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CurrentDevelopments inBiotechnologyand Bioengineering Production,IsolationandPurification ofIndustrialProducts Editedby
AshokPandey,SangeetaNegi, CarlosRicardoSoccol AMSTERDAM
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ListofContributors M.Adsul DBT-IOCCentreforAdvancedBioenergyResearch,IndianOilCorporation Limited
CristóbalN.Aguilar FoodResearchDepartment,SchoolofChemistry, AutonomousUniversityofCoahuila,Saltillo,Coahuila,México
A.Angel-Cuapio UniversidadAutónomaMetropolitana-Iztapalapa,MexicoCity, DF,Mexico
G.S.Anisha GovernmentCollegeforWomen,Trivandrum,Kerala,India
P.Binod CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology(NIIST), Trivandrum,India
J.Buenrostro-Figueroa DepartmentofBiotechnology,DivisionofHealthand BiologicalSciences,MetropolitanAutonomousUniversity,Iztapalapa,México
S.Chakraborty IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
M.L.ChávezGonzález FoodResearchDepartment,SchoolofChemistry, AutonomousUniversityofCoahuila,Saltillo,Coahuila,México
G.-Q.Chen TsinghuaUniversity,Beijing,China
S.Chen HubeiUniversity,Wuhan,PRChina
JuanC.Contreras-Esquivel FoodResearchDepartment,SchoolofChemistry, AutonomousUniversityofCoahuila,Saltillo,Coahuila,México
J.D.Coral BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
J.C.deCarvalho BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
J.deOliveira BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
A.Dhillon IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
M.J.Fernandes BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
R.Gaur IndianOilCorporationLimited,R&DCentre,Faridabad,India
A.Goyal IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
L.R.C.Guimarães BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
M.Haridas KannurUniversity,Kannur,India
R.Hemamalini IndianInstituteofTechnologyDelhi,NewDelhi,India
AyerimHernandez-Almanza FoodResearchDepartment,SchoolofChemistry, AutonomousUniversityofCoahuila,Saltillo,Coahuila,México
A.Illanes PontificiaUniversidadCatólicadeValparaíso,Valparaíso,Chile
J.Isar UniversityofDelhiSouthCampus,NewDelhi,India
A.Joseph KannurUniversity,Kannur,India
S.G.Karp BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
N.Karthik CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
R.Kaushik UniversityofDelhiSouthCampus,NewDelhi,India
S.K.Khare IndianInstituteofTechnologyDelhi,NewDelhi,India
P.C.S.Kirnev BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
D.Kothari IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
C.Larroche BlaisePascalUniversity,AubièreCedex,France
L.A.J.Letti BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
O.Loera-Corral UniversidadAutónomaMetropolitana-Iztapalapa,MexicoCity,DF, Mexico
A.I.Magalhães,Jr. BioprocessEngineeringandBiotechnologyDepartment, FederalUniversityofParaná(UFPR),Curitiba,PR,Brazil
A.B.P.Medeiros BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
J.D.C.Medina BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
F.Miranda-Hernández UniversidadAutónomaMetropolitana-Iztapalapa,Mexico City,DF,Mexico
N.R.Nair CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
S.Nair DowChemicalsGmBH,Dubai,UAE
K.M.Nampoothiri CSIR-NationalInstituteforInterdisciplinaryScienceand Technology(NIIST),Trivandrum,India
A.Nandan CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
S.Negi MotilalNehruNationalInstituteofTechnology,Allahabad,India
M.G.B.Pagnoncelli BioprocessEngineeringandBiotechnologyDepartment, FederalUniversityofParaná(UFPR),Curitiba,PR,Brazil;FederalTechnological UniversityofParana,DoisVizinhos,Brazil
A.Pandey CenterofInnovativeandAppliedBioprocessing,(anationalinstitute underDeptofBiotechnology,MinistryofS&T,GovtofIndia),Mohali,Punjab,India
A.K.Patel DBT-IOCCentreforAdvancedBioenergyResearch,IndianOilCorporation Limited
V.Rajulapati IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
S.Ramachandran InsightProfessionalInstitute,Dubai,UAE
A.Rani IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
C.Rodrigues BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
RosaM.Rodríguez-Jasso FoodResearchDepartment,SchoolofChemistry, AutonomousUniversityofCoahuila,Saltillo,Coahuila,México
R.Rodríguez FoodResearchDepartment,SchoolofChemistry,Autonomous UniversityofCoahuila,Saltillo,Coahuila,México
L.V.RodríguezDurán DepartmentofBiotechnology,DivisionofHealthand BiologicalSciences,MetropolitanAutonomousUniversity,Iztapalapa,México
HéctorA.Ruiz FoodResearchDepartment,SchoolofChemistry,Autonomous UniversityofCoahuila,Saltillo,Coahuila,México
A.Sabu KannurUniversity,Kannur,India
R.Saini DBT-IOCCentreforAdvancedBioenergyResearch,IndianOilCorporation Limited
S.Sajitha CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
S.Saran UniversityofDelhiSouthCampus,NewDelhi,India
R.K.Saxena UniversityofDelhiSouthCampus,NewDelhi,India
V.C.Sekhar CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
K.Sharma IndianInstituteofTechnologyGuwahati,Guwahati,Assam,India
R.Sindhu CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
R.P.Singh PunjabiUniversity,Patiala,Punjab,India
R.S.Singh PunjabiUniversity,Patiala,Punjab,India
ReetaR.Singhania DBT-IOCCentreforAdvancedBioenergyResearch,IndianOil CorporationLimited
C.R.Soccol BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
T.S.Swapna GovernmentVictoriaCollege,Palakkad,India
D.Tan XíanJiaotongUniversity,Xían,China
L.Thomas CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
M.V.Ushasree CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology (NIIST),Trivandrum,India
P.Valencia UniversidadTécnicaFedericoSantaMaría,Valparaíso,Chile
L.P.S.Vandenberghe BioprocessEngineeringandBiotechnologyDepartment, FederalUniversityofParaná(UFPR),Curitiba,PR,Brazil
K.Vibha MotilalNehruNationalInstituteofTechnology,Allahabad,India
J.Vidya CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology(NIIST), Trivandrum,India
N.Vijayan KannurUniversity,Kannur,India
N.Vivek CSIR-NationalInstituteforInterdisciplinaryScienceandTechnology(NIIST), Trivandrum,India
Q.Wang HubeiUniversity,Wuhan,PRChina
X.Wei HubeiUniversity,Wuhan,PRChina
A.L.Woiciechowski BioprocessEngineeringandBiotechnologyDepartment, FederalUniversityofParaná(UFPR),Curitiba,PR,Brazil
J.Yin TsinghuaUniversity,Beijing,China
A.ZandonáFilho BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
P.A.Zárate FoodResearchDepartment,SchoolofChemistry,Autonomous UniversityofCoahuila,Saltillo,Coahuila,México
S.F.Zawadzki BioprocessEngineeringandBiotechnologyDepartment,Federal UniversityofParaná(UFPR),Curitiba,PR,Brazil
AbouttheEditors AshokPandey
ProfessorAshokPandeyisEminentScientistattheCenterof InnovativeandAppliedBioprocessing,Mohali(anational instituteundertheDepartmentofBiotechnology,Ministry ofScienceandTechnology,GovernmentofIndia),and formerchiefscientistandheadoftheBiotechnology DivisionattheCSIR’sNationalInstituteforInterdisciplinary ScienceandTechnologyatTrivandrum.Heisanadjunct professoratMarAthanasiosCollegeforAdvancedStudies Thiruvalla,Kerala,andatKalasalingamUniversity,Krishnan Koil,TamilNadu.Hismajorresearchinterestsareinthe areasofmicrobial,enzyme,andbioprocesstechnology, whichspanvariousprograms,includingbiomasstofuels andchemicals,probioticsandnutraceuticals,industrial enzymes,solid-statefermentation,etc.Hehasmorethan 1100publicationsandcommunications,whichinclude16 patents,50+books,125bookchapters,and425originalandreviewpapers,withanhindex of75andmorethan23,500citations(GoogleScholar).Hehastransferredseveraltechnologiestoindustriesandhasbeenanindustrialconsultantforaboutadozenprojectsfor Indianandinternationalindustries.
ProfessorPandeyistherecipientofmanynationalandinternationalawards andfellowships,whichincludeElectedMemberoftheEuropeanAcademyofSciences andArts,Germany;FellowoftheInternationalSocietyforEnergy,Environmentand Sustainability;FellowoftheNationalAcademyofScience(India);FellowoftheBiotech ResearchSociety,India;FellowoftheInternationalOrganizationofBiotechnologyand Bioengineering;FellowoftheAssociationofMicrobiologistsofIndia;honorarydoctorate degreefromtheUniversite ´ BlaisePascal,France;ThomsonScientificIndiaCitation LaureateAward,UnitedStates;LupinVisitingFellowship;VisitingProfessoratthe Universite ´ BlaisePascal,France,theFederalUniversityofParana,Brazil,andtheE ´ cole PolytechniqueFe ´ de ´ raledeLausanne,Switzerland;BestScientificWorkAchievement Award,GovernmentofCuba;UNESCOProfessor;RamanResearchFellowshipAward, CSIR;GBF,Germany,andCNRS,Francefellowships;YoungScientistAward;andothers. HewaschairmanoftheInternationalSocietyofFood,AgricultureandEnvironment, Finland(Food&Health)during2003 04.HeistheFounderPresidentoftheBiotech
ResearchSociety,India(www.brsi.in);InternationalCoordinatoroftheInternational ForumonIndustrialBioprocesses,France(www.ifibiop.org);chairmanofthe InternationalSocietyforEnergy,Environment&Sustainability(www.isees.org);andvice presidentoftheAllIndiaBiotechAssociation(www.aibaonline.com).ProfessorPandey iseditor-in-chiefof BioresourceTechnology, HonoraryExecutiveAdvisorofthe Journalof WaterSustainability and JournalofEnergyandEnvironmentalSustainability, subject editorofthe ProceedingsoftheNationalAcademyofSciences(India), andeditorialboard memberofseveralinternationalandIndianjournals,andalsoamemberofseveral nationalandinternationalcommittees.
SangeetaNegi Dr.SangeetaNegiisanassistantprofessorintheDepartment ofBiotechnologyattheMotilalNehruNationalInstituteof Technology,India.ShehasaFirstClassMaster’sdegreein biochemistryandaPhDinbiotechnologyfromtheIndian InstituteofTechnology,Kharagpur.Shehasalsoworkedas anacademicguestattheBiologicalEngineeringDepartment, PolytechClermont-Ferrand;theUniversite ´ BlaisePascal, France;andtheBioenergyandEnergyPlanningResearch Group,SwissFederalInstituteofTechnology,Lausanne, Switzerland.Dr.Negi’scurrentresearchinterestsareinthe areasofbiofuels,industrialenzymes,andbioremediation.Sheisaneditorialboard memberofthe JournalofWasteConversion,BioproductsandBiotechnology andthe Journal ofEnvironmentalScienceandSustainability. ShehasbeenawardedasOutstanding ReviewerbyElsevierandhaswontheYoungScientistAwardbyDSTattheNational SeminaronBiologicalandAlternativeEnergiesPresentandFuture,organizedbyAndhra University,Visakhapatnam,in2009.ShehasalsowontheBestPosterAwardatthe InternationalCongressonBioprocessesinFoodIndustries(2008)atHyderabad.Dr.Negi hascontributedtonearly70publications,includingreviewarticles,originalpapers,and conferencecommunications.
CarlosRicardoSoccol
ProfessorCarlosRicardoSoccolistheresearchgroupleader oftheDepartmentofBioprocessesEngineeringand BiotechnologyattheFederalUniversityofParana ´ (UFPR), Brazil,with20yearsofexperienceinbiotechnological researchanddevelopmentofbioprocesseswithindustrial application.HegraduatedwithaBScinchemicalengineering(UFPR,1979),Master’sinfoodtechnology(UFPR, 1986),andPhDinGe ´ nieEnzymatique,Microbiologieet Bioconversion(Universite ´ deTechnologiedeCompie ` gne, France,1992).HedidhispostdoctoralworkattheInstitut ORSTOM/IRD(Montpellier,1994and1997)andatthe Universite ´ deProvenceetdelaMe ´ diterrane ´ e(Marseille, 2000).HeisanHDRProfessorattheE ´ coled’Inge ´ nieursSupe ´ riureofLuminy, Marseille France.Hehasexperienceintheareasofscienceandfoodtechnology,with emphasisonagro-industrialandagro-alimentarybiotechnology,actinginthefollowing areas:bioprocessengineeringandsolid-statefermentation,submergedfermentation, bioseparations,industrialbioprocesses,enzymetechnology,tissueculture,bioindustrialprojects,andbio-production.HeiscurrentlytheCoordinatorofMaster BIODEV-UNESCO,associateeditoroffiveinternationaljournals,andeditor-in-chiefof thejournal BrazilianArchivesofBiologyandTechnology.ProfessorSoccolhasreceived severalnationalandinternationalawards,whichincludetheScienceandTechnology AwardoftheGovernmentofParana ´ (1996);Scopus/ElsevierAward(2009);Dr.Honoris Causa,Universite ´ BlaisePascal,France(2010);OutstandingScientistatthe5th InternationalConferenceonIndustrialBioprocesses,Taipei,Taiwan(2012);andElected TitularMemberoftheBrazilianAcademyofSciences(2014).Heisatechnicalandscientificconsultantforseveralcompanies,agencies,andscientificjournalsinBraziland abroad.Hehassupervisedandmentored96MasterofSciencestudents,48PhDstudents,and14postdoctoralstudents.Hehas995publicationsandcommunications, whichinclude17books,107bookchapters,270originalresearchpapers,and557 researchcommunicationsininternationalandnationalconferencesandhasregistered 44patents.Hisresearcharticlesasofthiswritinghavebeencited(Scopusdatabase)5600 timeswithanhindexof36.
Preface Thisbookisapartofthecomprehensiveseries CurrentDevelopmentsinBiotechnologyand Bioengineering,comprisingninevolumes(Editor-in-chief:AshokPandey),anddealswiththe production,isolation,andpurificationofindustrialproductsproducedbybiotechnological processes.Thisbookcoversrecenttechnologicaladvancesofagreatnumberofbiotechnologicalproductsandisdividedintofourdifferentparts:ProductionofIndustrialand TherapeuticEnzymes,OrganicAcids,BiopolymersandOtherProducts,andProducts IsolationandPurification.
Part1isdevotedtotheproductionofindustrialandtherapeuticenzymes.Thefirst chapterdescribesthecurrentandfuturetrendsofproduction,application,andstrain improvementof a-amylases,oneofthemostimportantenzymesusedinindustry. a-Amylasesfindapplicationinseveralindustrialprocesses,suchasstarchliquefaction, desizingoftextiles,detergents,baking,bioethanolproduction,etc.Glucoamylaseisanother enzymeextensivelyusedinthefoodandfermentationindustries,mainlyforthesaccharificationofstarch,brewing,andproductionofhigh-fructosesyrup,whicharediscussedin Chapter2.Cellulases, b-glucosidases,andxylanasesarethesecondmostusedenzymesin industrybysalesvolume,withanincreasingdemandsince1995inseveralindustrialapplications,comprisingdetergentsandtextiles,animalfeed,food,paper,andbiofuels.These enzymesarediscussedinChapters4,5,and6ofthisbook.Chapter7discussesproteolytic enzymes,alsoknownas“proteases,”whichareusedtocleavethepeptidebondsconnecting twoaminoacids.Theyareproducedmainlybymicroorganismsandhavegreatcommercial value,beingusedinfood,dairy,detergents,andleatherprocessing.Lipolyticenzymesare hydrolasescomprising15familiesoflipases,asshowninChapter8ofthisbookthrougha studyoftheindustrialapplicationsandotherimportantaspectsoftheseenzymes.The purposeofChapters9and10istopresentanoverviewoflaccasesandperoxidases,covering theirproductionanduseinthepretreatmentoflignocellulosicbiomassandbiopulping,and alsoprojectingnewperspectivesonimprovingsuchprocessesandproductsusingthese enzymes.Sourcesofproduction,strategies,characteristics,applications,andindustrial importanceoftherapeuticenzymes,suchas L-glutaminase, L-asparaginase,andpenicillin acylase,arepresentedanddiscussedinChapters11,12,and13.Otherenzymes,suchas phytases,chitinases,keratinases,tannases,aminopeptidases,nattokinases,andpolysaccharidelyases,arereviewedinChapters14to23,coveringrecentadvances,production methods,potentialapplications,andtheglobalmarket.
Thesecondpartofthebookisdedicatedtoorganicacids.InChapters24and25,lactic acidandcitricacidproduction,synthesis(coveringfactorsthataffectbiochemicalpathways), andrecoveryareaddressed.Chapter26reviewsthemicrobialproductionofgluconicacid, propertiesofglucoseoxidase,production,recovery,andapplications.Succinicacidisan importantplatformmolecule,usedasanintermediateintheproductionofnumerous everydayproducts,amongwhicharepharmaceuticalsandadhesives,representingatotal immediateaddressablemarketofmorethan$7.2billion.Chapter27presentsananalysisof thecurrentmarket,biological-basedproductionprocesses,enzymaticregulation,and recoverysystemsofsuccinicacid.
Part3discussespolymerproductionandotherproducts.Polylactide(PLA),derived fromlacticacid,abiodegradablepolyester,hasapplicationsinpackaging,textiles,andthe biomedicalandpharmaceuticalindustries.Chapter28reviewsthepropertiesandapplicationsofPLA,focusingonrecenttechnologiesandimprovementofproductiontechniques. Polyhydroxyalkanoates(PHAs),afamilyofenvironmentallyfriendlypolyestersthatcanbe synthesizedbyawiderangeofmicroorganismsascarbonandenergyreserves,havebeen consideredanalternativetopetroleum-basedchemicals.Thecompositionandstructural diversityofPHAshaveledtovariouspropertiesandendlessapplicationstoformaPHAvalue chain.Chapter29brieflyintroducestheirproductionandapplication,highlightingthelaboratoryproductionbythemicrobialstrainsdevelopedusinggeneticand/ormetabolicengineeringorsyntheticbiologytechniques.Industrialproduction,recenttechnologies,and improvementofPHAproductionarealsodiscussed.Poly-g-glutamicacid(g-PGA)isanatural polymer,synthesizedbyvariousstrainsof Bacillus spp.,thatisusedinfood,cosmetics, agriculture,andthewastewaterindustry.Chapter30providesupdatedinformationonthe biosynthesis,fermentation,purification,andapplicationof g-PGA.InChapter31,recent developmentsinthebiologicalproductionof1,3-propanediolbyvariousnaturaland geneticallyengineeredmicroorganisms,nonnative1,3-propanediolproducers,aswellas mixedcultures,arediscussed.Importantaspectsofdownstreamprocessingandvarious methodsandstepsinvolvedintheextractionandpurificationof1,3-propanediolfromthe fermentationbrotharealsocoveredinthischapter.Theproductionofpetroleum-based plasticsisachallengingenvironmentalproblem,causingtheproductionandconsumption ofbiodegradableplasticstoreceiveconsiderableattentionnowadays.Chapter32providesan overviewofthedegradationmechanismsofbiodegradablepolymers,withparticular emphasisonthemainparametersaffectingthedegradationofthesepolymericbiomaterials. InChapter33thepotentialofbiologicalcontrolispresentedanddiscussedasapromising alternativetochemicalpesticides.Thefinaltwochaptersofthisbook,Chapters34and35, presentthemostrelevantdownstreamprocessestoextract,isolate,purify,andrefine fermentationproducts.
Weareconfidentthatthisbookwillbeprofitabletostudents,professors,researchers, andprofessionalsinterestedinstudyingbiotechnologyandbioengineering.Wethank Dr.KostasMarinakis,BookAcquisitionEditor;Ms.AnnekaHess;andentireproductionteam atElsevierfortheirhelpandsupportinbringingoutthisvolume.
a-Amylases R.Sindhu1, *,P.Binod1,A.Pandey2
1 CSIR-NATIONALINSTITUTEFORINTERDISCI PLINARYSCIENCEANDTECHNOLOGY(NIIST), TRIVANDRUM,INDIA; 2 CENTEROFINNOVATIVEANDAPPLIEDBIOPROCESSING, (ANATIONALINSTITUTEUNDERDEPTOFBI OTECHNOLOGY,MINISTRYOFS&T,GOVTOF INDIA),MOHALI,PUNJAB,INDIA
1.1Introduction
1.1.1Starch
Starchisthemajorpolysaccharidefoodreserveinnatureaftercellulose.Itservesasan importantsourceofnutritionforotherlivingorganisms [1].Itissynthesizedinthe plastidspresentinleavesandaccumulatesasinsolublegranulesinhigherandlower plants.Starchiscomposedofalargenumberofglucoseunitsjoinedbyglycosidicbonds. Itconsistsoftwotypesofmolecules:amyloseandamylopectin.Amyloseisalinear, water-insolublepolymerofglucosejoinedby a-1,4bonds,whereasamylopectinisa branched,water-solublepolysaccharidewithshort a-1,4-linkedlinearchainsof10 60 glucoseunitsand a-1,6-linkedsidechainswith15 45glucoseunits.Thelevelsof amylaseandamylopectinvaryamongdifferentstarches.Generally,starchiscomposed ofamyloseandamylopectinintherange25 28%and72 75%,respectively.
1.1.2Amylases Amylasesaretheenzymesthatbreakdownstarch,orglycogen.Theseenzymesare producedbyavarietyoflivingorganisms,rangingfrombacteriatoplantstohumans. Thoughamylasesareproducedbyseveralmicroorganisms,thoseproducedbyfungiand bacteriahavedominatedapplicationsintheindustrialsector [2].Bacteriaandfungi secreteamylasestotheoutsideoftheircellstocarryoutextracellulardigestion,which breaksdowntheinsolublestarch,andthenthesolubleendproducts(suchasglucoseor maltose)areabsorbedintothecells.
Amylasesconstituteaclassofindustrialenzymesoccupyingabout25%oftheenzyme market.Becauseoftheincreasingdemandfortheseenzymesinvariousindustries,there isenormousinterestindevelopingthemwithbetterproperties,suchasrawstarchdegradingamylasessuitableforindustrialapplications,andcost-effectiveproduction
*CorrespondingAuthor. CurrentDevelopmentsinBiotechnologyandBioengineering:Production,IsolationandPurificationofIndustrialProducts http://dx.doi.org/10.1016/B978-0-444-63662-1.00001-4 3 Copyright © 2017ElsevierB.V.Allrightsreserved.
techniques.Althoughamylasescanbederivedfromseveralsources,includingplants, animals,andmicroorganisms,microbialenzymesgenerallymeetindustrialdemands. Alargenumberofmicrobialamylasesareavailablecommerciallyandtheyhavealmost completelyreplacedthechemicalhydrolysisofstarchinthestarchprocessingindustry [3].Oneofthemostimportantadvantagesofusingmicrobesfortheproductionof amylasesisthebulkproductioncapacityandthefactthatmicrobescanbegenetically modifiedtoproduceenzymeswithdesiredcharacteristics [4].Theseenzymesareofgreat significanceinbiotechnology,withvariousapplicationsrangingfromfood,fermentation, andtextilestothepaperindustry.Eachapplicationof a-amylaserequiresunique propertieswithrespecttospecificity,stability,andtemperatureandpHdependence.
Moderntechnologiessuchascomputationalpackagesandonlineserversarethe currenttoolsusedinproteinsequenceanalysisandcharacterization.Thephysicochemicalandstructuralpropertiesoftheseproteinsarewellunderstoodwiththeuseof computationaltools.Theproteinsequenceprofile,suchasnumberofaminoacidsand sequencelength,andthephysicochemicalpropertiesoftheprotein,suchasmolecular weight,atomiccomposition,extinctioncoefficient,aliphaticindex,instabilityindex,etc., canbecomputedbyProtParam,andthesecondarystructureprediction,sequence similarity,evolutionaryrelationships,and3-Dstructureofvariousproteinscanbe computedusingtheESyPred3Dserver [5].
1.1.3Classi ficationofAmylases Basedonthemechanismofbreakdownofstarch,themoleculesareclassifiedintothree types: a-amylase, b-amylase,andamyloglucosidase. a-Amylasereducestheviscosityof starchbybreakingdownthebondsatrandom,therebyproducingvariablysizedchains ofglucose. b-Amylaseenzymebreakstheglucose glucosebondsbyremovingtwo glucoseunitsatatime,therebyproducingmaltose.Amyloglucosidaseistheenzymethat breakssuccessivebondsfromthenonreducingendofthestraightchain,producing glucose.Manymicrobialamylasesusuallycontainamixtureoftheseamylases.This chapterfocusesonlyon a-amylases.
a-Amylases(EC3.2.1.1)arestarch-degradingenzymesthatcatalyzethehydrolysisof internal a-1,4-O-glycosidicbondsinthepolysaccharideswiththeretentionofthe a-anomericconfigurationintheproducts.Mostofthe a-amylasesaremetalloenzymes, whichrequirecalciumions(Ca2þ)fortheiractivity,structuralintegrity,andstability. Theybelongtofamily13(GH-13)oftheglycosidehydrolasegroupofenzymes [6,7]. Basedontheend-productformation a-amylasesareclassifiedassaccharifyingand liquefyingamylases.Thesaccharifying a-amylasesarefurtherclassifiedasmaltose forming,maltotetraoseforming,maltopentaoseformingandmaltohexaoseforming basedontheendproductsformed [1].
The a -amylasefamilyisthelargestfamilyofgly cosidehydrolases,transferases,and isomerases,comprising30differentenzyme specificities.Theseenzymesareclassified intofourgroups:endoamylases,exoamylases,debranchingenzymes,andtransferases. Endoamylasesareenzymesthatcleaveinternal a-1,4bondsresultingin a-anomeric
Chapter1 a-Amylases5
products.Exoamylasesareenzymesthatcleave a-1,4,or a-1,6bondsoftheexternal glucoseresiduesresultingin a-or b -anomericproducts.Debranchingenzymesare enzymesthathydrolyze a -1,6bondsleavinglinearpolysaccharides.Transferasesare enzymesthatcleave a-1,4glycosidicbondsofthedonor moleculeandtransferpartof thedonormoleculetoaglycosidicacc eptor,forminganewglycosidicbond [7].
1.2Sourcesof a-Amylase a-Amylasesareuniversallydistributedthroughouttheplant,animal,andmicrobial kingdoms.Theenzymesfrommicrobialsourceshavedominatedapplicationsinindustrialprocesses [2].Though a-amylaseshavebeenderivedfromseveralmicrobial sources,includingbacteria,fungi,yeast,andactinomycetes,theenzymesproducedfrom bacterialandfungalsourceshavedominatedapplicationsinindustrialsectors.Because oftheirshortgrowthperiod,theirbiochemicaldiversity,andtheeasewithwhich enzymeconcentrationsmightbeincreasedbyenvironmentalandgeneticmanipulation, theenzymesfrommicrobialsourcesgenerallymeetindustrialdemands.
1.2.1Plant
a-Amylases Plantsstorecarbonpredominantlyasstarchandthemetabolismofstarchisessentialto alllife.Family1 a-amylasesarecharacterizedbyhavingasecretarysignalpeptide. Thisplaysanimportantroleinthedegradationofextracellularstarchincerealgrain endosperms.Family2 a-amylasesarecharacterizedbyhavingnopredictedtargeted peptideandarelocalizedinthecytoplasm.Theseamylaseshavebeenidentifiedfrom monocotyledons,dicotyledons,andgymnosperms.Theybecomemostactivewhenthe plastidialstarchreservesofleavesaremoredepleted.Theyareinvolvedingeneralstress responses.Family3 a-amylasesarecharacterizedbyhavingalargeN-terminaldomain, whichcontainsalargepredictedchloroplasttransitpeptide.Theseenzymesare responsiblefordegradingplastid-boundstarchinstoragetissuesandleaves [8].
1.2.2Bacterial a-Amylases a-Amylasesareproducedfromvariousbacterialsources,including Bacillus, Brevibacterium, Clostridium, Halomonas, Naxibacter, Nesterenkonia, Paenibacillus, Pseudomonas, Streptomyces sp.,etc.Amongthebacterialsources, Bacillus sp.iswidely used,especiallyfortheproductionofthermostable a-amylases. Bacillussubtilis, Bacillus stearothermophilus,Bacillusamyloliquefaciens,Bacilluslicheniformis,Bacillusacidocaldarius,Bifidobacteriumbifidum,and Bifidobacteriumacerans areimportantsources usedfor a-amylaseproduction [9].Alkalineandthermotolerantamylaseshavebeen reportedfrom Bacillus sp., B licheniformis,and Bacillushalodurans [10].Otherbacteria producing a-amylaseinclude Anoxybacillusbeppuensis [11], Bacilluslaterosporus [12], Bacillusacidicola [13], Chryseobacteriumtaeanense [14], Clostridium sp. [15], Microbacteriumfoliorum [16], Nesterenkonia sp. [17], Thermococcus sp. [18], Anoxybacillus flavithermus [19] etc.
1.2.3Fungal a-Amylases Severalfungalspeciesalsoproduce a-amylases,including Acremonium, Aspergillus, Penicillium , Mucor, Neurospora,and Thermomyces sp.Amongthefungalsources,the genus Aspergillus hasbeenwidelyusedfortheproductionof a-amylases. Aspergillus niger, Aspergillusflavus,and Aspergillusoryzae areimportantsourcesusedamongthe fungalsources [20,21].Otherfungalstrainsproducing a-amylaseinclude Thermomyces lanuginosus [22].
1.3Productionof a-Amylase 1.3.1ProductionMethods
Tomeettheindustrialdemand,itisessentialtodevelopalow-costmediumforthe productionof a-amylase.Itcanbeproducedbysubmergedfermentation(SmF)and solid-statefermentation(SSF).Theproductionisaffectedbyavarietyofphysiological factors,whichincludepH,temperature,aeration,inoculumconcentration,inoculum age,compositionofthegrowthmedium,surfactants,carbonsource,nitrogensource, etc. [23].Interactionsoftheseparametershaveasignificantinfluenceontheproduction oftheenzyme.Generally,SmFiscarriedoutusingsyntheticmedia,incorporatingmediumconstituentssuchasnutrientbrothandsolublestarch,aswellasothercomponents,whichareveryexpensive.Replacementofsuchconstituentsbycheapercarbon andnitrogensourcesaswellasnutrientswouldbenefittheprocessincostreduction. Agriculturalby-productsofferpotentialbenefitsinthisregard [7]
SSFisdefinedastheprocessinwhichthegrowthofmicroorganismsiscarriedouton solidsubstrateswithnegligiblefreewater,orfree-flowingwater [24].SSFplaysan importantroleintheproductionofenzymes.Agro-industrialsubstratesareconsidered thebestsubstratesforSSFprocesses.Itisofspecialinterestinthoseprocessesinwhicha crudefermentedproductmaybeuseddirectlyasanenzymesource.Thecommon substratesusedforSSFprocessesarewheatbran,ricebran,cassavawaste,palmoil waste,bananawaste,teawaste,coconutoilcake,coirpith,corncobs,etc.InSSF,itis importanttoprovideoptimizedwatercontentandtocontrolthewateractivityofthe fermentingsubstrate.Attimes,SSFispreferredtoSmFbecauseofitssimpletechnique, lowcapitalinvestment,lowerlevelsofcataboliterepressionandend-productinhibition, lowwastewateroutput,betterproductrecovery,andhigh-qualityproduction [25]
Continuousandfed-batchstudiesaremoreeffectivefortheproductionof a-amylase. ThestudyconductedbyLeeandParulekar [26] revealedthatthe a-amylaseproduction by B.subtilis TN106wasenhancedwhenbatchcultivationwasextendedwithfed-batch cultivation,andtheenzymeactivitywas54%higherinatwo-stagefed-batchoperation comparedtoasingle-stagebatchculture.MishraandMaheswari [27] reported a-amylasefromathermophilicfungus, T.lanuginosus;theenzymewasadimericprotein withamolecularmassof42kDawithoptimumpHandtemperatureof5.6and65 C,
respectively.Theenzymeproducedhighlevelsofmaltosefrompotatostarch,suggesting itsusefulnessinthecommercialproductionofmaltoseandglucosesyrups.Thestudy conductedbyKrishnaandChandrasekharan [28] revealedthatbananapeelcouldbe utilizedasapotentialsubstratefor a-amylaseproductionby A.niger.SaxenaandSingh [29] screenedvariousagro-industrialresiduesforamylaseproductionfrom Bacillus sp. andfoundmustardoilcaketobethebestsubstrate.Thestrainproduced5400U/gof amylaseat1:3moisturecontent,20%inoculum,andanincubationperiodof72h.Yang andWang [30] reported a-amylaseproductionby Streptomycesrimosus TM55using sweetpotatoresidueandpeanutmealresidueasasubstrate.Thestrainproduced 1903Uof a-amylaseafter96hofincubation.
Ramachandranetal. [20] usedcoconutoilcake(COC),aby-productofoilextraction fromdriedcopra,asasubstratefortheproductionof a-amylasefromfungi.COCsupplementedwith0.5%starchand1%peptoneenhanced a-amylaseproductionby A.oryzae.COCservesasasourceofsolubleproteinsandlipidsthusprovidingessential nutrientsforthegrowthofandenzymesynthesisbytheorganism.Productionof a-amylaseby B.amyloliquefaciens underSSFusingcornglutenmeal(CGM)wasreportedbySabanetal. [31].Thestudyrevealedthat a-amylaseproductioninamedium withCGMwasfivetimeshigherthanthatinamediumcontainingstarchandother components.UtilizationofCGMasasubstratemakestheprocesseconomicallyviable becauseCGMisaby-productofstarch-basedindustries.
Productionandoptimizationof a-amylasefrom A oryzae CBS819usingaby-product ofwheatgrinding(gruel)asthesolecarbonsourcewasdonebyKammounetal. [32]. Variousprocessparametersaffectingtheproductionwereoptimizedbyadoptinga Box Behnkendesign,whichincreasedtheenzymeproductionfrom40.1to151.1U/mL. Murthyetal. [33] reportedcoffeeby-productsassuitablesubstratefortheproductionof a-amylaseunderSSF.Coffeewastewasconvertedintovalue-addedproductsby fermentationusing Neurosporacrassa CFR308.Theoptimumconditionsfor a-amylase productionweremoisturecontentof60%,pH4.5,incubationtemperatureof27 C, particlesizeof1mm,andincubationtimeof5days.Underoptimizedconditionsthe strainproduced7084U/gdsof a-amylase.
Syedetal. [34] reportedextracellularamylaseproductionby Streptomycesgulbargensis DAS131bySmF.Thehighestamylaseproductionwasobservedwhenthemedium wassupplementedwith1%starch.TheenzymewasthermotolerantandstableatpH9.0. Starchandpeptoneweregoodsourcesofcarbonandnitrogen.Sharmaand Satyanarayana [13] reportedenhancedproductionofacidichigh-maltose-formingand Ca2þ-independent a-amylaseby B. acidicola;amaximumenzymetiterof366IU/Lwas attainedafter36hoffermentationatpH4.5,33 C,with0.5vvmaeration.Theenzyme titerwas10,100IU/Linfed-batchfermentation.Oneofthemainadvantagesoffedbatchfermentationoverthebatchfermentationisthattheconcentrationoflimiting substrateismaintainedatlowlevels,thusavoidingtherepressingeffectofhighsubstrate concentrationandtherebyminimizingtheaccumulationofinhibitorymetabolites.
Ahighlythermostableandcalcium-independent a-amylasefrom A.beppuensis TSSC-1wasreportedbyKikaniandSingh [11].Thisorganismproducedamonomeric a-amylasewithoptimalpHandtemperatureof7.0and55 C,respectively.Thekey findingsofthisstudywerecost-effectivepurification,highthermostability,andbroad pHstability.TheenzymeexhibitedCa2þ independenceandresistancetochemical denaturation,whichcouldmakeitsuitableformanyindustrialapplications.Another agro-industrialresidue,datewaste,hasalsobeenusedasthesubstratefortheproductionof a-amylaseusingyeast, Candidaguilliermondii CGL-A10 [35].Maximum enzymeproductionwasattainedinSmF(2056 mmol/L/min).Rajagopalanetal. [15] usedsugarcanebagassehydrolyzatefortheproductionof a-amylaseproducedbya solventogenic Clostridium sp.BOH3.Thestrainusedstarchdirectlywithoutanypretreatmentandproducedextracellularamylase(7.15U/mgprotein)andbutanolalmost equivalentto90%oftheyieldequivalenttoglucose.Sugarcanebagassewasusedby RoohiandKuddus [16] toproduceacold-active a-amylasefrom M.foliorum GA2. Maximumenzymeproduction(6610U)wasobservedwhenfermentationwascarried outinamediumcontaining40%bagasse,0.0003Mlactose,atpH8.0,withincubation temperatureof20 Cfor5dayatstaticconditions.Thiswasthefirstreportoncoldactive a-amylaseproductionfrom M.foliorum GA2. Table1.1 showsvariousmicroorganismsusedfortheproductionof a-amylase.
1.3.2FactorsInfl
uencingtheProductionof a-Amylase Productionof a-amylasebySSFandSmFisaffectedbyavarietyofphysicochemical factors [3].Theseincludemediacomposition,incubationtemperature,inoculumage, carbonsource,nitrogensource,pH,phosphateconcentration,aeration,andothers.
Table1.1 StrainsandStrategiesAdoptedfor a-AmylaseProduction
Microorganism Methodof ProductionSubstrateEnzymeYieldReferences
Bacillussubtilis TN106Fedbatch [26]
Streptomycesrimosus TM55SSFSweetpotatoresidue/ peanutresidue 2642.7U/gds [30]
Aspergillusoryzae SSFOilcake9196U/gds [20]
Aspergillusoryzae OBS819SmF151.1U/mL [32]
Neurosporacrassa CFR308SSFCoffeewaste7084U/gds [33]
Streptomycesgulbargensis DAS131SmFStarch [34]
Bacillusacidicola SmF366IU/L [13]
Anoxybacillusbeppuensis TSSC-1 [11]
Candidaguilliermondii CGL-A10SmF2056 mmol/L/min [35]
Clostridium sp.BOH3SmFSugarcanebagasse hydrolyzate 7.15U/mgprotein [15]
Microbacteriumfoliorum GA2SmFBagasse6610U/mL [16]
SmF,submergedfermentation; SSF,solid-statefermentation.
1.3.2.1IncubationTemperature
Theeffectoftemperatureon a-amylaseproductionisrelatedtothegrowthoftheorganism.Temperaturecontrolisveryimportantinfermentationprocessesbecause growthandproductionofenzymesaresensitivetotemperature.Hence,theoptimum temperaturevarieswiththeculture. a-Amylaseshavebeenproducedbyvariousmicrobesoverawiderangeoftemperature.ProductionsinSSFaswellasinSmFare usuallycarriedoutintherange25 37 C.However,psychrophilicandthermophilic temperatureshavealsobeenreportedfortheproduction.Forexample, a-amylase productionwasattainedat55 Cbythethermophilicfungi Thermomonosporafusca [36] and T.lanuginosus [27] andat80 Cbyahyperthermophilicbacterium, Thermococcus profundus [36].Apsychrophilicbacterium, Alteromonashaloplanktis,produced a-amylaseat4 C [37]
1.3.2.2pH
ThepHofthefermentationmediumplaysanimportantroleinenzymeproduction.It inducesmorphologicalchangesintheorganismsastheyaresensitivetotheconcentrationofhydrogenionspresentinthemedium.ApHchangeinthemediumaffectsthe growthaswellastheproductstability.UnlikeSmF,inwhichpHcontrolisalmost mandatoryfor a-amylaseproduction,inSSFprocesses,generallythereisnoneedtoset, orcontrol,thepH,asthesubstrates(agro-industrialresidues)mostlypossessexcellent bufferingcapacityandkeepthepHfavorableforthegrowthandactivityoftheculture. Mostofthe Bacillus strainsusedcommerciallyfortheproductionof a-amylaseshavean optimumpHof6.0or7.0.SomeofthemediumcomponentseliminatetheneedforpH control.Yabukietal. [38] reportedthat A.oryzae 557accumulated a-amylaseinthe myceliawhengrowninphosphate,orsulfate-deficient,mediumanditwasreleased whenthemyceliawereplacedinamediumwithpHabove7.2.BasedontheoptimalpH foractivity, a-amylasesareclassifiedasacidic,neutral,andalkaline [1].
1.3.2.3CarbonSources
a-Amylaseproductioncouldbeeitherconstitutiveorinducible.Galactose,inulin,and glycogenaresuitablesubstratesfor a-amylaseproductioninSmF.Supplementationwith lactose;ananalogofmaltose, a-methyl-D-glucoside;andyeastextractinducesthe production [7].Severalagro-residuessuchaswheatbran,ricebran,vegetablepeels,fruit peels,cassavabagasse,andvegetable-oil-extractedresiduesareusedassubstratesfor a-amylaseproductioninSSF.Moststudieson a-amylaseproductionby A. oryzae suggest thatthegeneralinducermoleculeismaltose.Erattetal. [39] observeda20-foldincrease inenzymeactivitywhenmaltoseandstarchwereusedasinducersin A.oryzae NRC 401013.Xyloseandfructosesupportgoodgrowth,buttheyarestronglyrepressive [40].
1.3.2.4NitrogenSources
Organicaswellinorganicnitrogensourcesareusedfortheproductionof a-amylases, althoughorganicsourceshavebeenpreferredoverinorganicnitrogensources.Commonly
usednitrogensourcesincludebactopeptone,ammoniumsulfate,ammoniumnitrate,Vogel salts,casein,meatextract,beefextract,yeastextract,cornsteepliquor,andsoybeanflour. Therearereportsontheuseofseveralothernitrogenoussourcesfor a-amylaseproduction. Forexample, L-asparaginewasreportedasthebetternitrogensourceforenzymeproductionby T.lanuginosus;caseinhydrolyzateandyeastextractimproved a-amylase productionseveralfoldandby110 156%,respectively,by A. oryzae [41].Complexnitrogen sourcesinthemediuminfluencetheproductionof a-amylases.Studiescarriedoutby Dettorietal. [42] revealedthatthesupplementationoftwoorganicnitrogensources enhancedamylaseproductionandthiswasbetterthanasingleorganicnitrogensource.
1.3.2.5MetalIons Supplementationofmetalionsinthefermentationmediumpromotesmicrobialgrowth, whichinturnacceleratestheenzymeproduction.Most a-amylasesareknowntobe metaldependentfordivalentions,e.g.,Ca2þ,Mg2þ,Mn2þ,andZn2þ [2]. SupplementationwithCa2þ isgenerallyrequiredforanincreasedin a-amylaseproductionbyseveralbacteria.Ca2þ impartsthermostabilityoftheenzymeduetosalting outofhydrophobicresiduesbyCa2þ intheprotein.Theproductionwasreducedto50% whenMg2þ wasomittedfromthemedium;Naþ andMg2þ showedcoordinatedstimulationofenzymeproductionby Bacillus sp.CRPstrain [43].However,somemetalions couldhaveanegativeimpactonthemicrobesfor a-amylaseproduction,e.g.,Li2þ and Hg2þ havenegativeeffectson a-amylaseproduction.Mg2þ alsoplaysanimportantrole in a-amylaseproduction.
1.3.2.6Surfactants Additionofsurfactantstothefermentationmediumisgenerallyknowntoincreasethe secretionofproteinsbyincreasingcellmembranepermeability.Thecommonlyused surfactantsareTween80,Tween40,TritonX-100,sodiumdodecylsulfate(SDS),polyethyleneglycol,andglycerol.Thesesurfactantsarereportedtoincreasecellpermeability,therebyenhancingenzymeyield.Arnesonetal. [44] reportedatwofoldincrease in a-amylaseproductionby T.lanuginosus.GoesandSheppard [45] reportedasignificantadvantageinusingthebio-surfactantsurfactintoenhancetheproductionof a-amylaseby B. subtilis inSSF.Inadditiontoincreasingtheenzymeactivity,surfactin offersotheradvantages,includingeco-friendliness,lesssensitivitytoextremesoftemperatureandpH,andbeingapotentialfungicide,therebyeliminatingcontaminationof theexposedsubstrate,comparedtosyntheticsurfactants.
1.3.2.7Agitation
Agitationinfluencesthemixingaswellastheoxygentransferrateinmostfermentations andthusinfluencescellmorphologyandproductformation [46,47].Itisgenerally believedthathigheragitationisdetrimentaltocellgrowth,whichinturncoulddecrease enzymeproduction.Agitationintensitiesupto300rpmarenormallyemployedforthe productionof a-amylaseinSmFfromvariousmicroorganisms.
Chapter1 a-Amylases11
1.4Assayof a-Amylases Activityof a-amylasesisquantifiedbymeasuringeithertheendproducts,likeglucoseor maltose,ortheamountofsubstratethatremainsafterenzymatichydrolysis. a-Amylases areassayedusingsolublestarchormodifiedstarchasthesubstrate.Theycatalyzethe hydrolysisof a-1,4glycosidiclinkagesinstarchtoproduceglucose,dextrins,andlimit dextrins.Thereactionismonitoredbyanincreaseinthereducingsugarlevelsora decreaseintheiodinecolorofthetreatedsubstrate.Variousmethodsareavailableforthe determinationof a-amylaseactivity [48].Thesearebasedonadecreaseinstarch iodine colorintensity,increaseinreducingsugars,degradationofcolor-complexedsubstrate, anddecreaseinviscosityofthestarchsuspension [3].Thecommonmethodsemployed forthedeterminationof a-amylaseactivityaretheiodinemethod [49];dextrinizingactivity [50];Sandstedt,Kneen,andBlishmethod [51];dinitrosalicylicacidmethod [52]; anddegradationofcolor-complexedsubstrate [53,54].
Thedinitrosalicylicacid(DNS)method [52] isamongthemostcommonlyused methodsforestimatingthereducingsugars.TheDNSreactswithreducingsugarunder boilingandturnstoredfromyellow.InthemethodofFuwaetal. [50],thestarchreacts withiodineandformsabluesolutionandtheintensityofthecolorisdirectlyproportionaltothestarchconcentration.Borondipyrromethene-labeledsubstratereleasesa fluorescentfragmentupondigestionwiththeenzymeandhasbeendevelopedfor determining a-amylaseactivityinfoods [55].
1.5 a-AmylaseInhibitors Proteinaceous a-amylaseinhibitorshavebeenisolatedfromplantsandmicroorganisms [56].Theseinhibitorscontrolendogenous a-amylaseactivityorworkindefenseagainst pestsandpathogens;someinhibitorsareantinutritionalfactors. a-Amylaseinhibitors belongtosevendifferentproteinstructuralfamilies.Sixtypesarefromhigherplantsand oneisfrom Streptomyces sp.High-resolutionstructuresareavailablefortarget a-amylaseandthesestructuresindicatemajordiversityandsomesimilaritiesinthe structuralbasisof a-amylaseinhibition.Varioustypesofinhibitorsinclude Streptomyces inhibitors,knottins, g-thionins,CMproteins,andkunitz-type,thaumatin-like,and lectin-likeinhibitors.Some a-amylaseinhibitorshaveadverseeffectsonnutritiondueto theirinhibitionofdigestiveenzymesinhumansandanimals. a-Amylaseinhibitorsfind applicationinobesityanddiabetictherapy.
1.6StrainImprovement Strainimprovementisusuallycarriedouttoincreaseproductionaswellastoimprove thepropertiesoftheenzyme.Thecatalyticpropertiesofenzymesaredeterminedby their3-Dstructure.Hence,enzymepropertiescanbealteredbysite-directedmutagenesis.Usingthismethod,thepropertiesofanenzymecanbeimproved,bymaking
Table1.2 SomeStrategiesAdoptedforStrainImprovement/Propertiesof a-Amylase
MicroorganismImprovedPropertyReferences
Bacillussubtilis BR151Thermostability [60]
Alternariatenuissima FCBP2522.39-foldincreasedproduction [62]
Thermobifidafusca NTU22Increasedproduction [63]
Bacillusamyloliquefaciens
Increasedproduction(1.4-fold) [71]
Anoxybacillus sp.HighstabilityinabsenceofCa2þ ionsat60 C andhighlevelsofmaltoseproduction [66]
Aspergillusoryzae IIB30Increasedproduction(2.1-fold) [70]
Paenibacillus sp.Highrateofmaltoseproduction [67]
Bacilluslicheniformis MSGSelf-inducible,cataboliterepressionfree,and glucose-activatedexpressionsystem [68]
B.subtilis ASO1aIncreasedproduction(7-fold)andhighstability inabsenceofCa2þ [69]
Thermotogamaritima Oxidativestability [72]
B.subtilis Improvedproteinstabilityandcatalyticefficiency [73]
Bacillus sp.AAH-31Increasedproduction [74]
itthermostable,reducingitsdependenceoncofactors,orincreasingitsactivityatlow temperature.Studiesonthecloningofthe a-amylasegenehavebeenextensivelycarried outforhyperproduction [7]. Table1.2 presentssomestrategiesthathavebeenadopted forstrainimprovementof a-amylase. a-Amylaseshavebeenengineeredforthe improvementofpropertiessuchaspH tolerance,thermotolerance,etc. [57 59].Barnettetal. [58] foundthattheintroduction ofdisulfidebondsintheenzymesandalterationofaminoacidspronetooxidationby anaminoacidresistanttooxidizingagentsimprovedthestabilityoftheenzyme. Suzukietal. [57] constructedhybridsofhomologousstrainsofthe B.licheniformis and B.amyloliquefaciens withimprovedthermostability.OzcanandOzcan [60] introduced thethermostableplasmidpC194Amy,ha rboringa5.2-kbDNAfragmentencodinga geneof B . stearothermophilus,into B . subtilis BR151byelectroporation.Therecombinantstrainsproducedmorethermostable a-amylasecomparedtothewild-type strain.Anewstrainof B.licheniformis CBBD302,carryingarecombinantplasmid, pHY-amyL,for B . licheniformis a-amylase(BLA)production,wasconstructedbyNiu etal. [61] .Thecombinationoftarget-directedscreeningandgeneticrecombinationled toanapproximately26-foldimprovementinBLAproductionandexportin B.licheniformis.Shafiqueetal. [62] reportedtheproductionofanextracellularamylase from Alternariatenuissima FCBP252inSSF.Chemicalmutagenesisusingethyl methanesulfonate(EMS)producedmutantswithahighlevelof a -amylaseactivity (2.39-fold)comparedtotheparentalstrain.Geneticcharacterizationofthemutants usingrandomamplifiedpolymorphicDNAPCRrevealedthattheexpressionpatterns ofthemutantswereisogenicvariantsoftheparentstrain.Yangetal. [63] expressed
an a-amylasegenefrom Thermobifidafusca NTU22in Pichiapastoris X33becauseof itspotentialapplicationasafoodsupplement.Recombinantexpressionresultedin higherlevelsofextracellularenzymeproduction(510U/L),indicatingconstitutive expressionandsecretionoftheprotein.T heamountofextracellularproteininthe cultureof P. pastoris transformantswaslessthanthatinthecell-freeextractof Escherichiacoli transformants,hencefacilitatingtheapplicationofcrudeamylasein industrywithoutpurification.
Thegeneencodingthe a-amylaseenzymein B . subtilis PY22wasamplifiedbyPCR, sequenced,andclonedinto P.pastoris KM71HstrainusingthevectorPpiczA,allowing methanol-inducedexpressionandsecretionoftheprotein [64].Recombinantexpressionresultedinhighlevelsofextracellularamylaseproduction(22mg/L).Thepresence ofCa2 þ ionsinthemediumresultedina41%increasein a-amylaseactivity.Expression in P.pastoris notonlyincreasedtheyieldofproductionbutalsopotentiallyhelped facilitatepurification.Genecloningandheterologousexpressionofthehigh-maltoseproducing a-amylaseof Rhizopusoryzae showedsuccessfulexpressionof R.oryzae a-amylasein P.pastoris atahighlevel(382mg/L) [65].Theenzymehadanextremely highaffinityformaltotrioseandnomaltotrioseremainedafterhydrolysis.Chaietal. [66] clonedtwogenesthatencoded a -amylasesfrom Anoxybacillus sp.andexpressed themin E.coli.Theenzymesproducedbytherecombinantstrainswerehighlystable evenintheabsenceofcalciumat60 Cfor48handtheyproducedhighlevelsof maltose.Proteinsequencingrevealedthattherecombinant a-amylasedifferedin17 aminoacidscomparedtotheamylaseproducedbythewild-typestrain.Agene encoding a-amylasefromthegenomicDNAof Paenibacillus sp.andtheheterologous expressionofrecombinantAmy1in E.coli BL21(DE3)facilitatedtherecoveryofthis proteininsolubleform.ThehighrateofmaltoseproductionduetotheactionofAmy1 couldbeexploitedfortheproductionofsimplesugarsasaby-productinfoodwaste processing [67] .
Theuseofanexpressionsystemtoovercomecataboliterepressionopensupan avenueforexploitingcheapcarbonsourcesfortheproductionofrecombinantenzyme. NathanandNair [68] developedarepression-freecatabolite-enhancedexpressionsystemforathermophilic a-amylasefrom B.licheniformis MSG.Aself-inducible,catabolite repression-free,andglucose-activatedexpressionsystemwasdevelopedusingathermophilic a-amylaseasamodel.The a-amylasegenefrom B.licheniformis MSGwithout any50 cre operatorproducedunimpededglucose-enhancedexpressionwhenfusedto thephosphatestarvation-induciblestrong pst promoterwithoptimumtranslationsignalsinaprotease-deficient B.subtilis.Theyieldwas18.5-foldhigherthanthatofnative promoter.Royetal. [69] clonedandoverexpressedaraw-starch-digesting a-amylase gene(AmyBS-I)from B.subtilis strainASO1ain E.coli BL21.Thegenealsoincludedits signalpeptidesequencefortheefficientextracellularexpressionofrecombinant a-amylaseincorrectlyfoldedform.TheextracellularsecretionofAmyBS-Iwassevenfold higheranditdidnotrequireCa2þ ionsforits a-amylaseactivity/thermostability,which wasanaddedadvantageforitsuseinthestarchindustry.
Randommutagenesishasalsobeenusedforenhancedproductionof a-amylase.A strainof A. oryzae IIB30wassubjectedtophysical(usingUVlight)andchemical mutagenesis(usingnitrousacidandEMS).MutationusingEMS-20showeda2.1-fold increasedamylaseactivitycomparedtothewild-typestrain [70].Anidenticalobservationwasearlierreportedfora B. amyloliquefaciens straininwhichmutationusingEMS improvedenzymeactivityby1.4-foldhigherthanthatoftheparentalstrain [71].Ozturk etal. [72] reportedsite-directedmutagenesisofmethionineresiduesforimprovingthe oxidativestabilityof a-amylasefrom Thermotogamaritima.Theoxidativestabilityof a-amylase(AmyC)wasimprovedbymutatingthemethionineresiduesatpositions43 and44,and55and62,tooxidative-resistantalanineresidues.Themutantexhibited improvedoxidativeproperties.TheengineeredAmyCcouldbeapotentialcandidatefor industrialapplications,especiallyinthepresenceofoxidizingagents.Thisisthefirst proteinengineeringattemptforAmyCfrom T.maritima.Yangetal. [73] carriedout structuralengineeringofhistidineresiduesinthecatalyticdomainof a-amylasefrom B.subtilis forimprovedproteinstabilityandcatalyticefficiencyunderacidicconditions bysite-directedmutagenesis.ThefourhistidineresiduesHis222,His275,His293,and His310inthecatalyticdomainwereselectedasthemutationsitesandwerefurther replacedwithacidicasparticacid,respectivelyyieldingfourmutantsH222D,H275D, H293D,andH310D.Theacidicstabilityoftheenzymewassignificantlyenhancedafter mutation,and45 92%oftheinitialactivityofthemutantswasretainedafterincubation atpH4.5and25 Cfor24h,whereasthatforthewildtypewasonly39.5%.Asrevealedby thestructuremodelsofthewild-typeandmutantenzymes,thehydrogenbondsandsalt bridgeswereincreasedaftermutation,andanobviousshiftofthebasiclimbtoward aciditywasobservedforthemutants.Thesechangesaroundthecatalyticdomain contributedtothesignificantlyimprovedproteinstabilityandcatalyticefficiencyatlow pH.Thisworkprovidedaneffectivestrategytoimprovethecatalyticactivityandstability of a-amylaseunderacidicconditions,andtheresultsindicatedpotentialapplicationfor theimprovementofacidresistanceofotherenzymes.
Thehydrolyticactivityofthermophilic,alkalophilic a-amylasecouldalsobe enhancedthroughtheoptimizationofaminoacidresiduessurroundingthesubstrate bindingsite [74].Twenty-fourselectedaminoacidresidueswerereplacedwithAla,and Gly429andGly550werealteredtoLysandGlu,respectively,basedoncomparisonof AmyL’saminoacidsequencewithrelatedenzymes.Y426A,H431A,I509A,andK549A showedhigheractivitythanthewildtypeat162 254%ofwild-typeactivity.Tyr426, His431,andIle509werepredictedtobelocatednearsubsite 2,andLys549wasnear subsite þ2.Ser,Ala,Ala,andMetwerethebestaminoacidresiduesforthepositionsof Tyr426,His431,Ile509,andLys549,respectively.Combinationsoftheoptimizedsingle mutationsatdistantpositionswereeffectiveinenhancingcatalyticactivity.ThedoublemutantenzymesY426S/K549M,H431A/K549M,andI509A/K549M,combiningtwoof theselectedsinglemutations,showed340%,252%,and271%ofwild-typeactivity, respectively.Triple-andquadruple-mutantenzymesoftheselectedmutationsdidnot showhigheractivitythanthebestdoublemutant,Y426S/K549M.
1.7PurificationandCharacterizationof a-Amylases a-Amylasesproducedbyfermentationarerelativelycrudepreparations.Mostofthe commercialuseof a-amylasedoesnotrequire100%purificationoftheenzyme.But, high-purityenzymesarerequiredwhentheyareusedinclinicalandpharmaceutical sectors.Thefirststepsinthepurificationinvolvetheisolationofcrudeenzymeafterthe fermentation.InSmF,thisisusuallydonebycentrifugingthefermentedmediumand takingthesupernatantasthesourceofcrudeenzyme;inthecaseofSSF,thefermented matterisusuallymixedwithwaterorbuffers,andaftersuitablemixingthecontentsare filtered,wherebythefiltratecontainsthecrudeenzyme.Then,theenzymeisconcentrated(inthesupernatant/filtrate),precipitated(usingsalts/solvents),andpurifiedusing variouschromatographictechniquessuchasion-exchangechromatography,gelfiltration,isoelectricfocusing,etc. Table1.3 presentsstrategiesadoptedforthepurificationof a-amylasefromvariousmicroorganisms.
Therearealargenumberofreportsonthepurificationandcharacterizationof a-amylasesproducedbybacterialorfungalsourcesinSmFandSSF [75 87].Anenzyme producedinSSFwaspartiallypurifiedbyammoniumsulfatefractionation.Theenzyme wasoptimallyactiveatpH5.0and50 Cwithamolecularmassof66kDa.Thepresenceof Mn2þ andFe2þ enhancedtheenzymeactivity,whereasinthepresenceofHg2þ andCu2þ theactivitywasreduced [76].Apartiallypurified a-amylasefrom Streptomyceserumpens MTCC7317showedamolecularmassof54,500Da [77]. a-Amylasefrom B.subtilis KIBGE-HASwaspurifiedbyultrafiltrationandammoniumsulfateprecipitationwith19.2foldpurificationandspecificactivityof4195U/mg.Theenzymewashighlystableinthe presenceofvarioussurfactantsanddetergents.MetalionssuchasMn2þ,Ca2þ,Mg2þ,Kþ , Co2þ,andFe3þ activatedtheenzyme,whereasBa2þ,Cu2þ,Naþ,andAl3þ strongly inhibitedtheactivity.Ahighlyefficientraw-starch-digesting a-amylasefrom B. licheniformis ATCC9945awaspurifiedbygel-filtrationchromatographywithasixfoldincrease inspecificactivityandrecoveryof38%withamolecularmassof31kDa [82].Thepurified enzymeshowedanoptimumpHandtemperatureof6.5and90 C,respectively.Co2þ , Ni2þ,andCa2þ slightlystimulated,whereasHg2þ completelyinhibited, a-amylaseactivity.An a-amylasefrom Brevibacteriumlinens DSM20158,purifiedbyion-exchange chromatographyonaDEAE Sephadexcolumn,showeda7.88-foldincreaseinpurity witha16.80%yield,anditappearedhomogeneousonSDS polyacrylamidegelelectrophoresiswithamolecularmassof58kDa.EDTAandHg2þ inhibitedtheenzyme activity,whereasMn2þ andCa2þ enhancedtheenzymeactivity [83].
AnovelSDS-andsurfactant-stable,raw-starch-digesting,andhalophilic a-amylase waspurifiedfrom Nesterenkonia sp. [17].Theextracellular a-amylasewaspurifiedto homogeneityby80%ethanolprecipitation,Q-Sepharoseanion-exchangechromatography,andSephacrylS-200gel-filtrationchromatography.Theoptimumtemperature andpHwere45.8 Cand7.5,respectively.Themolecularmasswasestimatedas100kDa. TheenzymewasinhibitedbyEDTA,butwasnotinhibitedbyphenylmethanesulfonyl fluorideand b-mercaptoethanol.Ca2þ stimulatedenzymeactivity,whereastheenzyme Chapter1 a-Amylases15
