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ガノノ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ルノガ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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