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Studies on cellulolytic enzymes
AlECHIVb:S
OF
HIO(‘HKMlST11~-
AND
BIOPHYSICS
Studies III. Isolation
of
123, 307-311 (1968)
on Cellulolytic a Cellulase
Enzymes
from
Penicillium
notatum
G. PETTERSSOK Irtstit~rie
of Biochemistry;
Received
July
Gnioersiiy
of Uppsaia,
11, 1967; wcepted
B cellldolytic enzyme has been isolnted PeniciZ2iunl notatunz. The purified enzyme electrophoresis.
AiYl)
23, 19G7
The analysis was performed as described by Miller et al. (5). h’:ztracfion oj’ crude material. To 100 gm of the crude mat#erial (3)” (precipitate from culture medium of Penicillium nofatum) w,s added 500 ml of 0.1 Y pyridine acetate buffer, pH 5. The mixture I\-as stirred for 4 hours at room temperat,urc and was t#hen centrifuged at 10,OOOgfor 20 minutes. The sediment was stirred with 250 ml of t’he same buffer for 2 hours and the suspension was centrifuged again at 10,OOOgfor 20 minutes. The supernatant fluids from both centrifugatiorls were pooled (A”). Ion - erchnge ckro,llatoglaplr.y. DEAESephadex) A-25,3 500 gm, was allowed to swell over night. The adsorbent was then washed with 3 liters of 0.5 nr hydrochloric acid and 3 liters of 0.5 M sodium hydroxide, as recommended by the manufact’urer, and subsequently \I-ith about 30 liters 0.1 31 pyridine acetate, pH 5. The gel was paclwd in :L c>,lindrical scparat’ory funnel (S X 30 cm), and the buffer was passed through the bed until the pH and the electrical conductance of the effluent’ remained constant. About 30 liters of buffer UYLSrequired for equilibration. The final bed volume was 2 liters. The ccllulase extract (about 700 ml) \~ns transferred to the top of the column :rnd
RESULTS
Enzyme assay. Cellulase act,ivity ~vas estimated from t#hereducing sugars formed upon incubation of enzyme nliquot,s (20-200 ~1) If-ith 2 ml of 1% CPIZC) (carboxymethylcellulose)’ in O.O;,M sodium acetate buffer at pH 5 for 10 minutes at 40”. 1 A gift from Mr. B. K’auclCr, Uddeholm AB, Uddeholm, Sweden. The degrees of stlbstitlltioll :rrrd polymerizatiolt nerc 0.73 ;rlrd 800, rcspec*lively.
Sweden
from the cldture filtrate of the mold is homogcneorw ill polyacrylamide gel
The earlier papers (I, 2) in this series described t’he purificat,ion and chsracterization of cellulases (EC 3.2.1.4) from the basidiomycete Poly~~o~~~s uekwdo~. Two cellulolytic enzymes were detect’ed in the culture medium from t)his fungus. The two components appeared to differ in t)heir nctivities t.oward cellulose degradation products of different molecular size. One of the components was surprisingly small; the molecular weight was about 11,000. These interesting findings prompted furt,her investigations of cellulases. Since of the Polypwus reasonable quantities erlzymes could not be obt#ained \vit,hout considerable effort, \ve decided t’o study cellulase from a more convenient source. The cellulase (3, 4) (p-1 ,&glucan glucanohydrolase EC 3.2.1.4) from Penicillluw notatum was selected because large quant,it,ies of quite active culture medium arc easily available. METHODS
September
Uppsala,
2 A gift from AB a Obtaiu:rblc from Swetler 1. 307
rlstr:l, SiidertBlje, -4B Pharm:lcia,
Sweden. Upps:ll:r,
PETTERSSON
FIG. 1. Distribution of ultraviolet-absorbing material :t~ld celllllase activity afler chromatography of a cellulase preparation (material Ao) from Penicilliwn not&~m 011 DEAE-Sephadex A-25. The bulfer WIS changed from 0.1 M pyriditre-acetate (pH 5) to 0.3 M pyridille-acetate (pH 5) at, the point indicated. (0) Absorb:lncy at, 280 -rnp; (0; cellulase activity (arbitrary Iwits).
allowed to penetrat’e into the bed. Elution bed by a perist*altic pump. The column was was begun with 0.1 M pyridine acetate buffer, operated at a flow rate of 50 ml/hour. After pH 5, and the effluent was collected in 100. about 1 liter of buffer had passed through ml fra,ctions. After 1 liter of buffer, including the column, the effluent was collected in 15the sample, had passed through t,he bed, the ml fract,ions. The absorbance at ‘BO mp and column was eluted wit,h 5 liters of 0.3 1~1 the cellulase act,ivit8y of each fraction was detcrmjned. The more retarded of t,hc two pyridine acet’ate, pH 5. The a,bsorbances of the fr:&ions were activity peaks show1 in Fig. 2 contained measured at 280 mp, and t.he distribution of most of the cellulase activity present. This cellulolytic activity was det#ermined. 111 the material (AZ) was concentratBed from 470 ml chromatogram shown in Fig. 1, the second to 5.4 ml by “vacuum dialysis” in collodium tubw6 The fract,ions corresponding to the peak accounts about S5’% of the ackivity recovered. The fractions corresponding to smaller, less retarded activity peak (Fig. 2) the unretarded activity peak were added to were added to the crude material in a new anot,her portion of the crude extract and experiment’. Colunln elecf~opho~esis. Electrophoretic rechromatographed. Fractions were pooled was performed with a prcas shown in Fig. 1 (fraction Al, tlbont 1200 fractionation ml), and solid ammonium sulfate was added parative zone electrophorcsis apparatus described elsewhere (7). Sephadex C&%,7 t,o 80% saturation. The sediment obtained upon cent,rifugation at 10,OOOg for 15 fine, \vas used as the anticonvec t,ion support. The Sephadex n-as allowed to sn-ell for at minutes \ras dissolved in 75 ml of 0.05 ai least 6 hours in 0.05 M pyridine acetate pyridine-acetate, pH 5.4. G-75. buffer, pH 5.4, and was packed into the flfolecula~ sieving on Sephadez Sephadex G-75,4 fine, n-as allowed to swell externally cooled column tube (2.2 X 43 cm). for 24 hours in 0.05 M pyridine-acetate buffer, The bed Ivas mashed thoroughly with buffer, pH 5.4. The gel was packed into a 7 X 100- and 2.7 ml of sample was applied on the column. After elect,rophoresis for 21 hours cm chromatography tube of the Recywith a potent,ial drop of 400 V across the chromes type (6). The bed volume was about 4 liters. The sample n-as introduced into the electrodes and with :L current of 34 mA, the 4 From AB Pharmacix, Uppsala @wedell). 5 A column of this type is now mam~facilwcd by LKB Produkter AB, Box 70, Brommn 1, Sweden.
6 C)bt:rilled from ~lcrnl~r:~~~fil~ergesellsch:~ft Giittingell (Germally). i From .4B Pharmacia, Tppsala (Swedell).
,
FIG. 2. I’Arltiolr p:rt~ertr nftt=r gel filtrxtiolr OII Sephadcs Gi5 of fractiorl A, (rn~ctcrial :tf(el ioweschange c:hronl:rtogr:lph?-). (0) Ahsorl~:~r~cy at 280 mp; (0) cell~~lnse activity (nr t)iir:iry
rlllitsj.
FIc. :3. I)islriblltioll of prc,teiil :il~ti ccllr11:~sc activity after zotlc,-clrctro~~horesis for 21 hours of fraction I\? imnlrrinl :Iftcxr gel filtrxliolr). Hrd dimetrsions, 2.2 X 4:3 cm; bilffcr, 0.05 11 pyridille-ncct:lle iprr 5.4); Prlrrcilt , 36 mA; w)ll:tgc, 100 1.; allodic direction t,o the Icft. (0) .\l)sc,rl):lllcy 280 rn,u; (0 I c*cllill:~c activity (arbitwry llllits).
.I rialyfical gd rlectroph0wsi.s. After Mach puritiwtiotl step aliyuots ww :ui:~lyzwd hjelectrophorcais ou ~)oI\~:Lcr!-l:Lr~iidegel (S, 9). The pd\mcrix:~tion \V:IS prrfornwd ill 0.0.5 nr Tris-h~drochlo~ic :kcl MFm-, pH S. Thcb
FIG. 1. l)istributioll of protein and cellulose activity after zone ~lcctrophoresis of fraction A-\:j (material from the first electrophorcsis). Red dimetlsiolls, buffer, current, nrld voltage RS irr Fig. 3.
the columns. Electrophoresis n-as begun with a potential difference of 50 V, which provided a current strength of 2 mA per column. After 15 minutes t’he voltage was increased to 100 V for a current of approximately 4 mA per column. One hour lat’er the clectrophoresis was stopped. The gel cylinders were removed from the glass tubes and were stained for one hour in a 1 % (w/v) solution of amido black in 7 % (v/v) attic acid. Figure 5 sho\vs the patterns obkiued at different stages of the purification procedure. The crude extract contains many compouents, but the material recovered aft)er the final electrophoresis shojvs only one band.
concentratjions of acrylamide and N, N-bisacrylamide were 5 % (w/v) and 0.25% (w/v), respectively. Ammonium persulfate (0.2 %, w/v) and dimethylaminopropionitril (0.02%, w/v) were used as catalysts. Portions (1 ml) of the deaerated mixt’ure were transferred to 0.5 X 7-cm glass tubes. Gel columns were arranged as described by Ornstein and Davies (9). Ten t,o 20 ~1 of the sample was layered on the upper surface of
*
I>ISCUSHION L
The results demonstrate that, a ccllulolytic enzyme from the mold Penicillium notatum can be purified by chromatographic aud eleckophoretic met(hods. The pooled fractions arc’ homogeneous upon electrophoresis in polyacrylamide gel. The data in Table I show that, the ionexchange chromat)ography step is part)icularly effectjive. This is also evident from the gel electrophoresis patt’ern. The second electrophoresis step has no effect, on the specific activity, probably because t,he assay procedure is not precise enough to reveal the small expected increase. Icungal as well as bnctcrial cellulases have
FIG. 5. l’olyacrylamide electrophoresis illus&rating the progressive purification of a cellulase from Penidium notaturn. Samples were taken after the following purification steps: Crltde extract, (to the right), ion-exchange chromatography, gel filtration, and the second electrophoresis step (t,o the left). Allodic migration downward. TABLE
I
PURIFICATION OF A CELLULASE FROM Penicillium step
\‘Chllle
Crude extract Chromat,ography UEAESephadex A25 and cont. Gel filtration on Sephadex G75 and cont. Electrophoresis I and cont. Elect,rophoresis
692 7-i
(ml)
Total A ‘1”
CellIhW activityU
Total cellulase activity
Specific activity0
56
150,000 4,140
33 197
22,800 14,600
0.152 3.51
8
146
1,168
1,280
10,200
8.77
5 .5
161
902
1,X0
9,680
75-l
80.6
8 ) 700
109
II
‘I Arbitrary * Cellulase
A*,”
notatum
lmits/ml. :trt,ivitJ- per :1 28,).
21i
6.92
45
i6
10.7
42
71
10.7
39
66
STUI)IES
ON CELLULOLYTIC
oft.en been reported to be present in multiple forms. It is therefore of interest that’ at least in the culture filtrat’e of I’. notatuw a single molecular species seems t’o be present after the purification procedures used. This is particularly noteworthy in view of earlier findings by the present aut’hor jvhere more than one component \\-a~ observed upon gel filtration (10). Likewise, in an introductory study undertaken before the final purification scheme ~-\-as s&led, electrophoresis of the crude exkact revealed t,he presence of several active components. However, when t#he gel filtration was performed after chromat~ography on DEAE-Sephadex, about X0% of the activity was recovered in :I narrow zone (see Fig. 2). These findings suggest’ t’hat t,he enzyme might be bound to inactive substances, such as prot,eins or carbohydrates, in the crude extract. If so, the cnzymically active components seem to be released in the adsorption step. The next paper in this series will deal \vith the chemical and physical characterizat’ion of this enzyme. ACKNOWLEDGMENTS I wish to thank my teacher Dr. J. Porath for mnlly stimulating discussions alld for this vnlu-
ENZYMES.
III
311
able criticism. I am also indebted to Professor A. Tiselilts for his kind interest in this work. For skilful technical assistalIce I want, to thank Miss Ulln-Bril t Asiii. This work was supported by a grant from the Swedish Natlu-al Bcieuce Research Comlcil. REFERENCES 1. PETTERSSON, (i., Cow~rw, IC. B., .INO PORATH, J., Niochim. Biophp. dcin 67, 1. (1963). 2. PETTERSSOS, (i., .\KI) PUR.\TH. .J., fZiochiru. Miophys. zlc/a 67, 9 (1963). 3. ERIKSSON, FREDR~K \-., .\SW LINI)V.\ I.,,, SX-EX, .I. Pharmacy Pharmacol. 11, 747 (1959j. 1. LINDVALL, SVEN, AND ERIKSSON, FREDRIK \-., J. Pharmacy Pharmacol. 11, 756 (1959). 5. MILLER, (;. L., BLUM, R., C~LENNON, W. IS., AND BURTON, A. L., Anal. Hiochevr. 2, 127 (1960). (i. POR.ZTH, J., .\NI) BENNICH, H., ~lrch. Bioche~. Hiophys. S7rppl. 1, 152 (1962). i. PORATH, J., .\KD T~JERT~X, S.,in Xc/hods biochern. =Inal. 9, 191 (1962). 8. HJERTI~N, S., JERSTEDT, S., AXD TISEI,IUR, A., Anal. Biochem. 11, 219 (1965). 9. ORNSTEIN, L., .au l).\vrs, B. J. “ljisc Electrophorcsis,” Preprinted prior to jorlrllal pllblicntioll by l>istillatiol~ Prodllcts IIIdllstries, Rorhest,er, New York (19(i2). Hioph!ys. .Ic/a 77, 10. PETTERSSON, (i., fjiochinl. tic,5 (19633.
OF
HIO(‘HKMlST11~-
AND
BIOPHYSICS
Studies III. Isolation
of
123, 307-311 (1968)
on Cellulolytic a Cellulase
Enzymes
from
Penicillium
notatum
G. PETTERSSOK Irtstit~rie
of Biochemistry;
Received
July
Gnioersiiy
of Uppsaia,
11, 1967; wcepted
B cellldolytic enzyme has been isolnted PeniciZ2iunl notatunz. The purified enzyme electrophoresis.
AiYl)
23, 19G7
The analysis was performed as described by Miller et al. (5). h’:ztracfion oj’ crude material. To 100 gm of the crude mat#erial (3)” (precipitate from culture medium of Penicillium nofatum) w,s added 500 ml of 0.1 Y pyridine acetate buffer, pH 5. The mixture I\-as stirred for 4 hours at room temperat,urc and was t#hen centrifuged at 10,OOOgfor 20 minutes. The sediment was stirred with 250 ml of t’he same buffer for 2 hours and the suspension was centrifuged again at 10,OOOgfor 20 minutes. The supernatant fluids from both centrifugatiorls were pooled (A”). Ion - erchnge ckro,llatoglaplr.y. DEAESephadex) A-25,3 500 gm, was allowed to swell over night. The adsorbent was then washed with 3 liters of 0.5 nr hydrochloric acid and 3 liters of 0.5 M sodium hydroxide, as recommended by the manufact’urer, and subsequently \I-ith about 30 liters 0.1 31 pyridine acetate, pH 5. The gel was paclwd in :L c>,lindrical scparat’ory funnel (S X 30 cm), and the buffer was passed through the bed until the pH and the electrical conductance of the effluent’ remained constant. About 30 liters of buffer UYLSrequired for equilibration. The final bed volume was 2 liters. The ccllulase extract (about 700 ml) \~ns transferred to the top of the column :rnd
RESULTS
Enzyme assay. Cellulase act,ivity ~vas estimated from t#hereducing sugars formed upon incubation of enzyme nliquot,s (20-200 ~1) If-ith 2 ml of 1% CPIZC) (carboxymethylcellulose)’ in O.O;,M sodium acetate buffer at pH 5 for 10 minutes at 40”. 1 A gift from Mr. B. K’auclCr, Uddeholm AB, Uddeholm, Sweden. The degrees of stlbstitlltioll :rrrd polymerizatiolt nerc 0.73 ;rlrd 800, rcspec*lively.
Sweden
from the cldture filtrate of the mold is homogcneorw ill polyacrylamide gel
The earlier papers (I, 2) in this series described t’he purificat,ion and chsracterization of cellulases (EC 3.2.1.4) from the basidiomycete Poly~~o~~~s uekwdo~. Two cellulolytic enzymes were detect’ed in the culture medium from t)his fungus. The two components appeared to differ in t)heir nctivities t.oward cellulose degradation products of different molecular size. One of the components was surprisingly small; the molecular weight was about 11,000. These interesting findings prompted furt,her investigations of cellulases. Since of the Polypwus reasonable quantities erlzymes could not be obt#ained \vit,hout considerable effort, \ve decided t’o study cellulase from a more convenient source. The cellulase (3, 4) (p-1 ,&glucan glucanohydrolase EC 3.2.1.4) from Penicillluw notatum was selected because large quant,it,ies of quite active culture medium arc easily available. METHODS
September
Uppsala,
2 A gift from AB a Obtaiu:rblc from Swetler 1. 307
rlstr:l, SiidertBlje, -4B Pharm:lcia,
Sweden. Upps:ll:r,
PETTERSSON
FIG. 1. Distribution of ultraviolet-absorbing material :t~ld celllllase activity afler chromatography of a cellulase preparation (material Ao) from Penicilliwn not&~m 011 DEAE-Sephadex A-25. The bulfer WIS changed from 0.1 M pyriditre-acetate (pH 5) to 0.3 M pyridille-acetate (pH 5) at, the point indicated. (0) Absorb:lncy at, 280 -rnp; (0; cellulase activity (arbitrary Iwits).
allowed to penetrat’e into the bed. Elution bed by a perist*altic pump. The column was was begun with 0.1 M pyridine acetate buffer, operated at a flow rate of 50 ml/hour. After pH 5, and the effluent was collected in 100. about 1 liter of buffer had passed through ml fra,ctions. After 1 liter of buffer, including the column, the effluent was collected in 15the sample, had passed through t,he bed, the ml fract,ions. The absorbance at ‘BO mp and column was eluted wit,h 5 liters of 0.3 1~1 the cellulase act,ivit8y of each fraction was detcrmjned. The more retarded of t,hc two pyridine acet’ate, pH 5. The a,bsorbances of the fr:&ions were activity peaks show1 in Fig. 2 contained measured at 280 mp, and t.he distribution of most of the cellulase activity present. This cellulolytic activity was det#ermined. 111 the material (AZ) was concentratBed from 470 ml chromatogram shown in Fig. 1, the second to 5.4 ml by “vacuum dialysis” in collodium tubw6 The fract,ions corresponding to the peak accounts about S5’% of the ackivity recovered. The fractions corresponding to smaller, less retarded activity peak (Fig. 2) the unretarded activity peak were added to were added to the crude material in a new anot,her portion of the crude extract and experiment’. Colunln elecf~opho~esis. Electrophoretic rechromatographed. Fractions were pooled was performed with a prcas shown in Fig. 1 (fraction Al, tlbont 1200 fractionation ml), and solid ammonium sulfate was added parative zone electrophorcsis apparatus described elsewhere (7). Sephadex C&%,7 t,o 80% saturation. The sediment obtained upon cent,rifugation at 10,OOOg for 15 fine, \vas used as the anticonvec t,ion support. The Sephadex n-as allowed to sn-ell for at minutes \ras dissolved in 75 ml of 0.05 ai least 6 hours in 0.05 M pyridine acetate pyridine-acetate, pH 5.4. G-75. buffer, pH 5.4, and was packed into the flfolecula~ sieving on Sephadez Sephadex G-75,4 fine, n-as allowed to swell externally cooled column tube (2.2 X 43 cm). for 24 hours in 0.05 M pyridine-acetate buffer, The bed Ivas mashed thoroughly with buffer, pH 5.4. The gel was packed into a 7 X 100- and 2.7 ml of sample was applied on the column. After elect,rophoresis for 21 hours cm chromatography tube of the Recywith a potent,ial drop of 400 V across the chromes type (6). The bed volume was about 4 liters. The sample n-as introduced into the electrodes and with :L current of 34 mA, the 4 From AB Pharmacix, Uppsala @wedell). 5 A column of this type is now mam~facilwcd by LKB Produkter AB, Box 70, Brommn 1, Sweden.
6 C)bt:rilled from ~lcrnl~r:~~~fil~ergesellsch:~ft Giittingell (Germally). i From .4B Pharmacia, Tppsala (Swedell).
,
FIG. 2. I’Arltiolr p:rt~ertr nftt=r gel filtrxtiolr OII Sephadcs Gi5 of fractiorl A, (rn~ctcrial :tf(el ioweschange c:hronl:rtogr:lph?-). (0) Ahsorl~:~r~cy at 280 mp; (0) cell~~lnse activity (nr t)iir:iry
rlllitsj.
FIc. :3. I)islriblltioll of prc,teiil :il~ti ccllr11:~sc activity after zotlc,-clrctro~~horesis for 21 hours of fraction I\? imnlrrinl :Iftcxr gel filtrxliolr). Hrd dimetrsions, 2.2 X 4:3 cm; bilffcr, 0.05 11 pyridille-ncct:lle iprr 5.4); Prlrrcilt , 36 mA; w)ll:tgc, 100 1.; allodic direction t,o the Icft. (0) .\l)sc,rl):lllcy 280 rn,u; (0 I c*cllill:~c activity (arbitwry llllits).
.I rialyfical gd rlectroph0wsi.s. After Mach puritiwtiotl step aliyuots ww :ui:~lyzwd hjelectrophorcais ou ~)oI\~:Lcr!-l:Lr~iidegel (S, 9). The pd\mcrix:~tion \V:IS prrfornwd ill 0.0.5 nr Tris-h~drochlo~ic :kcl MFm-, pH S. Thcb
FIG. 1. l)istributioll of protein and cellulose activity after zone ~lcctrophoresis of fraction A-\:j (material from the first electrophorcsis). Red dimetlsiolls, buffer, current, nrld voltage RS irr Fig. 3.
the columns. Electrophoresis n-as begun with a potential difference of 50 V, which provided a current strength of 2 mA per column. After 15 minutes t’he voltage was increased to 100 V for a current of approximately 4 mA per column. One hour lat’er the clectrophoresis was stopped. The gel cylinders were removed from the glass tubes and were stained for one hour in a 1 % (w/v) solution of amido black in 7 % (v/v) attic acid. Figure 5 sho\vs the patterns obkiued at different stages of the purification procedure. The crude extract contains many compouents, but the material recovered aft)er the final electrophoresis shojvs only one band.
concentratjions of acrylamide and N, N-bisacrylamide were 5 % (w/v) and 0.25% (w/v), respectively. Ammonium persulfate (0.2 %, w/v) and dimethylaminopropionitril (0.02%, w/v) were used as catalysts. Portions (1 ml) of the deaerated mixt’ure were transferred to 0.5 X 7-cm glass tubes. Gel columns were arranged as described by Ornstein and Davies (9). Ten t,o 20 ~1 of the sample was layered on the upper surface of
*
I>ISCUSHION L
The results demonstrate that, a ccllulolytic enzyme from the mold Penicillium notatum can be purified by chromatographic aud eleckophoretic met(hods. The pooled fractions arc’ homogeneous upon electrophoresis in polyacrylamide gel. The data in Table I show that, the ionexchange chromat)ography step is part)icularly effectjive. This is also evident from the gel electrophoresis patt’ern. The second electrophoresis step has no effect, on the specific activity, probably because t,he assay procedure is not precise enough to reveal the small expected increase. Icungal as well as bnctcrial cellulases have
FIG. 5. l’olyacrylamide electrophoresis illus&rating the progressive purification of a cellulase from Penidium notaturn. Samples were taken after the following purification steps: Crltde extract, (to the right), ion-exchange chromatography, gel filtration, and the second electrophoresis step (t,o the left). Allodic migration downward. TABLE
I
PURIFICATION OF A CELLULASE FROM Penicillium step
\‘Chllle
Crude extract Chromat,ography UEAESephadex A25 and cont. Gel filtration on Sephadex G75 and cont. Electrophoresis I and cont. Elect,rophoresis
692 7-i
(ml)
Total A ‘1”
CellIhW activityU
Total cellulase activity
Specific activity0
56
150,000 4,140
33 197
22,800 14,600
0.152 3.51
8
146
1,168
1,280
10,200
8.77
5 .5
161
902
1,X0
9,680
75-l
80.6
8 ) 700
109
II
‘I Arbitrary * Cellulase
A*,”
notatum
lmits/ml. :trt,ivitJ- per :1 28,).
21i
6.92
45
i6
10.7
42
71
10.7
39
66
STUI)IES
ON CELLULOLYTIC
oft.en been reported to be present in multiple forms. It is therefore of interest that’ at least in the culture filtrat’e of I’. notatuw a single molecular species seems t’o be present after the purification procedures used. This is particularly noteworthy in view of earlier findings by the present aut’hor jvhere more than one component \\-a~ observed upon gel filtration (10). Likewise, in an introductory study undertaken before the final purification scheme ~-\-as s&led, electrophoresis of the crude exkact revealed t,he presence of several active components. However, when t#he gel filtration was performed after chromat~ography on DEAE-Sephadex, about X0% of the activity was recovered in :I narrow zone (see Fig. 2). These findings suggest’ t’hat t,he enzyme might be bound to inactive substances, such as prot,eins or carbohydrates, in the crude extract. If so, the cnzymically active components seem to be released in the adsorption step. The next paper in this series will deal \vith the chemical and physical characterizat’ion of this enzyme. ACKNOWLEDGMENTS I wish to thank my teacher Dr. J. Porath for mnlly stimulating discussions alld for this vnlu-
ENZYMES.
III
311
able criticism. I am also indebted to Professor A. Tiselilts for his kind interest in this work. For skilful technical assistalIce I want, to thank Miss Ulln-Bril t Asiii. This work was supported by a grant from the Swedish Natlu-al Bcieuce Research Comlcil. REFERENCES 1. PETTERSSON, (i., Cow~rw, IC. B., .INO PORATH, J., Niochim. Biophp. dcin 67, 1. (1963). 2. PETTERSSOS, (i., .\KI) PUR.\TH. .J., fZiochiru. Miophys. zlc/a 67, 9 (1963). 3. ERIKSSON, FREDR~K \-., .\SW LINI)V.\ I.,,, SX-EX, .I. Pharmacy Pharmacol. 11, 747 (1959j. 1. LINDVALL, SVEN, AND ERIKSSON, FREDRIK \-., J. Pharmacy Pharmacol. 11, 756 (1959). 5. MILLER, (;. L., BLUM, R., C~LENNON, W. IS., AND BURTON, A. L., Anal. Hiochevr. 2, 127 (1960). (i. POR.ZTH, J., .\NI) BENNICH, H., ~lrch. Bioche~. Hiophys. S7rppl. 1, 152 (1962). i. PORATH, J., .\KD T~JERT~X, S.,in Xc/hods biochern. =Inal. 9, 191 (1962). 8. HJERTI~N, S., JERSTEDT, S., AXD TISEI,IUR, A., Anal. Biochem. 11, 219 (1965). 9. ORNSTEIN, L., .au l).\vrs, B. J. “ljisc Electrophorcsis,” Preprinted prior to jorlrllal pllblicntioll by l>istillatiol~ Prodllcts IIIdllstries, Rorhest,er, New York (19(i2). Hioph!ys. .Ic/a 77, 10. PETTERSSON, (i., fjiochinl. tic,5 (19633.