FLOUR AND BREADS AND THEIR FORTIFICATION IN HEALTH AND DISEASE PREVENTION FLOUR AND BREADS AND THEIR FORTIFICATION IN HEALTH AND DISEASE PREVENTION Second edition Edited By V R. P ICTOR REEDY R R W ONALD OSS ATSON AcademicPressisanimprintofElsevier 125LondonWall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101,UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates TheBoulevard,LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom ©2019ElsevierInc.Allrightsreserved. Nopartofthispublicationmaybereproducedortransmittedinanyformorbyanymeans,electronicormechanical, includingphotocopying,recording,oranyinformationstorageandretrievalsystem,withoutpermissioninwritingfromthe publisher.Detailsonhowtoseekpermission,furtherinformationaboutthePublisher’spermissionspoliciesandour arrangementswithorganizationssuchastheCopyrightClearanceCenterandtheCopyrightLicensingAgency,canbe foundatourwebsite:www.elsevier.com/permissions. 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LibraryofCongressCataloging-in-PublicationData AcatalogrecordforthisbookisavailablefromtheLibraryofCongress BritishLibraryCataloguing-in-PublicationData AcataloguerecordforthisbookisavailablefromtheBritishLibrary ISBN978-0-12-814639-2 ForinformationonallAcademicPresspublications visitourwebsiteathttps://www.elsevier.com/books-and-journals Publisher:CharlotteCockle AcquisitionEditor:MeganBall EditorialProjectManager:SusanIkeda ProductionProjectManager:NileshKumarShah CoverDesigner:MarkRogers TypesetbySPiGlobal,India Contributors MarijanaAcˇanski UniversityofNoviSad,NoviSad,Serbia EdithAgama-Acevedo InstitutoPolit(cid:1)ecnicoNacional,CentrodeDesarrollodeProductosBióticos,Yautepec,Morelos,Mexico MohamedA.Ahmed SchoolofEngineeringandPhysicalSciences,Heriot-WattUniversity,Edinburgh,Scotland;Departmentof FoodTechnology,FacultyofEngineeringandTechnology,SebhaUniversity,Sebha,Libya SaeedAkhtar InstituteofFoodScience&Nutrition,FacultyofAgriculturalSciences&Technology,BahauddinZakariya University,Multan,Pakistan RosarioAlonso-Dom´ınguez BiomedicalResearchInstituteofSalamanca(IBSAL),PrimaryCareResearchUnit,LaAlamedilla HealthCenter,CastileandLeonHealthService(SACYL),SpanishNetworkforPreventiveActivitiesandHealthPromotion (redIAPP),DepartmentofNursingandPhysiotherapy,UniversityofSalamanca,Salamanca,Spain JosephO.Anyango DepartmentofDairy,FoodScienceandTechnology,EgertonUniversity,Egerton-Njoro,Kenya FranklinB.Apea-Bah DepartmentofFoodandHumanNutritionalSciences,UniversityofManitoba,Winnipeg,MB,Canada VanessaCristinaArantes FaculdadedeNutric¸ão,UniversidadeFederaldeMatoGrosso,Cuiabá,Brasil AhmadArzani DepartmentofAgronomyandPlantBreeding,CollegeofAgriculture,IsfahanUniversityofTechnology,Isfahan, Iran MohamadF.Aslam DepartmentofNutritionalSciences,King’sCollegeLondon,London,UnitedKingdom GladysBarrera InstituteofNutritionandFoodTechnology,UniversityofChile,Santiago,Chile EmmaBeckett SchoolofMedicine&PublicHealth,TheUniversityofNewcastleandHunterMedicalResearchInstitute;Schoolof Environmental&LifeSciences,TheUniversityofNewcastle,Newcastle,NSW,Australia LuisA.Bello-P(cid:1)erez InstitutoPolit(cid:1)ecnicoNacional,CentrodeDesarrollodeProductosBióticos,Yautepec,Morelos,Mexico SarahE.Berry DepartmentofNutritionalSciences,King’sCollegeLondon,London,UnitedKingdom TrustBeta DepartmentofFoodandHumanNutritionalSciences,UniversityofManitoba,Winnipeg,MB,Canada AndreaBrandolini CREA—ResearchCentreforAnimalProductionandAquaculture,S.AngeloLodigiano,Italy DanielBunout InstituteofNutritionandFoodTechnology,UniversityofChile,Santiago,Chile AlmaC.Campa-Mada ResearchCenterforFoodandDevelopment,CIAD,Sonora,Mexico LydiaCampbell SchoolofEngineeringandPhysicalSciences,Heriot-WattUniversity,Edinburgh,Scotland ElizabethCarvajal-Millan ResearchCenterforFoodandDevelopment,CIAD,Sonora,Mexico PasqualeCatzeddu PortoConteRicercheSrl,Alghero(SS),Italy EmmaChiavaro DepartmentofFoodandDrug,UniversityofParma,Parma,Italy CristinaElizabethChuckHerna´ndez TecnológicodeMonterrey,CenterofBiotechnology—FEMSA,SchoolofEngineeringand Sciences,Monterrey,M(cid:1)exico RossanaCoda DepartmentofFoodandEnvironmentalScience,UniversityofHelsinki,Helsinki,Finland PrisciladaCostaRodrigues MestradoemNutric¸ão,AlimentoseMetabolismo,FaculdadedeNutric¸ão,UniversidadeFederalde MatoGrosso,Cuiabá,Brasil ChaianeAlinedaRosa MestradoemNutric¸ão,AlimentoseMetabolismo,FaculdadedeNutric¸ão,UniversidadeFederaldeMato Grosso,Cuiabá,Brasil MariseAuxiliadoradeBarrosReis FaculdadedeNutric¸ão,UniversidadeFederaldeMatoGrosso,Cuiabá,Brasil MariaPiadelaMaza InstituteofNutritionandFoodTechnology,UniversityofChile,Santiago,Chile PeterR.Ellis DepartmentofNutritionalSciences,King’sCollegeLondon,London,UnitedKingdom Ays¸eNaciyeErbakan DepartmentofInternalMedicine,NisaHospital,Istanbul,Turkey StephenR.Euston SchoolofEngineeringandPhysicalSciences,Heriot-WattUniversity,Edinburgh,Scotland AdrianaS.Franca MechanicalEngineeringDepartment,UniversidadeFederaldeMinasGerais,BeloHorizonte,Brazil xi xii CONTRIBUTORS JavierGonza´lez-Sa´lamo DepartamentodeQuímica,UnidadDepartamentaldeQuímicaAnalítica,FacultaddeCiencias, UniversidaddeLaLaguna(ULL),SanCristóbaldeLaLaguna,Spain DanielaGuardado-F(cid:1)elix TecnológicodeMonterrey,CenterofBiotechnology—FEMSA,SchoolofEngineeringandSciences, Monterrey;BiotechnologyPostgraduateRegionalProgram,FacultyofChemicalandBiologicalSciences,AutonomousUniversity ofSinaloa,FCQB-UAS,Culiacan,Sinaloa,M(cid:1)exico ElizabetJani(cid:1)cHajnal InstituteofFoodTechnology,UniversityofNoviSad,NoviSad,Serbia MehmetHayta ErciyesUniversity,FacultyofEngineering,DepartmentofFoodEngineering,Melikgazi,Kayseri,Turkey ErickHeredia-Olea TecnológicodeMonterrey,CenterofBiotechnology—FEMSA,SchoolofEngineeringandSciences,Monterrey, M(cid:1)exico JavierHerna´ndez-Borges DepartamentodeQuímica,UnidadDepartamentaldeQuímicaAnalítica,FacultaddeCiencias, UniversidaddeLaLaguna(ULL),SanCristóbaldeLaLaguna,Spain AntonioV.Herrera-Herrera InstitutoUniversitariodeBio-OrgánicaAntonioGonzález,UniversidaddeLaLaguna(ULL),San CristóbaldeLaLaguna,Spain AlyssaHidalgo DepartmentofFoodEnvironmentalandNutritionalSciences(DeFENS),UniversityofMilan,Milan,Italy SandraHirsch InstituteofNutritionandFoodTechnology,UniversityofChile,Santiago,Chile MajidHussain InstituteofFoodScience&Nutrition,FacultyofAgriculturalSciences&Technology,BahauddinZakariya University,Multan,Pakistan ElifMeltemI˙s¸c¸imen ErciyesUniversity,FacultyofEngineering,DepartmentofFoodEngineering,Melikgazi,Kayseri,Turkey TariqIsmail InstituteofFoodScience&Nutrition,FacultyofAgriculturalSciences&Technology,BahauddinZakariyaUniversity, Multan,Pakistan MartaS.Izydorczyk GrainResearchLaboratory,CanadianGrainCommission,Winnipeg,MB,Canada SiwapornJitngarmkusol DepartmentofFoodTechnology,FacultyofScience,ChulalongkornUniversity,Bangkok,Thailand SibelKacmaz FacultyofEngineering,DepartmentofFoodEngineering,GiresunUniversity,Giresun,Turkey HitomiKumagai DepartmentofChemistryandLifeScience,NihonUniversity,Fujisawa-shi,Japan Ma´rciaQueirozLatorraca FaculdadedeNutric¸ão,UniversidadeFederaldeMatoGrosso,Cuiabá,Brasil GladysO.Latunde-Dada DepartmentofNutritionalSciences,King’sCollegeLondon,London,UnitedKingdom MicheleCristianeLaux MestradoemNutric¸ão,AlimentoseMetabolismo,FaculdadedeNutric¸ão,UniversidadeFederaldeMato Grosso,Cuiabá,Brasil MarcoA.Lazo-V(cid:1)elez UniversityofAzuay,FoodEngineeringProgramResearchStrategicGroups(GEICA-UDA),Cuenca,Ecuador LauraLeiva InstituteofNutritionandFoodTechnology,UniversityofChile,Santiago,Chile WendeLi DepartmentofFoodandHumanNutritionalSciences,UniversityofManitoba,Winnipeg,MB,Canada GlauciaCarielloLima NutritionSchool-FederalUniversityofGoias,Goi^ania,Brazil MarkLucock SchoolofEnvironmental&LifeSciences,TheUniversityofNewcastle,Newcastle,NSW,Australia Ma´rioRobertoMaro´sticaJunior DepartmentofFoodandNutrition,FacultyofFoodEngineering,UniversityofCampinas,São Paulo,Brazil JorgeMarquez-Escalante ResearchCenterforFoodandDevelopment,CIAD,Sonora,Mexico JasnaMastilovi(cid:1)c InstituteofFoodTechnology,UniversityofNoviSad,NoviSad,Serbia TriciaMcMillan GrainResearchLaboratory,CanadianGrainCommission,Winnipeg,MB,Canada IlkemDemirkesenMert RepublicofTurkeyMinistryofFood,AgricultureandLivestock,FoodEnterprisesandCodexDepartment, Ankara,Turkey BanuMesci DepartmentofInternalMedicine,GoztepeTrainingandResearchHospital,MedeniyetUniversity,Istanbul,Turkey MarcoMontemurro DepartmentofSoil,PlantandFoodSciences,UniversityofBariAldoMoro,Bari,Italy DejanOrcˇi(cid:1)c DepartmentofChemistry,BiochemistryandEnvironmentalProtection,UniversityofNoviSad,NoviSad,Serbia MariaPaciulli FacultyofSciences,DepartmentofFoodandDrug,UniversityofParma,Parma,Italy AntonellaPasqualone DepartmentofSoil,PlantandFoodSciences,UniversityofBari‘AldoMoro’,Bari,Italy KristianPastor UniversityofNoviSad,NoviSad,Serbia RitaPaz-Samaniego ResearchCenterforFoodandDevelopment,CIAD,Sonora,Mexico EstherPerez-Carrillo TecnológicodeMonterrey,CenterofBiotechnology—FEMSA,SchoolofEngineeringandSciences, Monterrey,M(cid:1)exico EricaPontonio DepartmentofSoil,PlantandFoodSciences,UniversityofBariAldoMoro,Bari,Italy CONTRIBUTORS xiii AlessandroPugliese DepartmentofFoodandDrug,UniversityofParma,Parma,Italy Agust´ınRascon-Chu ResearchCenterforFoodandDevelopment,CIAD,Sonora,Mexico Jos(cid:1)eI.Recio-Rodr´ıguez BiomedicalResearchInstituteofSalamanca(IBSAL),PrimaryCareResearchUnit,LaAlamedillaHealth Center,CastileandLeonHealthService(SACYL),SpanishNetworkforPreventiveActivitiesandHealthPromotion(redIAPP), DepartmentofNursingandPhysiotherapy,UniversityofSalamanca,Salamanca,Spain La´ısM.Resende FoodScienceGraduateProgram,UniversidadeFederaldeMinasGerais,BeloHorizonte,Brazil MassimilianoRinaldi DepartmentofFoodandDrug,UniversityofParma,Parma,Italy CarloGiuseppeRizzello DepartmentofSoil,PlantandFoodSciences,UniversityofBariAldoMoro,Bari,Italy CristinaM.Rosell DepartmentofFoodScience,InstituteofAgrochemistryandFoodTechnology,SpanishScientificResearch Council(CSIC),Valencia,Spain NataliaSa´nchez-Aguadero BiomedicalResearchInstituteofSalamanca(IBSAL),PrimaryCareResearchUnit,LaAlamedillaHealth Center,CastileandLeonHealthService(SACYL),SpanishNetworkforPreventiveActivitiesandHealthPromotion(redIAPP), DepartmentofNursingandPhysiotherapy,UniversityofSalamanca,Spain SergioO.Serna-Saldivar TecnológicodeMonterrey,CenterofBiotechnology—FEMSA,SchoolofEngineeringandSciences, Monterrey,M(cid:1)exico PaulA.Sharp DepartmentofNutritionalSciences,King’sCollegeLondon,London,UnitedKingdom KhetanShevkani DepartmentofAppliedAgriculture,CentralUniversityofPunjab,Bathinda,India NarpinderSingh DepartmentofFoodScienceandTechnology,GuruNanakDevUniversity,Amritsar,India SandeepSingh DepartmentofFoodScienceandTechnology,KhalsaCollege,Amritsar,India PrabhjeetSingh DepartmentofBiotechnology,GuruNanakDevUniversity,Amritsar,India DarrylM.Small SchoolofScience,AppliedChemistryandEnvironmentalScienceDiscipline,RMITUniversity,Melbourne,VIC, Australia Ba´rbaraSocas-Rodr´ıguez DepartamentodeQuímica,UnidadDepartamentaldeQuímicaAnalítica,FacultaddeCiencias, UniversidaddeLaLaguna(ULL),SanCristóbaldeLaLaguna,Spain NorbertoSotelo-Cruz DepartmentofMedicine,UniversityofSonora,Sonora,Mexico ValentinaStojceska CentreforSustainableEnergyinFoodChains,BrunelUniversityLondon,CollegeofEngineering,Designand PhysicalSciences,Uxbridge,UnitedKingdom WilliamR.Sullivan SchoolofScience,AppliedChemistryandEnvironmentalScienceDiscipline,RMITUniversity,Melbourne, VIC,Australia KanithaTananuwong DepartmentofFoodTechnology,FacultyofScience,ChulalongkornUniversity,Bangkok,Thailand JohnR.N.Taylor DepartmentofConsumerandFoodSciences,UniversityofPretoria,Pretoria,SouthAfrica ReikoUrade InstituteforIntegratedRadiationandNuclearScience,KyotoUniversity,Osaka,Japan MichelaVerni DepartmentofSoil,PlantandFoodSciences,UniversityofBariAldoMoro,Bari,Italy AmardeepSinghVirdi DepartmentofFoodScienceandTechnology,GuruNanakDevUniversity,Amritsar,India DjuraVuji(cid:1)c UniversityofNoviSad,NoviSad,Serbia MilenaMorandiVuolo DepartmentofFoodandNutrition,FacultyofFoodEngineering,UniversityofCampinas,SãoPaulo,Brazil Ku€braYıldız DepartmentofNutritionandDietetics,FacultyofHealthSciences,MedeniyetUniversity,Istanbul,Turkey C H A P T E R 1 Deamidation of Gluten Proteins as a Tool for Improving the Properties of Bread Hitomi Kumagai*, and Reiko Urade† *Department of Chemistryand Life Science, Nihon University,Fujisawa-shi, Japan †Institutefor Integrated Radiation and Nuclear Science, Kyoto University, Osaka, Japan O U T L I N E Introduction 3 FunctionsChanged by Deamidation 6 Physicochemical Functions 6 Methods for Deamidation 4 PhysiologicalFunctions 7 Acid/Alkali 4 Enzyme 5 References 9 Anion/Cation-Exchange Resin 6 Abbreviations HMGglutenin high-molecular-weightgluteninsubunit pI isoelectricpoint RAST radioallergosorbenttest SDS sodiumdodecylsulphate TGase transglutaminase WDEIA wheat-dependentexercise-inducedanaphylaxis INTRODUCTION Wheatglutenformedfromgliadinandglutenin,twomajortypesofprotein,playsacriticalroleinbread-making.1–3 Regardingtherheologicalpropertiesofbreaddough,gliadincontributestoitsviscosity,whereasglutenincontributes toitselasticityandstrength.Gliadinisatypicalmonomericprolaminproteinthatissolublein60%–90%aqueousalco- hol, whereas glutenin is a large polymer comprised of high- and low-molecular weight subunits cross-linked with disulfidebondsthatisalcohol-insolublebutsolubleindilutedacidoralkalinesolutions.Thephysicalandchemical propertiesofglutenproteins,includingmolecularweight,gliadin:gluteninratio,anddegreeofprimaryamidation,are reportedlyfactorsthatcanaffectbakingquality.4–9Furthermore,thepoorsolubilityandemulsificationofglutenpro- teins under most food-processing conditions often preclude the additionof theseproteins to a variety of foods. Physiologically,itiswellknownthatgliadinistheprincipalallergenthatinducesseriousallergicreactionsinsus- ceptible individuals. Allergic manifestations of reactions to gliadin include atopic eczema/dermatitis syndrome (AEDS), which often occurs in children,10–12 and wheat-dependent, exercise-induced anaphylaxis (WDEIA), which mainly occurs in adults.13,14 Certain tandem sequencing sites with glutamine residues of ω-5 gliadin and a high-molecular-weightgluteninsubunit(HMWglutenin),whicharethemostpotentantigensforWDEIA,havebeen identifiedasthe primary structure ofimmunoglobulin E(IgE)-binding epitopes.15–17 Oneapproachtomitigatingtheallergenicityofglutenproteinsistochemicallymodifytheproteinsbyalkylation, acylation, or deamidation. Alkylation and acylation are sometimes used to change the functionality of proteins.18 FlourandBreadsandtheirFortificationinHealthandDiseasePrevention 3 ©2019ElsevierInc.Allrightsreserved. https://doi.org/10.1016/B978-0-12-814639-2.00001-0 4 1. DEAMIDATIONOFGLUTENPROTEINSASATOOLFORIMPROVINGTHEPROPERTIESOFBREAD FIG.1 Deamidationofprotein.Anamidegroupinthesidechainofglutamineorasparagineisconvertedtocarboxylgroupbytypicaldeami- dation.Asaresult,glutamineorasparagineisconvertedtoglutamicacidorasparticacid. TABLE1 PercentagesofAcid-AmidicandAcidicAminoAcidsinFoodProteins Glutamine Asparagine Glutamicacid Asparticacid Wheatgliadin 33.8 2.6 1.9 0.4 Soy11Sglobulin 10.4 8.6 7.6 3.8 Milkα -casein 8.3 4.3 10.1 2.4 s1 However,thesemodificationsrequirechemicalsthatarenotallowedinfoodintendedforhumanconsumption.Con- versely, deamidation, a reaction that converts glutamine and asparagine residue to glutamic-acid and aspartic-acid residue, respectively (Fig. 1), can be achieved under mild conditions that are approved for food processing. Conse- quently,proteindeamidationisrecognizedinthefoodindustryasamethodtoimprovethefunctionalityofproteins infoodsystems.19–21Deamidationisoftenappliedtocerealandpulseproteinsbecausethepercentageofglutamine andasparagineresidueintheseproteinsishigherthanthecorrespondinglevelsinanimalproteins.Notably,approx- imately one-third of the amino acids in wheat gliadin are comprised of glutamine residue (Table 1). Thereisampleevidencesuggestingthatdeamidationimprovessolubilityandsurfaceproperties.Also,physiolog- icalfunctionssuchasallergenicityandcalciumbioavailabilitycanbealteredbydeamidation.Thepopularmethodsfor proteindeamidationincludeacid,enzyme,andcation-exchange-resintreatment.Areviewofthecurrentdeamidation methods and the resulting functional improvementsare presented in this chapter. METHODS FOR DEAMIDATION Acid/Alkali Amongtheestablishedchemicaldeamidationmethods,treatmentwithanacidisthemostpopular.22–39Deamida- tionunderacidicconditionscausesthedirecthydrolysisofanamidetoacarboxylategroup.40Tothisend,theamidein asparagine residues is much more amenable to deamidation than the amide in glutamine residues.41 Furthermore, conductingthereactioninanacidsolutionatahightemperatureachievesahighdegreeofdeamidation,butacertain level of peptide-bond hydrolysis inevitably occurs, which produces bitter-tasting peptides and reduces processing properties such as gelation and solubility. Thedegreeofproteindeamidationinanacidsolutionincreases asthetemperatureincreases.Forexample,upto 90% of wheat gluten was deamidated following the addition of 1.0M hydrochloric acid (HCl) solution at 95°C for 30min,whereasonlyupto30%wasdeamidatedin1.0MHClsolutionat50°Cfor30min.22AlthoughHClisthemost commonacidused,organicacidssuchasacetic,malic,tartaric,andcitricacidcanalsobeusedfordeamidation.Wheat glutenwasdeamidatedtoapproximately60%in0.034–0.133Maceticacidat121°Cfor15min,whichwassimilarto the deamidation level observed in 0.055–0.137M HCl at 121°C for 15 min.33 However, the degree of deamidation observed at the 0-min treatment time was already quite high (24%–52%), which might be explained by the 12-h reaction time, which is required to release ammonia from the deamidated samples during the determination of the deamidation level. Therefore, the actual degree of deamidation by acetic acid might be much lower. Although the degree of deamidation was not measured, wheat gluten was deamidated in 0.082M acetic acid at 121°C for 1.INTRODUCTORYCHAPTERS METHODSFORDEAMIDATION 5 15min,andvariouspropertieswerecomparedwiththoseofunmodified,succinylated,anddeamidatedandsucciny- latedgluten.39Wheatglutenwasdeamidatedin1.6Mmalicacid,0.8Mtartaricacid,or1.0Mcitricacidatupto30%at 70°Cfor 20h,whichwas equivalent to the deamidation level observed in 0.08MHCl.38 Alkalideamidationislesspopularthanothermethods,27,42,43likelybecauseacross-linkingreactionresultinginthe formationoflysinoalanineoccursreadilyunderalkalineconditions,44reducingthenutritionalvalueduetothelossof lysine and of protein-processing properties due to polymerization. Both lysinoalanine and lanthionine production increasewithelevationsinthereactiontemperaturefrom50°Cto90°C,aswellaswithincreasesinthesodiumhydrox- ide (NaOH) concentration from 0 to1.5%.In addition,the formationof anintramolecular cyclicimideintermediate that produces β-carboxyl linkageoccurs in alkali deamidation more predominantly than direct hydrolysis.40 Thepresenceofanionsenhancesthedeamidationrateofproteins.Forexample,thedeamidationofsoyproteinat 100°Cfor3hinapH8.0bicarbonateandphosphatesolutionenhanceddeamidationlevelsupto40%and25%,respec- tively.However,theadditionofacetate,sulfate,andchlorideanionswasineffective.45Sodiumdodecylsulfate(SDS), ananionicsurfactant,also increasesdeamidation rates.The degree of cottonseedproteindeamidation in 0.4MHCl heated to 70°C for 5h increased from 30% to 100% when SDS was increased from 0 to 0.08M.46 These anions may promotethedeamidationrateviaacatalyticfunctioninducedbyheatingorHClbecauseproteinscanbedeamidated to acertain degree justby heating47–49 or by treatmentwithacid. Enzyme Aspreviouslynoted,chemicaldeamidationoftencausesundesiredmodifications,suchasthecleavageofpeptide bonds;however,deamidationbytheadditionofenzymesisanalternativemethodthatdoesnotcausepeptide-bond hydrolysis.Enzymaticdeamidationhasreportedlybeenachievedusingprotease,transglutaminase(TGase),peptido- glutaminase,andproteinglutaminase.Katoetal.developedamethodtodeamidatefoodproteinsbytreatmentwith proteasesatpH10.50,51Theauthorsnotedthatapproximately20%oftheasparagineorglutamineresidueinovalbu- min,lysozyme,andsoyproteins(7Sglobulinand11Sglobulin)weredeamidatedbytreatmentwithpapain,pronase, and chymotrypsin without (or with only slight) proteolysis. In addition, chymotrypsin immobilized on controlled- pore glass was effective at deamidating ovalbumin, lysozyme, 7S globulin, 11S globulin, and gluten at pH 10 and 20°C. The percentages of deamidated ovalbumin, lysozyme, 7S globulin, 11S globulin, and gluten were 10.0, 8.4, 6.0,5.0,and8.0,respectively.Inaddition,thelargermolecular-weightfractionsofsoyproteinsandglutendissociated into smaller molecular-weight fractions on SDS-polyacrylamide gels, suggesting the occurrence of partial peptide- bond cleavage.52 TGase (glutaminyl-peptide: amine γ-glutamyltransferase, EC 2.3.2.13) is an enzyme that catalyzes amine- incorporating and cross-linking reactions via the transfer of an acyl group between a γ-carboxyamide of the peptide-boundglutamineandaprimaryamineand/orlysylsidechain.Inaddition,TGasecatalyzesthedeamidation ofglutamineresidues.TheTGasesderivedfrombloodplasmaanderythrocytesandfromStreptomycesmobaraensisare used in commercial products, as these enzymes can be produced in larger quantities. Generally, cross-linking will dominatethedeamidationreaction.However,iftheproteincontainsalargenumberofglutamineresiduesandonly afewlysineresidues,suchasingliadinsandglutenins,waterwillreactasanucleophile,resultinginthedeamidation ofglutamines.53Inastructuralanalysis,Mazzeoetal.determinedthat19of94glutamineresiduespresentinrecom- binant α-gliadin were converted to glutamic-acid residues by the addition of guinea-pig liver tissue TGase.54 The experiments were conducted on a recombinant α-gliadin, and on a panel of 26 synthetic peptides, overlapping the completeproteinsequencebytheuseofadvancedmatrix-assistedlaserdesorption/ionization(MALDI)time-of-flight mass spectrometry (TOF-MS) and tandem MS methodologies. Bacilluscirculanspeptidoglutaminase(EC3.5.1.43)iswell-knownenzymethatcatalyzesthehydrolysisofglutamine residuesinpeptides.55Theisolatedenzymeconsistsoftwodistinctisozymes:peptidoglutaminaseIandII.Further- more,thepeptidoglutaminasesaredimerscomprisedofsubunitswithmolecularweightsof42kDaand51kDa,and isoelectricpoint(pI)valuesof4.1and4.0,respectively.56,57Peptidoglutaminasesarelocatedintracellularly.Peptido- glutaminaseIdeamidates aC-terminalglutamine residue, and peptidoglutaminase IIdeamidates aninternalgluta- mine residue, as well as a C-terminal residue. The peptidoglutaminases are inactive against higher-molecular-mass peptidesandproteins.58,59Also,theactivityofpeptidoglutaminaseisgenerallylimitedtothedeamidationofgluta- mine residue on theshort peptide chain; therefore,thedeamidation ofnative casein andwhey proteins by peptido- glutaminaseisminimal.59Hamadaetal.investigatedthedeamidationofmodifiedfoodproteinsbypeptidoglutaminase andfoundthatpriorproteolysisanddisruptionofthecompactstructureofintactproteinsarerequiredtoincreaseprotein deamidation.60Therefore,anewenzymethatcatalyzesthedeamidationofnativeproteinswithouttherequirementfor pretreatmentisdesirable. 1.INTRODUCTORYCHAPTERS