Biochimica et BiophvsicaActa 840 (1985) 255-263 255 Elsevier

BBA22061

Purif icat ion of endo- ( l ~ 3)-~8-D-glucanases iysing yeast cell walls from

R h i z o c t o n i a s o l a n i

T a i c h i Usu i , K a z u h i d e T o t a n i , A t s u s h i T o t s u k a a n d M a s a o O g u c h i

Department of Agricultural Chemistry, College of Agriculture, Shizuoka UniL,ersio'. Oh!'a 836. Shi=uoka (Japan)

(Received August 21st, 1984) (Revised manuscript received February 19th, 1985)

Key words: Endoglucanase; Cell wall lysis; (Rhizoctonia)

Three forms of endo-(l ---> 3)-fl-D-glucanases lysing yeast cell walls from Rhizoctonia solani were separated by precipitation with ammonium sulfate and by successive chromatographies on CM Bio-Gel A and Bio-Gel P-60 or P-30, and were finally purified by substrate affinity, chromatography on short-chain pachyman-AH- Sepharose CL 6B column. Each preparation was found to be homogeneous on gel filtration and by electrophoresis on acrylamide gel with sodium dodecyl sulfate. They exhibit high activity against insoluble pachyman, but only restricted activity against soluble short-chain pachyman. In the affinity chromatography, three enzymes were found to be strongly adsorbed on the column, so that they could be easily eluted with substrate solution using biospecific counter-ligand. It was thus revealed that covalent binding of such a soluble glucan to aminohexyI-Sepharose provides a useful carrier for separation of endo-(l ~ 3)-fl-D- glucanases lysing yeast cell walls.

Introduction

Purification of (1 -~ 3)-fl-D-glucanase has been of great interest because of their action on yeast cell walls and a very useful ability for studies on the structure. Because isolated cell walls can be solubilized almost entirely by a purified (1 --* 3)-/3- D-glucanase [1-4]. The use of specific highly puri- fied glucanase is, therefore, important in order to clarify the hydrolytic action on the yeast cell wall. In most cases, however, the major objective of the researcher was to apply the crude glucanase solu- tions to cell suspensions in order to prepare proto- plasts rather than for the analysis of cell wall structure.

Considerable difficulties have been encountered in obtaining highly purified lyric (1 ~ 3)-fl-D- glucanase devoid of interfering activities such as nonlytic (1 ~ 3)-fl-D-glucanase, (1 ~ 6)-fl-D- glucanase by affinity chromatography have either

been done with the use of water-insoluble polysac- charides consisting mainly of (1 ---, 3)-fl-linked D- glucose such as pachyman from Poria cocos [5] or cell wall glucan of Candida utilis [6], or glycopro- tein from the cell walls of baker's yeast attached to Sepharose [7]. However, in these experiments, the immobilization of the glucan on a matrix gel has been restricted in use because of highly insoluble polysaccharides. Recently, it has been reported that a proteinase-free (1 -~ 3)-fl-D-glucanase from Arthrobacter sp. has been purified by affinity chro- matography on a2-macroglobulin-Sepharose [4], We have already reported that the use of water- soluble (1 ~ 3)-fl-D-glucan immobilized on Se- pharose is a useful method for separation of the exo- and endo-(1 --* 3)fl-D-glucanases from Rhizoctonia solani and of the exo(1--* 3)-fl-D- glucanase from Basidiomycetes sp. QM 806 [8]. However, it does not lyse yeast cells. Further in- vestigation was carried out to determine whether

0304-4165/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

256

such chemically degraded glucan covalently bound to an insoluble carrier serves as a biospecific ligand for separation of (1 ~ 3)-fl-D-glucanase lysing yeast cell walls or not.

The present paper describes a procedure incor- porating affinity chromatography on soluble (1 -* 3)-]3-D-glucan-aminohexyl-Sepharose for the puri- fication of endo-(1--* 3)-]3-o-glucanases lysing yeast cell walls as well as the nonlytic glucanases, from an enzyme mixture present in the culture filtrates of Rhizoctonia solani.

Material and Methods

Enzyme and substrates A commercially available yeast cell lytic enzyme

preparation, Kitalase (Kumiai Chemicals Co.) pre- pared from the culture filtrates of Rhizoctonia solani [9], was used as the source of lyric and nonlytic (1--* 3)-fl-D-glucanases. Kitalase (12 g) was suspended in 60 ml of 50 mM acetate buffer at pH 5.5 with stirring for 1 h, and insoluble material was removed by centrifugation. To the solution, solid ammonium sulfate was added to give 80% saturation. After centrifugation of the precipitate formed, it was dissolved in 50 ml of 10 mM acetate buffer at pH 4.5 and then dialyzed against 10 1 of the same buffer. The crude enzyme solution was subjected to purification in order to obtain (1 ---, 3)-/~-D-glucanases.

Pachyman was prepared from air-dried Bukuryo (Poria cocos) by the method of Saito et al. [10]. Short-chain pachyman (average degree of poly- merization of 20) was prepared by formic acid treatment of the pachyman [11]. Cibachron blue pachyman was prepared according to the method of Philpott and Chapman [12]. All other substrates were obtained from commercial sources.

Enzyme assays Assay A. The mixture containing 1.0 mg of

short-chain pachyman in 0.1 ml, 50 mM acetate buffer (pH 5.5) and enzyme solution in a total volume of 1.0 ml was incubated at 40°C for 20 rain. The amount of reducing activity produced was determined by the Somogyi-Nelson method [13,14]. 1 unit of activity was defined as that amount of enzyme liberating 1 /~mol of reducing

sugar equivalent as glucose, per min under the assay conditions.

Assay B. The assay was similar to assay A except for the substrate (0.5% pachyman) and the incubation time (30 min). A definition of the activ- ity was the same with that of assay A.

Assay C. Lytic activity was assayed with lyophilized preparation of Baker's viable yeast (Oriental Yeast Co.) according to Kobayashi et al. [9]. 1 unit was defined as the amount which caused 20% decrease of the absorbance at 660 nm of the cell suspension, per 1 h under the standard assay conditions.

Assay D. Viable yeast cells of Saccharomyces cerevisiae IFO 0259 in the stationary growth phase were used as a substrate and the percentage de- crease in the turbidity was determined by the same reaction system with assay C. Yeast cells at sta- tionary growth phase were prepared as follows. The yeast was cultured using a medium containing 4 g glucose, 1 g peptone, and tap water 100 ml (sterilized at 120°C for 20 rain) at 30°C for 40 h in the flasks by shaking. After cultivation, the yeast was collected by centrifugation (4200 × g for 10 min) and it was washed two times with cold saline solution (0.8% sodium chloride).

Proteinase was assayed with casein as a sub- strate as previously described [9]. 1 unit of pro- teinase activity was defined as the amount of the enzyme liberating into supernatant 1 /~mol of tyrosine equivalent per 1 h. (1 --* 6)-/~-D-Glucanase and /~-glucosidase activities were determined using pustulan (ex. P. papullosa, Calbiochem) and sali- cine as substrates, respectively, according to the procedure of assay A. Cellulase activity was made with carboxymethyl-cellulose and Avicel as sub- strates as previously described [15].

Protein determination Protein was measured by the method of Lowry

et al. [16] with bovine serum albumin as a stan- dard. For the routine assay of column fractions, protein was estimated from the extinction at 280 r i m .

Gel electrophoresis on sodium dodecyl sulfate (SDS) To examine the homogeneity of the purified

enzyme preparations, SDS-polyacrylamide gel electrophoresis was carried out according to the

procedure of Laemmli with discontinuous buffer system [17].

The molecular weight of the purified enzyme was estimated by SDS polyacrylamide slab gel electrophoresis according to the procedure of Weber and Osborn with continuous buffer system [18]. MW-Marker (Oriental Yeast Co.) containing an oligomer series of cytochrome c was used as molecular weight standards.

Molecular weight estimation of gel filtration The molecular weight of the enzyme was also

estimated by the gel filtration [19]. A 1.5 by 85 cm column of Bio-Gel P-60 was calibrated with each of 2 mg/0.5 ml solutions of bovine serum al- bumin, egg albumin, chymotrypsinogen A and cy- tochrome c, respectively, in 50 mM acetate buffer (pH 5.5). The void volume was determined with a similar solution of Blue dextran 2000.

Hydrolysis of glucans The relative rates of attack of the purified lyric

enzyme on glucans were determined by incubating 0.25% solutions of substrates in 50 mM acetate buffer (pH 5.5) with 0.5 units (assay C) per ml of enzyme. The rate on pachyman was arbitrarily set at 100. The relative rates of the nonlytic enzyme on glucans compared to short-chain pachyman at 100 were also determined with 1 unit (assay A) per ml of enzyme under the conditions of assay A. These comparisons are based on the assumption that the rate was first order with respect to enzyme throughout the course of the determination.

The action patterns of the purified lytic en- zymes toward pachyman were analyzed as follows: Pachyman (10 mg) was digested with each 5 units of L-l, L-2 and L-4 in a final volume of 1.0 ml of 50 mM acetate buffer (pH 5.5) at 40°C, and aliquots were removed at 0.5, 1 and 48 h for paper chromatography and gel-permeation chromatogra- phy. Paper chromatography was performed on Toyo No 50 filter paper by the ascending method at 50°C with the following solvent system (v/v): (A) n-butanol/pyridine/water (6 : 4 : 3), and (B) n-propanol /e thyl aceta te /water (7 : 1 : 2). Sugars were detected by the alkaline silver nitrate reagent [19]. Glucose, 1,3-fl-glucooligosaccharides (degree of polymerization, 2-7) [20], 1,6-fl-glucooligosac- charides (degree of polymerization, 2-4) [21] were

257

used as authentic sugars. Gel filtration was per- formed on a Bio-Gel P-2 column (1.5 × 95 cm). Fractions of 2 ml were collected and each fraction was assayed for the total sugar content by the phenol-sulfuric acid method [22].

Results and Discussion

Purification of lytic and nonlytic (1 ~ 3)-fl-D- glucanases

All operations were prepared at 4°C unless otherwise stated.

CM Bio-Gel A ion-exchange chromatography. The desalted enzyme solution obtained by solid ammonium sulfate precipitation was directly ap- plied to a CM Bio-Gel A column equilibrated with 10 mM acetate buffer at pH 4.5, washed with same buffer, and eluted with a linear gradient of 0-0.1 M NaC1 as shown in Fig. 1. The fraction showing lyric activity with assay C was designated L (lytic enzyme). On the other hand, the fraction showing the (1--, 3)-fl-D-glucanase activity with assay A but not showing the lytic activity with assay C was designated N (nonlytic enzyme). The elution pat- tern showed the enzyme mixture to fractionate into four highly lytic peaks and two highly non- lytic peaks; highly lytic activity (L-l) with a low level of nonlytic enzyme firstly emerged from the column a little behind the major protein peak during washing with several volumes of the start- ing buffer. More tightly bound other enzyme frac- tions N-l, L-2, N-2, L-3 and L-4 were eluted from the column by a salt gradient in buffer, according to the order of elution. Eluates of the correspond- ing lytic and nonlytic fractions were combined in each case and concentrated to low volume (4-5 ml) using an Amicon Diaflo unit equipped with a YM-5 membrane operating at 50 lbs / in 2 pressure, and then dialyzed overnight against 10 1 of 50 mM acetate buffer at pH 5.5 (buffer A).

Bio-Gel P-60 or P-30 chromatography. A portion of each of the four enzyme fractions (L-l, L-2, N-1 and N-2) obtained from the CM Bio-Gel A col- umn was loaded onto a Bio-Gel P-60 column, equilibrated with buffer A. Elution was performed with the same buffer as shown in Fig. 2. Gel filtration of the L-1 fraction resulted in complete separation of lyric and nonlytic enzymes. In the case of fraction L-2 with contaminations of frac-

258

10-

E

S a l

4_

?

S O-

N-1 L-2 I I I

10 30

L

, t i

50 70 90

Tube no, (20 ml each) - - j ~ Tube no (10 ml each)

L -3 L -4

C 110 130

6O

40

b • 20

Fig. 1. CM Bio-Gel'A chromatography of yeast cell-lytic activity (L) and short-chain pachyman degrading activity (N) present in the culture filtrates from Rhizoctonia solani. The enzyme solution (50 ml with a fraction L activity of 68 950 U and a fraction N activity of 20 500 U) was placed on a column (2.6 x 19 cm) of CM Bio-Gel A. The column was washed with 1 1 of equilibration buffer at a flow rate of 20 m[/h. Elution was done with a linear gradient from 0 (500 ml) to 0.1 M NaC1 (500 ml) in equilibration buffer (the arrow shows the position of the buffer change). Fractions were analyzed for protein A2~ o ( ) and for lytic activity (L, e) on lyophilized yeast cells and (1 ~ 3)-/3-D- glucanase (N. ©) on short-chain pachyman.

tions N-1 and N-2 as evident from Fig. 1, a peak showing highly lytic activity was completely sep- arated from the N-1 contaminant peak, but it overlapped the small N-2 contaminant peak. These contaminant peaks certainly coincide in the rela- tive elution positions of the corresponding peaks of fractions N-1 and N-2 (Fig. 2C and D). Por- tions of fractions L-3 and L-4 were also subjected to a Bio-Gel P-30 column, as shown in Fig. 3. A small overlap of fractions L-3 and L-4 is evident f rom Fig. h F rom compar ison of the two elution profiles, it was evident that the L-3 and L-4 peaks were separated from each other on the column, albeit not completely. In this case, the activity obtained with assay B instead of assay A was used for monitoring the (1--* 3)-fl-D-glucanase, since neither lytic peak showed the activity of assay A. Thus, combinat ion of assays B and C is useful to distinguish between fraction L-4 showing glucanase activity and fraction L-3 not showing the activity.

A f f i n i t y c h r o m a t o g r a p h y . Each enzyme solution partially purified from the Bio-Gel P-60 or P-30 was directly loaded onto a column of short-chain pachyman-aminohexyl-Sepharose CL 6B, equi- librated with buffer A containing 1 M NaC1. The affinity gel was prepared according to our previ- ous report [8]. Elution was done as shown in Fig. 4. With respect to fractions L-1 and L-2, we found that none of lyric activity was eluted when the column was sequentially washed with high and low

salt solutions. Under such conditions it was strongly adsorbed on the column, but could be cleanly eluted with biospecific counterligands. In the case of the L-2 fraction with a low level of N-2 contaminant as ment ioned above, elution with the low salt solution was effective for the removal of the contaminant . This finding was supported by the affinity behavior of the N-2 fraction. In frac- tion L-4, the elution behavior of most of the lyric activity was the same with those of L-1 and L-2 enzyme activities, al though a low level of lyric activity (probably due to L-3 contaminant) was firstly washed with high salt solution. From these results, it should be noted that nonlytic enzyme or proteinase contaminant inseparable on CM Bio- Gel A and Bio-Gel P-60 or P-30 chromatographies could be removed completely from these fractions by this step. Furthermore, the lytic enzyme frac- tions L-l , L-2 and L-4 obtained were regarded as lytic (1 ~ 3)-/~-D-glucanase, since they also exhibit the pachyman degrading activity with assay B. On the other hand, most of the lyric activity of L-3 fraction together with very high proteinase activity showed no detectable affinity for the gel and was recovered by elution with high salt solution. The nonadsorbed fraction did not show the activity with assay B as expected from Fig. 3A. Therefore, this lytic enzyme should be distinguished from the lytic (1 ~ 3)-/%D-glucanases. Further studies of L-3 with such high proteinase activity will be reported

4 -

2

0

4 - E :D 2-

~n c ° O- O u

2 C~ i

n 7 0 - 4_ 03

l 3 5 -

Y, - - O -

Z 10-

5"

A

L _ ¸ |

04~ ~ ~ ~

2 -

1.-

2

O f l "

0 5

O - 0 - ~ ' 4 0

L - 2 I I

C

N - 2

D

J 8 0 120

E l u t i o n v o l u m e ( m l )

40

20

O

4 0

2 0

0

*6 O

1 0 u

5 -1

,O

- 2 0

-10

- 0

Fig. 2. Bio-Gel P-60 chromatography of fractions L-l, L-2, N-1 and N-2 obtained from CM Bio-Gel A. The enzyme solution (2 ml) was placed on a column (1.5 × 85 cm) of Bio-Gel P-60 at a flow rate of 10 ml /h . A, B, C and D are elution profiles of fractions L-1 (an L activity of 1000 U), L-2 (an L activity of 1700 U), N-1 (an N activity of 500 U) and N-2 (an N activity of 130 U), respectively. The void volume was determined with Blue Dextran 2000.

259

C © a0 O4

O '

u

/21

JD

0.SH

oJ

L - 3 ! !

A

L- P

4 0 80 120

E lu t i on v o l u m e ( m l )

.10( - 2

E

• 50 -1

>_

0 -O

721 u (3 E

• 10( u -15 >,

g 0_

'50 -7.5

0 -0

Fig. 3. Bio-Gel P-30 chromatography of L-3 and L-4 fractions obtained from CM Bio-Gel A. Conditions were the same as those for Fig. 2. A and B are the elution profiles of fractions L-3 (an L activity of 3400 U) and L-4 (an activity of 1000 U), respectively, C3, pachyman degrading activity.

in a future paper. In nonlytic enzyme fractions, the chromatographic behavior of fraction N-I resem- bled those of fractions L-l , L-2 and L-4, but that of fraction N-2 was distinguishable by the bio- specific nature of the adsorption of other glucanases, as expected from our previous report [81.

Thus, the eluates forming the corresponding enzyme peak were cobmined in each case and concentrated to a low volume (1-2 ml) using an Amicon Diaflo unit as mentioned above. The en- zyme solutions other than fraction N-2 were again subjected to a Bio-Gel P-60 column (L-l, L-2 and N - l ) or a Bio-Gel P-30 column (E-4) according to the same procedure used for enzyme purification, in order to remove short-chain pachyman con- tained in the eluant. Thus, enzyme preparations (L-l , L-2, L-4, N-1 and N-2) purified from affinity chromatography were completely devoid of other activities, such as those of (1 --* 6)-/~-D-glucanase, cellulase and fl-glucosidase. Each enzyme also gave

260

a single band on SDS-gel electrophoresis, as shown in Fig. 5, and was found to be homogeneous on gel filtration of Bio-Gel P-60 or P-30. Molecular weights were estimated by electrophoresis on SDS

0 2- D4 10 2 (1) (2)

O- 0 ~,,I ; -- ' -- ' 0 -0 i . - I4

4-_ 04 ~ B j ~ 20 _-8

2- 02 (1) (2) 10 -4

O. - . . . . . . . 0 . 0

100- 08 40 -4 -~ C

-

50- 0.,4 20 -2 (1) "2; u °

- ~, - m

o- o~ --_-~_.*. o -~. -o

A 20 ~ -4 20- o D ~ ~ _ ~_ ~.o ~ ~_.

10- 10 u -2 H

- g

0- O- .0 0

-4 -40

03- E ~ " 02- ) ~ .2 -20

f ~ (1) (2 - 0 1 -

O- 0 * ~r ' 0 -0

20- 04- ~ -4 -5.0

10 02- -2 25

( ~

0 O- -- 0 -0 20 60 100 120

Elution volume (mJ)

Fig. 4. Short-chain pachyman-aminohexyl-Sepharose CL 6B chromatography of lyric and nonlytic enzyme fractions par- tially purified by gel filtration. The column was washed with several volumes of buffer A containing 1 M NaC1 at a flow rate of 10 ml /h . Elution solutions were (1) buffer A; (2) 0.2% short-chain pachyman in buffer A. A - F are elution profiles of fractions L-1 (18 ml with a lyric activity of 370 U), L-2 (20 ml with a lyric activity of 800 U), L-3 (20 ml with a l!¢tic activity of 1200 U), L-4 (18 ml with a lyric activity of 350 U), N-1 (24 ml with a nonlytic activity of 420 U) and N-2 (18 ml with a nonlytic activity of 100 U), respectively. Fractions were analyzed for proteinase activity zx on casein.

O r i g i n

~ i , 4 [ l ! I D

- F r o n t

, , . , i Z Z -J -J

Fig. 5. Polyacrylamide gel electrophoresis of purified lytic and nonlytic (1---, 3)-,8-D-glucanases. Lanes 1 5 are enzymes of fractions N-l, N-2, L-I. L-2 and L-4, respectively. Purified enzyme preparations were electrophoresed in 12.5% poly- acrylamide gel in the presence of 0.1% SDS. Protein bands were detected with Coomassie Brilliant Blue R-250.

gels and by Bio-Gel P-60 gel filtration. SDS-gel electrophoresis gave values of 22000 for fraction L-l, 23000 for L-2, 20000 for L-4, 70000 for N-1 and 29000 for N-2; gel filtration gave values of 16000 for fraction L-l, 15000 for L-2, 12500 for L-4, 58000 for N-1 and 18500 for N-2. This indicates that these enzymes do not possess a subunit structure.

A summary of the purification procedures of purified lytic (1 ~ 3)-/~-D-glucanases is presented in Table I. It was difficult to compare specific activity with assay C and that with D on enzyme fractions during the fractionation procedure due to possible synergism. It is thought that the process of lysis of yeast cells requires involvement of more enzymes than just lytic glucanases. Some re- searchers have shown that the lytic activity of microbial glucanases on viable yeast cells is greatly dependent on the presence of proteinases [3,23,24]. Jeffries and Macmillan have also reported that purified lytic and nonlytic (1 ~ 3)-,8-D-glucanases from Oerskovia act synergistically to hydrolyze the walls of viable yeast [25]. Our studies on the

T A B L E I

P U R I F I C A T I O N O F T H R E E LYTIC ( l -~ 3 ) - f l -D-GLUCANASES

Three enzymes were prepared from 12 g of Kitalase: a by assay method C: b by assay method D; ~ by assay method B.

261

Step Protein Lysis " Specific activity (1 ~ 3)-fl-o-

(mg) (U) lysis ~ lysis b Glucanase ~

( U / m g ) ( U / r a g ) (U)

Specific activi ty

( U / r a g )

L-1

0 -80% A m S O 4 884 68 950 78 66 20 500 23

CM Bio-Gel A 59.5 2 200 37 2 250 38

Bio-Gel P-60 23.5 730 31

Aff in i ty chromatography 14.9 580 39 35 1 475 99

L-2 0 -80% A m S O 4 884 68950 78 66 20500 23

CM Bio-Gel A 109.8 7470 68 4273 39

Bio-Gel P-60 23.4 3 540 151 Affinity chromatography 12.8 1 740 137 49 2773 217

L-4 0 -80% A m S O 4 884 68950 78 66 20500 23 CM Bio-Gel A 75.2 1 580 21 920 15

Bio-Gel P-30 14.0 630 45 - -

A ffi ni ty ch romatography 9.0 100 11 16 360 51

possible synergism of the present lytic glucanases with other enzymes such as fraction L-3 with proteinase activity or nonlytic glucanases (frac- tions N-1 and N-2) will be presented in a subse- quent paper. Results of purification of nonlytic enzymes were similarly represented in the activity with assay A for initial step (AmSO 4 fractiona- tion). Fraction N-1 was purified 90-fold with a 35.5% yield, while fraction N-2 was purified 37-fold with a 10.7% yield.

Action of lytic (1 ~ 3)-fl-D-glucanase toward pachy- man

Three lytic glycanase preparations hydrolyzed predominantly water-insoluble (1--, 3)-fl-glucans such as pachyman (degree of polymerization of 255) and curdran (degree of polymerization of 455). Curdran produced by Alcaligenes faecalis var. rnyxogenes 10C3, is essentially linear polysac- charides consisting mainly of (1 ~ 3)-fl-linked D- glucose residues, and is closely related in chemical nature to pachyman [26,27 ]. Thus, the relative rates of attack of fractions L-l, L-2 and L-4 on short-chain pachyman compared to pachyman at 100 were 1.6, 1.0 and 2.2, respectively, and in the

Vo G7 06 G5 G4 G3 G2 G1

I I I I I I I I

~ 4 0 0 -

o

o

2oo

3

5- ©

o

A B .~

/ / , , i i4

30 50 70 Tube no. ( 2 rnl e o c h

Fig. 6. Time course of the hydrolysis of pachyman by L-1 enzyme. (A) Gel filtration through Bio-Gel P-2. The elution positions of Blue Dextran 2000 (V o), 1,3-fl-glucooligosac- char ides (G2-GT) and glucose are shown. (B) Paper chro- matography with solvent system A. Lam: authentic glucose (1) and 1,3-fl-glucooligosaccharides (2-7) .

262

TABLE 11

ACTION PATTERNS OF LYTIC (1 --, 3)-fi-D-GLUCANASES T O W A R D PACHYMAN

GIc, glucose: G 2-Gv, a series of glucooligosaccharides (degree of polymerization, 2-7).

Enzyme fraction Initial product (1 h incubation) Final product (48 h incubation)

L-I G 5 >> G 4, G 6, G~, higher oligo G 5 >> G 4, G 6, G 7, higher oligo > G 2, G~ L-2 Gs >~ G4, G 6, GT, higher oligo G 5 >> G 4, G 6, G 7, higher oligo > G 2, G3 L-4 G 2-Gv, higher oligo G 2 Gv, higher oligo > Glc

same way the rates of fractions L-l, L-2 and L-4 on curdran were 110, 104 and 90, respectively. While nonlytic glucanases exhibited high affinity to short-chain pachyman, the relative rates of frac- tions N-1 and N-2 on pachyman compared to short-chain pachyman at 100 were 21 and 18, respectively. Thus, lyric enzymes show much more affinity for insoluble glucan than for soluble glucan. The modes of action of the lyric enzymes toward pachyman were further examined by analyzing the hydrolysis products using paper chromatography and gel filtration, as summarized in Table II. L-1 and L-2 enzymes released pre- dominantly pentaose from pachyman during the entire course of hydrolysis and attacked the linear (1 ~ 3)-fl-D-glucan in an endo-fashion, as shown in Fig. 6. The hydrolysis products were estimated by paper chromatography to be 1,3-fi-glucose- oligomers. Arthrobacter lytic (1 -~ 3)-fi-D- glucanase showed a similar pattern of action on glucans [2,3]. Hydrolysis of fraction L-4 was also found to be by endoglucanase, yielding a mixture of products ranging from biose to heptaose or more. The action pattern of the enzyme was obvi- ously different from that of fractions L-1 and L-2 in that, in general, a variety of oligomer endo- products was found.

Cibachron blue pachyman has been used as a specific substrate for endo-activity, because it re- leases dye in the presence of the enzyme [12]. This method was certainly specific for endo-enzymes (fraction N-2), but not for endolytic ones (frac- tions L-l, L-2 and L-4). Fractions N-1 and N-2 have been shown to be of exo- and endo-nature, respectively, based on the action toward short- chain pachyman [4]. Such endolytic glucanases seem to be substantially different from the non- lytic one in the mode of attack against glucan.

In conclusion, short-chain pachyman covalently bound to aminohexyl-Sepharose was found to be a very useful biospecific ligand for separation of yeast lyric (1 ~ 3)-fi-D-glucanases as well as non- lytic ones, in spite of the very low specificity of the lytic enzymes toward the glucan. It is also possible that a wider application of this procedure will lead to innovations and modifications, and produce an improved method for preparation of lytic and nonlytic (1 ---, 3)-fi-D-glucanases from most sources by affinity chromatography.

References

1 Bacon, J.S,D., Gordon, A,H., Jones, D., Taylor, I.F. and Welbey, D.M. (1970) Biochem. J. 120, 67-78

2 Kitamura, K., Kaneko, T. and Yamamoto, Y. (1974) J. Gen. Appl. Microbiol. 20, 323-344

3 Vr~.anska, M., Biely, P. and Kraltk~, Z. (1977) Z. Allg. Microbiol. 17, 465-480

4 Zlomik, H., Fernandez, M.P., Bowers, B, and Cabib, E, (1984) J. Bacteriol. 159, 1018-1026

5 Clark, A.E. and Stone, B.A. (1968) Biochem. J. 110, 7-8 6 Villa, T.G., Natario, V. and Villanueva, J,R. (1976) Appl,

Environ. Microbiol. 32, 185-187 7 Edward, M. and Sturgeon, R.J. (1977) Carbohydr. Res. 57,

3 13 8 Totani, K., Harumiya, S., Nanjo, S. and Usui, T. (1983)

Agric. Biol. Chem. 47, 1159-1162 9 Kobayashi, R., Miwa, T., Yamamoto, S. and Nagasaki, S,

(1980) J. Ferment. Technol. 58, 311-317 10 Saito, H., Misaki, A. and Harada, T. (1968) Agric. Biol.

Chem. 32, 1261 1269 11 Ogura, K., Tsurugi, J. and Watanabe, T. (1973) Carbohydr.

Res. 29, 397 403 12 Philpott, N.A. and Chapman, J.M. (1977) Anal. Biochem.

79, 257 262 13 Somogyi, M. (1952) J. Biol. Chem. 195, 19-23 14 Nelson, N. (1944) J. Biol. Chem. 153, 375-380 15 Hirayama, T., Sudo, T., Nagayama, H., Matsuda, K. and

Tamari, K. (1976) Agric. Biol. Chem. 40, 2137 2142 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall,

R.J. (1951) J. Biol. Chem. 193, 265-275

17 Laemmli, U.K. (1970) Nature 277, 680-685 18 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244,

4406-4412 19 Trevelyan, W.E,, Procter, D.P. and Harrison, J.S. (1950)

Nature 166, 144-145 20 Barker, S.A., Foster, A.B., Stacey, M. and Webber, J.M.

(1958) J. Chem. Soc. 2218-2227 21 Lindberg, B. and McPherson, J. (1954) Acta Chem. Scand.

8, 985-988 22 Dubois, M., Gilles, K.A., Hamilton, J.K., Revers, P.A. and

Smith, R. (1956) Anal. Chem. 28, 350-356

263

23 Obata, T., lwata, H. and Namba, Y. (1977) Agric. Biol. Chem. 41, 2389-2394

24 Scott, J.H. and Schekman, R. (1980) J. Bacteriol. 142, 414-423

25 Jeffries, T.W. and Macmillan, J.D. (1968) Carbohydr. Res. 95, 87-10

26 Harada, T., Misaki, A. and Saito, H. (1968) Arch. Biochem. Biophys. 124, 292-298

27 Warsi, S.A. and Whelan, W.J. (1957) Chem. Ind. (Lond.) 1573