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1、NewBiotechnologyVolume28,Number6October2011RESEARCHPAPERExpressionandcharacterizationofanextremelythermostableb-glycosidase(mannosidase)fromthehyperthermophilicarchaeonPyrococcusfuriosusDSM3638ResearchPaper12314SungH.Park,KwanH.Park,ByungC.Oh,InteazAllia
2、ndByongH.Lee1DepartmentofFoodScienceandAgriculturalChemistry,McGillUniversity,Ste-Anne-de-Bellevue,QuebecH9X3V9,Canada2DepartmentofBiology,UniversityofIncheon,Incheon402-749,SouthKorea3LeeGilYaCancerandDiabetesInstitute,GachonUniversityofMedicineandScience
3、,Incheon406-840,SouthKorea4DepartmentofMicrobiologyandImmunology,McGillUniversity,Montreal,QuebecH3A2B4/AAFC,CanadaGenomicanalysisofthehyperthermophilicarchaeonPyrococcusfuriosusrevealedthepresenceofanopenreadingframe(ORFPF0356)similartotheenzymesinglycosi
4、dehydrolasefamily1.Thisb-glycosidase,designatedPFTG(P.furiosusthermostableglycosidase),wasclonedandexpressedinEscherichiacoli.TheexpressedenzymewaspurifiedbyheattreatmentandNi-NTAaffinitychromatography.Thegenewascomposedof1452bpencoding483aminoacidsforaprote
5、inwithapredictedmolecularmassof56,326Da.ThetemperatureandpHoptimawere1008Cand5.0insodiumcitratebuffer,respectively.ThesubstratespecificityofPFTGsuggeststhatitpossessescharacteristicsofbothb-galactosidaseandb-mannosidaseactivities.However,throughkineticstudi
6、esbyITC(IsothermalTitrationColorimetry)whichisverysensitivemethodforenzymekinetics,PF0356enzymerevealedthehighestcatalyticefficiencytowardp-nitrophenyl-b-D-mannopyranoside(3.02kcat/Km)andmannobiose(4.32kcat/Km).Theenzymeshowedtransglycosylationandtransgalac
7、tosylationactivitiestowardcellobiose,lactoseandmannooligosaccharidesthatcouldproduceGOS(galactooligosaccharides)andMOS(maltooligosaccharides).Thisnovelhyperthermostableb-glycosidasemaybeusefulforfoodandpharmaceuticalapplications.Introductionconditionssuita
8、bleforbiocatalysis.ThishasproventobethecaseExtremophilesareagroupofmicroorganismsthatcansurviveandvariousapplicationsinsugarchemistry,detergentproduction,andeventhriveinextremeenvironments.Theyareclassifiedlip