he smiled, too.
“Now for the steel mill, and the last of our giants.”
“Is the mill deserted?” asked Dr. Crandon, as they went in.
“It’s much easier,” said Mr. Prescott, “to find the giants in a steel mill than it is to find the men. If you look around you’ll find a few, but they’ll be in most unexpected places.”
“I see a man,” exclaimed Billy, “up in a cage!”
“He’s controlling that crane,” said Mr. Prescott. “See it carry that ingot of red-hot iron!”
“This,” said Dr. Crandon, “passes belief. There’s a boy over there, in a reclining chair, who is opening a furnace down on this side.”
“Look at that!” exclaimed John Bradford, pointing to a crane like a huge thumb and forefinger, which had picked up a red-hot ingot, tons in weight, and was dropping it on a waiting car.
“Let’s follow it,” said Mr. Prescott, pleased to see John Bradford so excited.
They followed it to a room filled with clanking rolls.
Another crane swung the red-hot iron into the jaws of rollers.
On went the fiery bolt, sometimes up on one roller, then down on another, till at last they found that it had come out a finished rail.
Then a huge, round steel magnet, lowered by a man in a derrick house, picked up half a dozen rails; another lever sent the crane down the overhead tracks; and the rails were dropped in order on waiting cars.
“It used,” said Mr. Prescott, “to take a dozen men to load a single rail.
“Giants or not, Billy Bradford?”
“Giants for sure,” replied Billy.
“Fire-eaters!” exclaimed Dr. Crandon. “Let’s go!”
“I’m ready,” said Mr. Prescott. “I’m glad that the work is so much easier for the men, but I must confess that I don’t care to watch red-
hot iron shooting, almost flying around.”
“I’m ready to go,” said Billy.
“Joseph,” said Mr. Prescott, a few minutes later, “drive till you find a country road.”
That evening, as they sat together on the hotel veranda, Mr. Prescott said:
“I’ve been thinking,” then he stopped a moment to see whether Billy was listening, “how much iron has done to make the world smaller.”
Then, seeing that Billy’s eyes were opening wider and wider, he said:
“The world is so much smaller than it used to be that I sometimes wonder how much smaller it may grow.”
“Isn’t it just as far around the world as it always was?” asked Billy, looking first at Mr. Prescott, then at his Uncle John, and then back at Mr. Prescott.
“It’s of no use, Billy,” said Dr. Crandon, “to expect this man to tell us anything straight out. He’s trying to train our minds. If we’re going around with him, we shall have to submit to indirect methods of obtaining information.”
“If you’ll excuse me, Crandon,” said Mr. Prescott, “I’m not sure that Billy won’t learn as fast by my ‘indirect methods’ as he will by the kind of words that you are using.”
“Even, I think,” said Dr. Crandon.
Then the three men smiled, each in his own way.
Billy didn’t smile. All his best heroes seemed to be showing “disagreeable spots” at the same time.
But Billy had only a minute of thinking that, for Dr. Crandon said, in his most friendly tone:
“I think I know what he’s driving at, so I’ll lend you a hand. It would take a long time to sail around the world, wouldn’t it?”
“Sure,” answered Billy, quite like himself.
“But, if we were to start in an automobile, and drive to a train that would take us to San Francisco——”
“And then,” said Uncle John, “take a steamer across the ocean ——”
“And,” finished Mr. Prescott, “get back home in less than forty days, wouldn’t that make the world smaller than if we had to sail and sail and sail?”
“Of course,” answered Billy. “Anybody can see that.”
“And, if you were to go alone, Billy,” continued Mr. Prescott, in his very friendliest tone, “you could wire me or ‘phone me or cable me almost anywhere along the route. Wouldn’t that make the world seem very small?
“And what do all these things mean but iron—iron engines and iron rails and iron wires and watches with steel springs and magnetic steel needles in compasses that guide the great steamers through the paths of the sea?”
“Sometimes,” said Billy, in a half-discouraged tone, “I think there’s no end to knowing about iron.”
“That’s not very far from true, Billy,” said Mr. Prescott. “We could sit here till to-morrow morning trying to mention things made of iron, or by means of iron, and then we should be likely to forget many of them.
“If it weren’t for iron and steel implements and tools, men would have hard work to earn a living.
“Dr. Crandon, what does it seem to you that we should lose if we were to lose iron?”
“I’ve been thinking about the arts—surgery, too. We need iron for sculpture, for music, for printing books and papers. We need iron, I should say, for art’s sake.”
“And you, Bradford?”
