Studies of macerating enzyme acting on middle lamella pectin. III. Separation of the macerating enzyme by duolite CS-101-香川大学学術情報リポジトリ

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ユ41 Vol9,No3(1959)

STUD‡ES OF MACERAT王NG ENZYME ACTING

ON MIDDLE LAMELLA PECTIN

Ⅱ Separation of the MaceratingIEnzymeby Duolite CS−101.

Aki工a KA丁Ⅰ

(Laboratory of TechIlicalMicrobiolog−y) (Received October30,1957)

The author announcedin paItI′1)that macerating enzyme(ME)was sepaIated from the mixed enzyme solution by the chromatography of AmberliteIRC−50・By that method only a smal1amount of the

enzymes could be treatedin one experiment,and the concentration of ME solution thus obtained was

verylowIf the amounts of polygalactuIOnaSe(PG)and MEincIeaSedin charged enzymes solution,PG

was also elutedinto ME solution,and the experiment was resultless

ln this report,the author descIibes the experiments whichwere carried out with Duolite CS−10lME

coufd be surely separated from the mixed enzyme solutioll by Ihe simpfe procedure using Duolite

CS−10l,and the concentratration of ME was found to be higher than that of the experimentsin wich

AmberliteIRC−50was used

Expe≧r…ments and Results

I Mき⊂rOOrganism and⊂uttiYat;on method

The microorganismusedinthis工epOItWaS the same asinthe previous studies,Cl‖榊sineumvaz sikohianumThe cultivation of the bacteria was carried out with semi”Synthetic medi11m,mOdified Speakman/s mediumThe culturesolution ofユCOcc,WaS COmpOSed ofOl75g pectin,0”05gK2HPO4, 0C5g KH2FO4,0‖02g MgSO4・フH20,0C〇ユgFeSO4r,Ol001g NaCl,000ユg MnSOも,5ccyeast extract,and hyd‡01ysed solution of peptone containing64∈mgtotalN“Its pHwas adjusted to6・8Corn mash ofthe

bacteIia wasinoculated to the medium,andincubation proceeded foIフ2hrsat3フOC.Afterincubation, the cl】1ture董1uid was cooled,filtered thIOughcloths,Centrifuged(3,000rpm)for.15min in order to remove the bacterialcell.The centrifuged solution thl】S Obtained was storedin anice chamber at OOC.

This solution had deep yellowish br’own colour

Activities of PG and ME are shown in Tabie 1 Activity of PG was expressed by the decrease of

Viscosity caused by the action of enzyme on C)5%pectic acid solution forlho11r(3) Activity of ME was shown by the degr’ee Of separation of fibres caused by the action of enzyme on a piece 05 Ganpi barks of the Size oflxユcm,and each volume of the enzyme solution employed was noted,aS WaS mentionedin the previous paper(1) ∬”Adsorp七;on of po[yga雇acturonase and

Tablel… Activities of PG and MEin the

Centrifuged solution.

ME(Degree of separation of fibres)

PG unit/ml

95 附(1.0)十冊(0…3)−≠(0,1)

macerating enzyme on丘⊃uo=te CS・ヱ01。

The author reportedin the previous paper(4)that the differencein behavior of the two enzymes was recogllized on the adsorption by using the buffered resin of p‡‡60ln this study,the resin was buffered at the pモIvalue of5O to88and H−,Na−forms of the resin were also used for’the adsorption of PG

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Tech.Bu11‖Fac.Agr.Kagawa U王1iv”

142

and ME”These resins were buffered by the method of HIRS,STEIN and MooRE(5),Twenty mlof the

resin was employedin each experiment.The size of the column was2.3×5hCcm

The centrifuged solution was passed through a coiumn of Duolite A−7 for the purpose of removing impuritiesThe passed solution of300mi,WaS takenin a beaker and the pH of this enzyme solution

was adiustedtothevalue of the resin on which the solution was added”Theenzymesolution was charged on the top of the column which consisted of 2C)mlof the buまfered resin as described above‖ The flow

Iate(SV)0まthe enzyme solution was adjusted to4,Alloperations were carried olユt at8O to100C When the adsorption of the enzymes on each resin was finished,the activities of PG and ME were determined by using each passed solutionThe results are shownin Table2

Table2… AdsoIption of PG and ME on Duolite CS−10lofvarious pH val一」eS

Activity of ME in passed solution (Degree of separation of fij⊃reS)

Ratio o董 adsoIption of PG (%) p‡王 of bu董fered resin PG unit/ml in passed so】n H一重orm 5.0 60 64 68 フ2 フり6 80 8.8 Na一重orm 100 1CO 66.3 3〕け5 305 30“5 26.3 20 O 55 8 45.3 0 0 32 56 56 56 フ○ フ6 42 52 一一士 + + 帖刷冊此⋮拙

PGand ME were completely adsorbed on the column of pH below 5.0,and PG first passed throughthe resin of6“0,While the adsorption of ME on the same resin was observed to be almost complete.And then,the amounts of PG and ME adsorbed on the columns dec工eaSed corresporLdingly with the ascent of

pH value f工Om 64 to8..0‖ The adsorption ratio of PG agalnincreasedin case of theIeSin of pH8小8 and Na−・form,but the adsorption of ME did notincreasein these columns

Ⅱ、EIu七ion of po‡yga‡ac七uronase and macerating en王yme adsor・bed on Duolite CS∴ヱ01

The王eSin which adsorbed PG and ME was washed with water,and half volume of each resin,namely lOml,WaS takeninto a smallglass tube,and then N/10hydrochloIic acidsolutionwas added on the top Of the column,at a flowIate(SV)of4The elution of the enzymes was optlrated at 80 to100C,and lO mlof each effluent was takeninto a test tube as one fIaCtion。The activities of PG and MEin each

fraction are shown in Table 3

As described above,these operations we工e nOt COmplete chromatography,but a degree of elution of the two enzymes was observed as shownin Table3

Table3h Elution of PG and ME adsorbed on Duolite CS−10l

Activity of MEin eluate(Degree PG unit/ml

uff’ered

i 】Fractionno

adsorption d of eluate

pH of

eluate in eluate l Of separation of fibres)

榔(1い0) 一片.(0一2)

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Vol.9,Nol3(195∈∋)

88︵○︵○︵0

55555 ︹O﹁⊥

、⊥

±榊十±± ±一一一l

When the enzymes were adsorbed on the bu董fered resins at pH above60,the activity of MEin the effluentincreased coITeSPOnding to the rising of pH value of bufferedIeSin.Thisincrease of ME activ・

ity continued untillpH value of the buffered resin reached toフ.6。No activity of ME was recognized in the eluate obtained from the columns o董pH8O and pH value aboveit,for ME was not adsorbed on

these columns

工t was found that the column of pH7h2was suitable for the removalof PG from the miⅩed enzyme SOlution,for the activity of PGin the eluate was observed to below while ME revealed noticeable

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Tech.Bull.Fac.Agr.Kagawa Univ..

144

Ⅳ‖h州uen⊂eOfYOlume oforig盲na】enzyme sohtion on七he separat盲on o†macera血唱enZyme

A procedure,aS menthioned above,WaS eStablished for the sepaIation of ME,and a requisite volume

of the mixed enzymesolution must be taken to a definite volume of Duolite CS−101asin case of

Ambe王litelRC・50

20mlof the buffered resin of pH7‖2was takenin a glass tube,and the mixed enzyme solution of20 to3COmi,WaS paSSed through the column,and then the elution of ME waspe工formedusingN/10hydro・ chloric acidsolution.Asshownin Table4,the activity of ME was only detectedin each 董raction of

Table4小Influence ofvolume of the mixed enzyme solution on the separation of ME

eluate untillthe added enzyme solutionincz・eaSed to200mi,While PG was also eluted when300miof

the mixedenzyme solution was charged.Therefore,uSing20 mlof Duolite CS・10l,PGin the mixed enzyme solution colユ1d be completely removed untillthe amount of PGincreased to16000 unitsh And

thus,PG couldbe removed more effectively from the mixed enzyme solution and ME richsolution would

be obtained moresecurely bythechromatography of Duolite CS・10l,than by AmberliteIRC・5Cl

Maceration enzyme,thus prepared,decomposed middlelamella pectin,but not acted on citrus pectin

The author described elsewhere the action of ME on middlelame11a pectin S u m m a r y

Cl。jusineum vaI・.sikokianum produces PG and MEin the culture media小 A method was established for the sepaIation of MEin the′culture solution‖It was found thatthemiⅩedenzymesolutionwas char−

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Vol.9,Noり3(1958) 145

ged on a column ofDuoliteCS・10lof which pH valuewas adjusted to7。2and MEwas eluted from the

COlumnaddingN/lOhydrochloric acidsolutionaseluant..Thisprocedure was found to be superior to the method of AmberliteIRC・50described alreadyin partI,(1)

A⊂know】edgment

The authorwishes toexpress his sincere thanks to Prof.H.KATAGIRIOf Kyoto Univ。for his gui−

dance and encouIagementHeis alsoindebted to Mr..Y.ANABUKIOf thislaboratory for his assistance in this experiment R e f e r e n ⊂e S (1)KA7Ⅰ,A.:BuLl..Agr.Chem…Soc,Japan,20,(4)ⅩAJI,A.,ANA】∋UKI,Y.:Tech.Bull.Kaga・ 8(1956) ぴαAg㌢巾CoJJ…,7,222(1956) (2)KAJI,A..,SAILTO,H.:J‖Ferm‖Tech.Japan,(5)HIRS,C.Hn W.,STEIN,W.Hり,MooRE S.: 30,242(1952) /.βわJl.C如桝.リ200,493(ユ953) (3)KAJI,Aい,ANABUKI,Y,.:],,Agr.Chem 50C./α♪α乃,29,フプ5(1955ト 中葉ペクチン溶解酵素に関する研究 Ⅱ Duolite CS−101に.よる中葉ぺクチソ溶解酵素の分離 梶 DuoliteCS−101を使用して,Cl.juSineum varlSikokianumの発酵液中の中葉ぺクチ・y溶解酵素の分離法を確 立したり先ず,各値のpH価に理研化したDuoliteCS・ユ01のカラムを準備し,ポリガラクチ.ユ.ロナ・−ゼ,中葉ぺクチン 溶解酵素の混合酵素液を通過せしめて,両酵素の吸着状況を検討した、次に.Ⅳ/10塩酸を溶出剤として,これ等のカ ラムに吸着された酵素の溶出を行い,両酵素の溶出の羞を観察した1.こ.れ等の実験結果より,中葉ぺクチン溶解酵素 を分離する方法を次のように.決定した.発酵液を遠沈した後,DuoliteA−フを通過させて爽雑物を除去して,この通 過液を酵素原液とする、別に.Duolite CS一・l01をpHフ.2に緩衝化してカラムを作激する.このカラムの上端より.pH フ.2に調節した酵素原液をSV,4で添加し,酵素の吸着を行う.カラムを水洗後,Ⅳ/10塩酸を以て,同一・の流速で酵 素の溶出を行い,中農ぺクチッ溶解酵素のみを含有する溶出液を得るこれ等の操作はすぺて8−100Cで遂行する− DuoliteCS・10l,20mlのカラム(2.3×5,.Ccm)を使用するとき,原酵素液の添加容量ほ2(刀mlが限度であり,そ の中に含有されるポリガラクチ.ユロナ・−・ゼの全立ほ16000単位であった.添加鼠が3COmlに増加したと.きは溶出液中 に・ポリガラクチ.1ロナ・−ゼが混入した.以上述べた中英ぺクテン溶解酵素の分離方法ほ第一報に記載したAmberlite IRC・50を使用する方法より優れていた

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