lsolationandCharacterizationo左anExtracellular

SerineProteasewithStrongFibrinolyticActivity

丘ｒｏｍＢａｃ川"ｓ肌脆iJisCIR110anditsImmobilization

ontoCelluloseBeads

SachikoSAToH＊，KoichiTAKAKuRA＊，TakenoriYAMAsAKI＊＊

YasunoriORII＊*，TakashiHATTA＊

NoboruTAKIzAwA＊＊andHohzohKIYoHARA＊＊

＊Ｂ/ＯＧ'Zg/"Ce〃"ｇＬａｂｏｍ/ｏｍ

Ｒｃｓｍ”/z伽伽ｔｅｃｌ／Ｔｅｃｈ"oﾉ09〕ノ

α"‘Ｂ/otecﾉi"o/qgyLa6omtoXy，

＊*Ｄ"α汀ｍｅ"ｔｑ／Ａ〃/〃CAC加加ｿqy，

Rzzc"/ｔｙｑ／Ｅ"ｇｉ"eemog

OAB〔Zy[ｚｍａＵ)z/"cぶりｑ／Ｓｃｊe"Ce，

尺加ﾉｰc〃０１－１，OhcZy[z”αｍ0-0005,ノZZPα〃

（ReceivedOctober6,1997）

Ｂｔｚｃｊ/伽ｓ"6ﾉﾉﾉﾉＳＣＩＲｌｌＯ，whichsecretedapotentfibrinolyticserineprotease，was

isolatedfromfermentedfoodTheenzymewaspurifiedtohomogeneityfromthe

culturesupernatantinhighyieldsbyhydrophobicinteractionchromatography,cation‐

exchangechromatography,andgelpermeationchromatography・Themolecular

weightoftheenzymeestimatedbySDS-polyacrylamidegelelectrophoresisandgel

filtratiｏｎｗａｓ２８ｋＤａ・Theisoelectricpoint（pI）ｏｆｔｈｅｅｎｚｙｍｅｗａｓ８９５、ThepH

optimumofcaseinhydrolysisｗａｓ10.5,ａｎｄｔｈｅｅｎｚｙｍｅｗａｓｓｔａｂｌｅａｔｐＨ６－１２ａｎｄｕｐ

ｔｏ５５ｏＣＴｈｉｓｅｎｚｙｍｅｈａｄｓｔｒｏngcaseinolyticandfibrinolyticactivity,anditsactivity

wasevenmorestabilizedbyCa2+，butinhibitedbyphenylmethanesulfonylfluoride

(PMSF）ａｎｄａ２－ｍａcroglobulinOfthesyntheticsubstrates,themostsensitivesub-,

stratewasz-Ala-Ala-Leu-PNAforsubtilisin,Onthebasisoftheresultsobtained,it

wasdemonstratedthatthisenzymewasasubtilisin-likeserineprotease､Thepurified

enzymewascovalentlyｉmmobilizedontoporouscellulosebｅａｄｓｂｙｔｈｅＩＶ‐

hydroxysuccinimideactivationmethodTheimmobilizedenzymemaintained

caseinolyticandfibrinolyticactivities､Thisprovidespromiseforpotentialapplications

inantithrombogenicbiomaterialsusedinblood-contactingmedicaldevicesandartifi‐

cialorgansaswellasthrombolyticagents．

lntroduction

Thrombolyticenzymesarebecomingmoreimportantinpracticalapplicationsof

therapy、Thrombolyticenzymesdigestfibri､，themainproteincomponentofblood

clotsVariousthrombolyticenzymessuchasurokinase，streptokinase，andtissue

106SachikoSAToH・KoichiTAKAKuRA・TakenoriYAMAsAKl・YasunoriOR肥TakashiHATTA・NoboruTAKIzAwAandHohzobKl)DHARA

plasminogenactivatorhavebeenwidelyusedinthetreatmentofthrombosisl)．

However,theseenzymesarenotonlyveryexpensive,butalsostillhaveproblemssuch

astheirshorthalflivesintheblood,antigenicity,andlackofstability・Thefibrinolytic

serineprotease,BrinasefromASPe“/伽０噸αehasbeenknownforalongtime,and

assessedintherapeuticattemptｓａｓａｔｈｒｏｍｂolyticagent2)3)4)．Unlikeurokinaseand

streptokinasewhichactasplasminogenactivators,itcandirectlylysefibrinclots・In

recentyearsthethrombolytictherapybyoraladministrationoffibrinolyticserine

proteases，nattokinasefromvegetablecheesenatto5)6)，ａｎｄlumbrokinasefromthe

earthworm,Ｌ""6戒"ｓ'w6e"zzshasbeeninvestigatedbySumiandhiscoworkers7)8)9）

Thesetwoserineproteasesalsodemonstratedfibrinolyticactivityirrespectiveofthe

presenceofplasminogenTheimmobilizationoflumbrokinaseonpolyurethanesurface

hasalsobeenreportedmorerecentlybyRyuc/czLlo)．

Withadvancesinmedicaltechnology,therehasbeenanincreasinglygreatdemand

forthedevelopmentofantithrombogenicbiomaterialswhichcanbeusedforblood

contactingmedicaldevicesandartificialorgansAsapracticalapproachtoantithrom‐

bogenicbiomaterials,theimmobilizationofsuchserineproteasesseemsofinterestand

significanceforobtaininganantithrombogenicsurfacewhichiscapableofdissolving

anythrombusthatmightbedevelopedonthesurface・

Inthisinvestigationweisolatedthebacteriawhichwerecapableofproducinga

strongfibrinolyticenzyｍｅｆｒｏｍｆｅｒｍentedfoods，andobtainedpurifiedenzyme

preparationsinhighyieldsbythesequentialchromatographicprocedures・Thepurified

enzymehasbeencharacterizedandprovedtobeasubtilisin-IikeserineproteaseThe

enzymedemonstratedstrongfibrinolyticactivityinthepresenceorabsenceofplas‐

minogenFurthermore,thisenzymewascovalentlyimmobilizedontocellulosebeads

asamodelsupport,andthefibrinolyticactivityoftheimmobilizedenzymewasbrieflyexamined

MaterialsandMethods

Mz蛇γjZzZs-MilkcaseinandbovinefibrinogenwerepurchasedfromNacalaitesque

(Tokyo,Japan);soymilkwasfromHonenCorp(Tokyo,Japan);pepstatin,ZV-tosyl-L‐

lysyLphloromethylketone（TLCＫ)，ZV-tosyl-L-phenylalanyl-chloromethylketone

(ＴPCK),trypsininhibitor,leupeptinandbovineplasmaa2-macroglobulin(α2Ｍ)were

obtainedfromBoehringerMannｈｅｉｍＧｍｂｈ(Mannheim,Germany);PMSFwasfrom

SigmaChemicalCo．（StLouis,ＭＯ)；Peptidyl-4-methyl-coumaryl-7-amides（MCA-

substrates)andz-AIa-Ala-Leu-PNAwerefromPeptidelnstitute,Inc.(Osaka,Japan)．

Activatedcellulosebeads(dia.,Ｃａ200匹、)ｗｅｒｅobtainedasagiftfromKurarayCo.，

Ltd,MedicalProductsDivision,Kurashiki,JapanThegelbeadscarried8atomspacer

armsattachedtothematrixbyepichlorohydrinandactivatedbyjV‐

hydroxysuccinimide・ThesubstitutionlevelwasapproximatelｙｌＯﾉumolNHS-groups／

mlgelPhenyl-ToyopearlandCM-ToyopearlweretheproductsofTosoh（Tokyo，

Japan)．TheFPLC-systemandaSuperosel2columnfromPharmacia（Uppsala，

Sweden)wereusedforisolationoftheenzyｍｅ

lsolaIionandCba｢acte｢izationolanExtracellula『SerineB｢OteasewithSt｢ongFib｢inolyticAclivityi｢omBlrilllIsslblMiSCIRllOanditslmmobilizationontoCelluloseBeads lO7

Ｓ〃/"ｓα"cノ〃CCCﾉﾉＺｚ－Ｂ．ｓ"6ﾉﾉﾉｊＳＣＩＲｌｌＯａｎｄＣＩＲｌ２０ｗｅｒｅｉｓｏｌａｔｅｄｆromfermented

vegetables（cookedpumpkinandsoybean）duringthisstudy・Thecharacteristicsof

thesestrainsaredescribedbelow.Ｂ､ｓ"肱/ｄｓｌＦＯ３９３６１１)ａｎｄＩＦＯ３３３５１２)werefromthe

lnstituteforFermentation,Osaka,Japan・Ｔｈｅｙwereusedasthesubtilisin-producing

strainsforcomparisonwithB．ｓ"6ｶﾞﾉﾉＳＣＩＲ１１０・Allthesestrainsweregrownin

L-medium（1％peptone，０５％yeastextract,0.1％glucose，0.5％ＮａCl，ｐＨ6.5）or

soymilk-medium(2％soymilk,0.5％yeastextract,0.1％glucose,0.5％ＮａCl,ｐＨ6.5)．

肋祓ｃａｊｊ０〃Cl／伽励""0ﾙﾉｶﾞＷ"gy"ｅ－Ｂ.ｓ"6t/比CIR110wasgrownaerobicallyat

30oCinL-medium・Afterl5hrofincubation,cell-freeenzymesolutionswereprepared

bycentrifugationatlO,ＯＯＯｘｇｆｏｒｌＯｍｉｎＴｈｅｃｕｌｔｕｒｅｂｒｏｔｈｗａｓｂｒｏｕｇｈｔｔｏＬ５Ｍ

(ＮＨ４)２Ｓ０４，３０ｍＭＴｒｉｓ－ＨＣｌ（ｐＨ8.0)，５mMCaCl2，andwasappliedtoaPhenyl‐

Toyopearlcolumnchromatographypreviouslyequilibratedwithabufferconsistingof

1.5Ｍ（ＮＨ４)２Ｓ０４，３０ｍＭＴｒｉｓ－ＨＣｌ（ｐＨ8.0),ａｎｄ５ｍＭＣａＣｌ２､Proteinswereeluted

withalineargradientfrom１．５ｔＣＯＭ（ＮＨ４ＬＳＯ４ｉｎ３０ｍＭＴｒｉｓ－ＨＣｌ（ｐＨ8.0）and5mMCaCl2Theactivefractionswerepooledandwerebroughtto75％saturation

withammoniumsulfateaｎｄｋｅｐｔａｔ４･Cforovernight・Theprecipitateswerecollected

bycentrifugationatlO,ＯＯＯｘｇｆｏｒ２０ｍｉｎ,thendissolvedinanequilibrationbufferfor

CM-Toyopearl，consistingoflOmMTris-ＨCｌ（ｐＨ7.0)，５mMCaCl2、Thesample

solutionwasdialyzedagainstthesamebufferforovernight，ｔｈｅｎａｐｐｌｉｅｄｔｏａＣＭ‐

ToyopearlcolumnElutionofproteinswasconductedwiththesamebuffer,theactive

fractionswerepooled,broughttoOl5MNaCl,concentrated,andappliedtoaSuperose

l2columnpreviouslyequilibratedwithabufferconsistingof20mMTrisHCl(ｐＨ7.5)，

５ｍＭＣａＣｌ２，０．１５MNaClThefinalpreparationwasconcentrated,ｓｔｏｒｅｄａｔ－２０ｏＣ，

andusedforfurtherexperiments

E"」aWllcczssCZys-Caseinolyticactivitywasassayedasfollows：anenzymesolution

(200ﾉul）suitablydilutedinl5mlmicrotubewasmixedwith200/ｚｌｏｆ1％casein

solutioninO1Mglycine-ＮａＯＨ,ｐＨ10.5at37oCAfter5minincubation,600/ｚｌｏｆ５％

trichloroaceticacid(ＴＣＡ)solutionwasaddedtothereactionmixture・Themixture

wasfurtherincubatedatroomtemperaturefor30minandthencentrifugedat

l2,OOOrpmforl5minThen,500匹ｌｏｆｔｈｅｓｕｐｅｒｎａｔａｎｔｗａｓｍｉｘｅｄｗｉｔｈ２､５ｍｌｏｆ０．４Ｍ

Ｎａ２ＣＯ３ａｎｄ0.5mlofFolin-Ciocalteauphenolreagent・Thereactionmixturewas

incubatedｆｏｒｌＯｍｉｎａｔ４０ｏＣ，ａｎｄａｎａｂｓｏｒｂａｎｃｅｏｆｔｈｅｍｉｘｔｕｒｅｗａｓmeasuredat

660ｎｍ､Oneunitoftheproteaseactivityisｄｅｆｉｎｅｄａｓｔｈｅａｍｏｕｎｔｏｆｔｈｅｅｎｚｙｍｅｔo

producethedigestwhichisnotprecipitatedbyTCAsolutionandgivesabsorbance

valueequivalenttolﾉｕｍｏｌｅｏｆｔｙｒｏｓｉｎｅｐｅｒｍｉｎａｔ３７゜CThefibrinolyticactivitywas

measuredusingstandardbovinefibrinｐｌａｔｅｓｉｎｔｈｅａｂsenceofplasminogenl3)．

Measurementofamidolyticactivitywasdonebyfluorogenicandchromogenicassays

usingMCAsubstratesandz-Ala-Ala-Leu-PNA,respectively・Thefluorometricassay

wasconductedinthefollowingmanner;enzyme,125ﾉuMsubstrate,0.1ＭＮａＣｌ,５０mM

Tris-HClbuffer（ｐＨ8.0),ｉｎａｆｉｎａｌｖｏｌｕｍｅｏｆ1.35mlwasincubatedat37oCAfter

adｄｉｔｉｏｎｏｆＬ５ｍｌｏｆ１７％ＣＨ３ＣＯＯＨ，theamountof7-amino-4-methyl-coumarin

(ＡＭＣ）liberatedwasmeasuredbyaHitachi２０００fluorescencespectrophotometer

108SachikoSAToH・KoichiTAKAKuRA・TakenoriYAMAsAKl・YasunoriORll・TakashiHATTA・NoboruTAKlzAwAandHohzohKIYCHARA

(Tokyo,Japan).Theexcitationandemissionwavelengthswere380nmand460nm，

respectively・Theassayusingz-Ala-Ala-Leu-PNAwasdoneaccordingtothemethod

ofStepanovetczL14)．

〃""０６ﾉﾉﾉ>ｇａｊｊｏ〃ｑ／伽C"`ay伽c-Theenzymewascovalentlyimmobilizedonthe

porouscellulosebeadsactivatedbyjV-hydroxysuccinimideester､Atypicalimmobili‐

zationprocedurewasasfollows:ｌOOmgoftheactivatedbeadsweresuspendedinlml

lOmMphosphatebuffer（PB）atpH74Tothesuspension,ａｇｉｖｅｎａｍｏｕｎｔｏｆｔｈｅ

ｅｎｚｙｍｅｗａｓａｄｄｅdundershakinginthecoldroom，ａｎｄｔｈｅｍｉｘｔｕｒｅｗａｓｋｅｐｔａｔ４ｏＣ

forindicatedtime､Then,ｔｈｅｂｅａｄｓｗｅｒｅｗａｓｈｅｄｗｉｔｈｌＯｍＭｐｈｏｓphatebuffersaline

(PBS),andthebufferconsistingofO・lMTrisHCl(ｐＨ8.0),0.5ＭＮａCl,wasaddedto

blocktheremainingactivegroups

A"αMcczノ，”cc〃”s-Proteinconcentrationwasdeterminedbythemethodof

BradfordwithIgGasastandardl5).Theisoelectricpoints(ｐＤｏｆｔｈｅｅｎｚｙｍｅｗａｓ

ｍｅａｓｕｒｅｄｂｙｉｓｏｅlectricfocusingonAmpholinePAGplatefrompH3,5-9.5（Phar‐

macia)．SDS-polyacrylamidegelelectrophoresis（SDS-PAGE）ｗａｓｄｏｎｅｕｓｉｎｇ15％

polyacrylamidegelbythemethodofLaemmlil6)．Inaddition，ＭＡＬＤＩＴＯＦ－ＭＳ

(MatrixAssistedLaserDesorptionlonization,TimeofFlightMassSpectrometry）

analysiswasperformedforthemeasurementsofmolecularweightoftheenzymeusing

KompactMALDIImassspectrometer(Shimadzu-Kratos,Kyoto,Japan）

ResultsandDiScussion

Cﾉicz?zzc陀沈α"o〃ｑ／jSMztes-StrainsCIR110andCIR120wereidentifiedasazcﾉﾉﾉﾉUS

S"伽ノヵattheNationalCollectionsofIndustrialandMarineBacteriaLimited(ＮＣＩＭＢ

Ｌｔｄ.),Aberdeen,ScotlandBothstrainswerestrictlyaerobic,gram-positive,catalase

producing，andendosporeformingThemaximumtemperatureforthegrowthwas

55oCThebiotinrequirementwastesteｄｉｎｔｈｉｓｗｏｒｋＢｏｔｈｓｔｒａｉｎｓｗｅｒｅｓｈｏｗnto

requirebiotinforgrowth,thereforethesewereclassifiedintｏａｚｃﾉﾉﾉZ`ss"ＭＪｊＭｏα/ねﾉ'7)．

ＧγDzuﾉﾉｶα"cノｅ"2Ｍ,ｚｅＰ”〃ｃｔｉｏ〃－Toexaminetheproductionoffibrinolytic

enzyme(s)ｂｙＢ.ｓ"肋比ＣＩＲ110,ｔｈｅｔｉｍｅｃｏｕｒｓｅｏｆｇｒｏｗｔｈａｎｄｅｎｚｙmeactivitiesin

culturebrothofCIR110andCIR120strainsweremeasuredFurthermoretwotypical

B.ｓ"6ﾉﾉﾉｊＭＭｔｏﾉstrains,IFO3936andIFO3335wereinvestigatedforcomparison・ＡＩｌ

ｓｔｒａｉｎｓｓｈｏｗｅｄａｌｍｏｓｔｔｈｅｓａｍｅｐatternsofcellgrowthinL-medium・Figurelshows

enzymeproductionbyeachstrainsintheL-medium(Ａ)orsoymilk-medium(B).Itis

noteworthythatofthestrainstested,Ｂ・ｓｚ`Ｍ/fsCIR110producedtheproteasewiththe

highestcaseinolyticandfibrinolyticactivityinbothmedia，whileotherstrains

producedthefibrinolyticenzymeonlywhentheywereculturedinsoymilk-medium・

Generally，８ｓ"肱比仇α伽strainshavebeenculturedinthemediumcontaining

soybeancakeextract（soymilk)'8)．However，ｉｎｔｈｉｓｃａｓｅ，theenzymepurification

becomesquitedifficultbecausetheseparationofthelipidsandinsolublesmallparti‐

clesofsoymilkisnecessary，Therefore，itisinterestingthatB．ｓ"6/"たCIRllOwas

capableofproducingthehighlyfibrinolyticproteasewithoutsoymilkThisadvantage

mayofferpromiseforpossibleapplicationsinthelarge-scaleenzymeproduction．

lsolationandC11a｢acterizationo(anExlracellularSe『ineP｢oteasewithStron8FibrinolvticActivityITom比(１１/llsslIMljsCIRllOanditslmmobilizationontoCelluloseBeads lO9け

100600

Ａ-１

ｍ００４

２

（ｌＥう）ご一シ一一・回り三一．巨一①⑩ロ。

００

０８

６４

（同ＥＥ）量夛冒：三一．■定ロ匡

,ｴﾆｰさデー５

△一Z-器-２

2０

旦二〒･-百－百T-5-cRcFo声-５

●

●

０ ５ ＯＴｌｍｅｍ

５ 2０ ０５１０１５２０ＴＩｍｅ(hr）

100600Ｂ-２Ｂ-１

窪／△

･壜ア

００

０８

６４

（ｐＥＥ）ら一シ一万回。｜哀一．巨一』口匡

００

００

４２

（ｌＥョ）ａ一畠・ロ⑨一員一．巨一①③尺〕

5.ﾉ。ロ

ロ

△

●ロ△△

◎し元

2０◎△ロ

。。ロ

０５１０１５２００５１０１５２０Ｔｉｍｅ(h｢）Ｔｉｍｅ化r）

Fig.１．TimecoursesofcaseinolyticandfibrinolyticenzymesproductioniｎＬ－ｍｅｄｉｕｍａｎｄｓｏｙｍｉｌｋ

ｍｅｄｉｕｍｂｙＢ・szz6t伽ＣＩＲ110（●),Ｂ､ｓ"6ﾉﾉﾉjｓＣＩＲ１２０（○),Bszz6ﾉﾉﾉjSIFO3936（△),ａnｄＢ．

ｓ"6ﾉﾉﾉﾉＳＩＦＯ３３３５（□).Ｃｅｌｌｓｗｅｒｅｇｒｏｗｎａｔ３０ｏＣｉｎＬ－medium(A)orsoymilkmedium(B)ona

rotaryshaker・Theculturesupernatantswereusedforcaseinolyticactivityassay(Ａ-1,Ｂ-1）

andfibrinolyticactivityassay(Ａ-2,Ｂ-2)．

Ｐｂ‘γi/１Ｍ'０〃ｑﾊﾙｅ/if伽"ｏＭｃｃ"Zjwze-Astrongfibrinolyticserineproteasewas

purifiedfromB．ｓ"6t"たCIR110throughPhenyl-Toyopearl，CM-Toyopearl，and

Superorel2chromatography・Crystallizationwasperformedbyultrafiltrationof

CM-Toyopearlfraction(L2mgprotein/ｍｌ)toa3-foldconcentrationandallowedto

standforfewdays・ＡｓｓｕｍｍａｒｉｚｅｄｉｎＴａｂｌｅｌ，proteasewasabout35-foldenriched

with88％yield,ａｎｄｈａｄａspecificcaseinolyticactivityof３．８×103Ｕ/mgproteinThis

purificationmethoｄｗａｓｅａｓｉｌｙｓｃａｌｅｄｕｐｔｏａｔｌｅａｓｔｌＯｔimesinthelaboratory・

Previously,Ｓｕｍｉｅｊα/,6)ａｎｄＦＵｊｉｔａｅＭ/､'9)demonstratedthepresenceofafibrinolytic

enzyme(namednattokinase)inthevegetablecheesenattoButtheyhavenotdescribed

aboutthepurificationyieldoftheenzyme・Wehaveachievedthepurificationofthe

strongfibrinolyticenzymefromtheculturebrothofB.ｓ"伽/ｊＳＣＩＲ１１０ｉｎｈｉｇｈｙｉｅｌｄｓ

(80-95％),andthispurificationprocedurecouldeasilybescaleduptotheindustrial

preparationsystems、

Figure2showsSDS-polyacrylamidegelelectrophoretogramsoftheeachsampleof

thepurificationstepsandthemicrophotographofthecrystallineprotease、Thefinal

preparationshowsthesinglepolypeptidewithmolecularmassof28kＤａ(lane6inFig．

110SachikoSAToHKoichiTAKAKuRA・TakenoriYAMAsAKl・YasunoriOMl・TakashiHATrANoboruTAKlzAwAandHohzohKlYoHARA

Table１．PurificationofthefibrinolyticserineproteasefromBs"6ﾉﾉﾉﾉＳＣＩＲ１１０ａ

total

volume

（ｍｌ）

total

protein

（ｍg）

total

act．

(units）

ＳＰａＣｔｂ．

(units/mg

ofprotein）

Purificationstep yield

(％）

purification

（x・fold）

Crudefraction

Phenyl-Toyopearl

Ammoniumsulfate

precipitation

CM-toyopearl

Superosel2

●●●

『ⅡⅡ（（叩〃〈】《叩く囮）

2420

１６７

784８．６×１０５1.1×１０３１００

２６８８．３×１０５３．１×１０３９７

２５０８．０×１０５３．２×１０３９３

２２０７．７×１０５３．５×１０３９０

２００７．６×１０５３．８×１０３８８

１

２－８

2７ 2.9

□の

。。、、一一一，民、四）

178

５０

3.2

3.5

Enzymeactivitywasassayedateachstepofpurificationprocedureinglycine-NaOHbuffer，pHlq5usingcaseinassubstrate・

Specificactivitywasmeasuredinmicromolesoftyrosineperminutepermiligramofprotein

ａ３Ｄ

ＢＡ

４■

４７００４

９６３２１

霜繍鶴辨～認

１２３４５６－－５０１A、

Fig.２．ＳＤＳＰＡＧＥ(A)andmicrophotograph(B)ofthecrystallineprotease・ACoomassiestain(15％gel):Ｌａｎｅｌ,molecularmassstandards(inkilodaltons;Pharmaciaelectrophoresiscalibration

kits);lane２，crudefraction;lane３，Phenyl-Toyopearlfraction;lane４，(NH4LSO4precipitate；ｌａｎｅ５，CM-Toyopearlfraction；ｌａｎｅ６，Superosel2fraction．

2Ａ)Inaddition,themolecularweightmeasuredbyTOF-MSanalysiswas27,683．

Qmzlbﾉ此Ｐ叩e伽ｓｑプ伽ｅ"こ)wze-Thephysicalcharacterizationwasmainly

studiedbycaseinhydrolysis．

（A)ｐＨｏｐｔｉｍｕｍ、ＴｈｅｐＨｐｒｏｆｉｌｅｏｆｅｎｚｙｍｅａｃｔｉｖｉｔｙｉｓｓｈｏｗｎｉｎＦｉｇ３、ＴｈｅｐＨ

ｏｐｔｉｍｕｍｗａｓｌＯ､5,andtheenzymewasstablebetweenpH6-12atleast20hoursat25。Ｃ

(datanotshown)．

（B)Substratespecificity・Table2showedthesubstratespecificityoftheenzymefor

varioussyntheticsubstrates・Thehighlysensitivesubstratesfortheenzymewere

z-Ala-Ala-Leu-PNAforsubtilisinandSuc-AIa-Ala-Pro-Phe-MCAforchymotrypsin、

However,ｉｔｈａｄｌｉｔｔｌｅｏｒｎｏａｃｔＭｔｙｔｏｗａｒｄｔｈｅthrombinandurokinasesubstrate．

（C)Effectofinhibitorsonenzymeactivity・Effectofinhibitorsonenzymeactivity

wasstudiedbothincaseinolysisandfibrinolysis、Theresultsobtainedwithvarious

inhibitorsaresummarizedinTable3．Ｂ､ｓ"6ﾉﾉﾉﾉSCIR110proteasewasfullyinhibiteｄ

ｂｙＰＭＳＦ,aninhibitorofserineproteases・a2-Macroglobulin(α2Ｍ),amajorprotease

inhibitorofbloodplasma，alsoinhibitedcaseinolyticandfibrinolyticactivitytoa

significantextent・However，ＴＰＣＫ，ＴＬＣＫ，andLeupeptinhadnoeffectonboth

onontoCelluloseBeadso1ationandChaTacterizationo(anExlracellu1arSe｢ineProteasewithStTongFibrinolyticActivitY(r0mBhciⅢlssIbMsCIRIlOanditslmmob １１１

００

００

００

０ｓ

１

［ｌＥへ。］勇窒シ一一○回。－昌一ｏＥ－①吻呵。

●Ｐ１ＰＥＳ－Ｔｎｓ

ｏＣＡＰＳ－ＮａＯＨ

０

６７８９１０１１１２

ｐＨ

Fig.３．pHProfileoftheenzymeactivity・Analiquot（6ﾉugprotein）wasassayedforproteolytic

activitywithcaseinasthesubstrateatvariousｐＨｖａｌｕｅｓＥａｃｈｖａｌｕｅｗａｓｔｈｅｍｅａｎｏｆ

ｔriplicatedeterminationsSymbols:●,Pipes-Trisbuffer;○,CAPS-NaOHbuffer．

Table2Substratespecificityofthepurifiedenzymea

Rateofhydrolysis

(nmol/minmgprotein）Substrate Ｋｍ(ｍＭ）

Suc-AIa-Ala-Pro-Phe-MCA

Boc-lleGlu-Gly-Arg-MCA

BocPhe-Ser-ArgMCA

Boc-Glu-Lys-Lys-MCA

Boc-Leu-Ser-Thr-Arg-MCA

Boc-Val-Leu-Lys-ＭＣＡ

Ｂｏｃ－Ｖａｌ・Pro-Arg-MCA

Boc-Gln-Ala-Arg-MCA

LeuMCA

z-Phe-Arg-MCA

Bz-Arg-MCA

Glt-Gly-ArgMCA

z-Ala-AlaLeu-PNA

(Chymotrypsin）

(FactorXa）

(Trypsin）

(Plasmin）

(ActivatedProteinC）

(Plasmin）

(a-Thrombin）

(Trypsin）

(Aminopeptidase）

(KallikTein）

(Trypsin）

(Urokinase）

(SubtilisinA）

●●■●

（、宝画一）戸》〃ＪＤｌ，【口⑭一・．【Ⅱ】ロー【叩】。●【叩】。．【｜叩】●●●■らｐＢ

（０（００）〈０７０戸‐【』７．４。ｎｎｎｎ（０

（叩〃公】【』『四〉（．、叩》一’『〉（宅く四）

（皿）（血》●幻扣》。｜勺■■△

〈】三ｍ〉（叩クハ】

０．１７

０．２５

０．５

aTheenzymeactivitywasassayedbythemethoddescribedinmaterialsandmethods．

Table･aEffectsofvariousinhibitorsonthecaseinolyticandfibrinolyticactivityofpurified

enZymea

RelativeActivity(％）

caseinhydrolysis fibrinhydrolysiｓConcentrationlnhibitor

None

PMSF

a2-Macroglobulin

Pepstatin

TLCK

TPCK

Trypsininhibitor

Leupeptin

０２６２７５０８

０２９９００９

１１１

０００３４６０４

０９９８０９

１１

ＭＭＭＭＭＭＭ

ｍ浬浜浜“瓜区

２０２０００２

３７７１

２５

Thepreincubationoftheenzymewitheachinhibitorwascarriedoutａｔ３７ｏＣｆｏｒ３０ｍｉｎ,andthe

aliquotwasassayedforcaseinandfibrinhydrolysis、Eachvaluewasthemeanoftriplicatelneasurernents．

ａ

112SachikoSAToH・KoichiTAKAKuRA・TakenoriYAMAsAKl・YasunoriORII・TakashiHA1TA,NobomTAⅢzAwAandHohzohKlYoHARA

０

100

０５

訳）萱シ一一Ｕ江一回ゴロ｜②ロ区

Ｂ２◆

０

2５３５４５５５６５Temperature(℃）

Fig.４．ＥｆｆｅｃｔｏｆＣａ２＋andtemperatureonthestabilityoftheenzymeTheenzymedissolvedinthe

Tris-HCIbuffer（ｐＨ7.5)，ｉｎｔｈｅｐｒｅｓｅｎｃｅｏｒａｂｓｅｎｃｅｏｆ５ｍＭＣａ２＋wasincubatedatthe

indicatedtemperaturesforlhr，andtheresidualproteolyticactivitiesweremeasuredwith

caseinassubstrateSymbols:○,inthepresenceofCa2+;●,intheabsenceofCa2十．

Ｏ

鑿ﾄ゜雫…州M…c比…ＩＩｉ二:!：１Ｍ野…… Ｏ

篝ﾄ｡-qI2-9…~NH-c…c朏州ⅡⅢ｡-m>C写H‘ＯｌＣＨ２oＨ

Fig.５．Covalentcouplingoftheenzymetocellulosebeads

activatedwithjV-hydroxysuccinimideester．

activitiesAsawhole，theseresultsindicatedthattheenzymewasasubtilisin-like

protease,beingconsistentwithitsabilitytocleavez-Ala-Ala-Leu-PNA,Theinhibition

ofserineproteasesｂｙａ２Ｍｉｓｗｅｌｌｋｎｏｗｎａｎｄｅｘｐｌａｉｎｅｄａsduetotheentrapmentof

theproteaseswithinthea2Mmoleculetoformthea2M-proteasecomplex2o)．This

inhibitionshouldbetakenintｏａｃｃｏｕｎｔｆｏｒｕｓｅｏｆｔｈｅｅｎｚｙｍｅａｓａｔｈrombolyticagent、

ＤＥｆｆｅｃｔｏｆＣａ２＋onenzymeactivityandstability,Theenzymewasconsiderably

stabilｉｚｅｄｂｙ５ｍＭＣａ２ｔＡｓｓｈｏｗｎｉｎＦｉｇ４,theresidualactivitywaslOO％when

incubatedat25-50oCforlhourinthepresenceofCa2+,ａｎｄ０％at45oCintheabsence

ofCa2+、

ＩＳＯ山c/γｊｃＰｏｊ"/ｑ／ノルｅ"ｚＷ'zc-Thepurifiedenzymewashomogeneouson

isoelectricfocusingandtheisoelectricpointwas895(datanotshown).Thisvaluewas

differentfrom9､８ofsubtilisinNAT21）and85ofsubtilisinAM22)．

加加０６雌atjo〃ｃＷﾉｉｃｅ"jajwce-Thefibrinolyticenzymewascovalentlyimmobilized

ontheporouscellulosebeadsthroughtherｅａｃｔｉｏｎｓｃｈｅｍｅａｓｓｈｏｗｎｉｎＦｉｇ５・The

immobilizationreactionwascompletewithin7h,independentoftheconcentrationof

theenzyme,whilethesaturatedcaseinolyticandfibrionolyticactivityoftheimmobil

izedenzymewasdependentontheinitialenzymeconcentrationofthereaction

lsoliltionandCharacte『izationo[anExt｢acellularSerinelﾔoteasewithSt『ongFib｢inolyticActivityl｢omBbMMisl（lWjsCIRllOanditslmmobilizationontoCellulo雛Beads ll3

mixture・Ｔｈｅａｍｏｕｎｔｏｆｉｍｍｏｂｉｌｉｚｅｄｅｎｚｙｍｅｗａsalmostproportionaltotheinitial

enzymeconcentration，ａｔleast，inthelowconcentrationrangeexamined（Ｆｉｇ６)．

Figure7showedthefibrinolyticactivityoftheimmobilizedandfreeenzyme、The

immobilizedenzymewaslessaffectedbyproteaseinhibitorsthannon-immobilized

enzymeintermsofcaseinolysisandfibrinolysis(datanotshown）Wehavefoundthat

theimmobilizationand/orchemicalmodificationsoftheenzymecouldplayanimpor‐

tantroleinretainingitsfibrinolyticactivity・Thepropertiesoftheenzymeimmobil

izedonseveraldifferentcarriersarenowunderinvestigation・Thepreliminaryexperi‐

mentsofenzymeimmobilizationshoｗｅｄｔｈｅｇｏｏｄｒｅｓｕｌｔｓｆｏｒｔｈｅｅｎｚｙｍｅｓtabilityand

theresistancetotheproteaseinhibitors23)．Thedetailedstudywillbereportedelse‐

where・ThefibrinolyticenzymeofB.ｓ"6/ﾉﾉﾉＳＣＩR110mayhavepromiseforpotential

８ ０００６ ０

０４ ０

２ ００

（｜Ｅへ。〉ら』シーーロ⑩。一員一．Ｅ①⑩⑮。

（剣ＥＥ〉らＥ←：。』且一．Ｅ』Ｑ正

〔Ｕ（Ｕ

釦、》

【。ワ』

■Ｉ

５０１３o

Enzymeconcn.（ｍgノｍＩ）

Fig.６．FibrinolyticandcaseinolyticactｉｖｉｔｙｏｆｔｈｅｅｎｚｙｍｅｉｍｍｏｂｉｌｉｚｅｄｏｎｃellulosebeadsThe

activatedcellulosebeadswereincubatedwithagivｅｎａｍｏｕｎｔｏｆｔｈｅｅｎｚｙｍｅｉｎｌＯｍＭ

ｐｈｏｓｐhatebuffer(ｐＨ7.4)ａｔ４.Cunderslowconstantshakingfor8hoursThen,４０ﾉulofbeads

suspensionwereusedforenzymeassaySymbols:○,caseinolyticactivity；●,fibrinolytic

activity．

Fig.７．Fibrinolyticactivityofthefree（left）andimmobilizedenzymes(right)．Thepurifiedfree

enzyme(10Ｕ)andimmobilizedenzyme(15Ｕ/25mgbeads)wereseparatelyplacedonafibrinplate，andthefibrinolyticactivitywasmeasuredafterincubationforlhrat３７°Ｃ．

114SacbikoSA10HKoichiTAKAKuRA・TakenoriYAMAsAKl・YasunoriORlI・TakashiHAT『A・NoboruTAKlzAwAandHohzohKIYCHARA

Table､４．ComparisonoftheN-terminalaminoacidsequencesoftheenzymewiththatofbacterial

serineproteases

Subtilisinsa １ １０ 2０ 3０ Homology

ＣＩＲ１１０

ＮＡＴ

ＡＭ

ＢＰＮ，

Carlsberg

ＡＱＳＶＰＹＧＩＳＱＩＫＡＰＡＬＨＳＱＧＹＴＧＳＮＶＫＶＡＶ

ＡＱＳＶＰＹＧＩＳＱＩＫＡＰＡＬＨＳＱＧＹＴＧＳＮＶＫＶＡＶ

ＡＱＳＶＰＹＧＩＳＱＩＫＡＰＡＬＨＳＱＧＹＴＧＳＮＶＫＶＡＶ

ＡＱＳＶＰＹＧＶＳＱＩＫＡＰＡＬＨＳＱＧＹＴＧＳＮＶＫＶＡＶ

AＱＴＶＰＹＧＩＳＬＩＫＡＤＫＶＱＡＱＧＦＫＧＡＮＶＫＶＡＶ

100％'5）

100％25）

９７％23）

６７％8）

Subtilisinsshownabovewereproducedby:CIRllO,Ｂ,ｓ"6'伽ＣＩＲ110;ＮＡＴ,ES肋"/ｉｓ(natto)；ＡＭ,ＥＳ"6t伽var.α”y/0sαccﾉzα伽c"s;ＢＰＮ，,βα”/ひﾉﾉ9勿沈c/e"s;Carlsberg,ＢⅢChe"加”た．

ａ

applicationsinantithrombogenicbiomaterialsandthrombolyticagents．

Acknowledgements

ThesupportofaportionofthisworkbyYaegakiZymotechnicsCo.，andKuraray

Co.，LtdisgratefullyacknowledgedWewｉｓｈｔｏｔｈａｎｋＭｒ・SHirata（Okayama

AgricultureDevelopmentInstitute)forthemicrophotographoftheenzymecrystals．

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