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1、NewBiotechnologyVolume28,Number6October2011RESEARCHPAPERExpressionandcharacterizationofanextremelythermostableb-glycosidase(mannosidase)fromthehyperthermophilicarchaeonPyrococcusfuriosusDSM3638ResearchPaper12314SungH.Park,KwanH.Park,ByungC.Oh,InteazAlliandByong
2、H.Lee1DepartmentofFoodScienceandAgriculturalChemistry,McGillUniversity,Ste-Anne-de-Bellevue,QuebecH9X3V9,Canada2DepartmentofBiology,UniversityofIncheon,Incheon402-749,SouthKorea3LeeGilYaCancerandDiabetesInstitute,GachonUniversityofMedicineandScience,Incheon406-84
3、0,SouthKorea4DepartmentofMicrobiologyandImmunology,McGillUniversity,Montreal,QuebecH3A2B4/AAFC,CanadaGenomicanalysisofthehyperthermophilicarchaeonPyrococcusfuriosusrevealedthepresenceofanopenreadingframe(ORFPF0356)similartotheenzymesinglycosidehydrolasefamily1.Th
4、isb-glycosidase,designatedPFTG(P.furiosusthermostableglycosidase),wasclonedandexpressedinEscherichiacoli.TheexpressedenzymewaspurifiedbyheattreatmentandNi-NTAaffinitychromatography.Thegenewascomposedof1452bpencoding483aminoacidsforaproteinwithapredictedmolecularmas
5、sof56,326Da.ThetemperatureandpHoptimawere1008Cand5.0insodiumcitratebuffer,respectively.ThesubstratespecificityofPFTGsuggeststhatitpossessescharacteristicsofbothb-galactosidaseandb-mannosidaseactivities.However,throughkineticstudiesbyITC(IsothermalTitrationColorime
6、try)whichisverysensitivemethodforenzymekinetics,PF0356enzymerevealedthehighestcatalyticefficiencytowardp-nitrophenyl-b-D-mannopyranoside(3.02kcat/Km)andmannobiose(4.32kcat/Km).Theenzymeshowedtransglycosylationandtransgalactosylationactivitiestowardcellobiose,lacto
7、seandmannooligosaccharidesthatcouldproduceGOS(galactooligosaccharides)andMOS(maltooligosaccharides).Thisnovelhyperthermostableb-glycosidasemaybeusefulforfoodandpharmaceuticalapplications.Introductionconditionssuitableforbiocatalysis.ThishasproventobethecaseExtrem
8、ophilesareagroupofmicroorganismsthatcansurviveandvariousapplicationsinsugarchemistry,detergentproduction,andeventhriveinextremeenvironments.Theyareclassifiedlip
