ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Comparative Isolation
and Characterization
167, 165-175 (1975)
Studies
on P-Glucan
Hydrolases
of an Exo(l-+3)-/3-Glucanase Helix pomatia
J. J. MARSHALL3
AND
from the Snail,
‘* ’
R. J. A. GRAND’
Department of Chemistry, Royal Holloway College (University of London), Englefield Green, Surrey TW20 OEX, England., Laboratories for Biochemical Research, Howard Hughes Medical Institute, and Department of Biochemistry, University of Miami School of Medicine, Miami, Florida 33152 Received September
13, 1974
An exo-fi-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-ZOO, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades p-(1 -3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1+3; 1+4)-j3-glucans are also extensively degraded and j3-(l-6)- and &(l-4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-@-glucanases, reported previously, in the broadness of its substrate specificity. The K, values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50-55°C.
The highly complex nature of the /3glucanase mixture present in snail digestive juice has long been recognized (2, 3). Use has been made of the unpurified mixture of glucanases for the formation of yeast protoplasts and related purposes (4), but the nature and role of the enzymes functional in the degradation of microbial cell walls has not been established. In view of the importance of purified &glucanases, r Supported m part by a grant from the Science Research Council. ’ This paper is the third in the series “Comparative Studies on B-Glucan Hydrolases.” Paper II appeared in Comp. Biochem. Ph&iol. B., in press (1). - 3 Investigator of the Howard Hughes Medical Institute. Address enquiries and reprint requests to J. J. Marshall, Department of Biochemistry, University of Miami School of Medicine, P.O. Box 520875, Biscayne Annex, Miami, FL 33152. ’ Present address: Department of Biochemistry, University of Birmingham, Birmingham, Warwicks, England.
with rigidly established specificities, for the examination of the molecular architecture of microbial cell walls, and the mechanism of their enzymolysis, as well as for the examination of the molecular structures of isolated polysaccharides (5, 6), we have undertaken the separation and purification of the constituent ,f3-glucanases of snail juice. We now report on the purification and properties of a new type of exo(l&3)-P-giucanase [( l-3)+glucan glucohydrolase, EC 3.2.1.581 from this source. MATERIALS
165 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved
AND
METHODS
Glusulase, a crude mixture of snail digestive enzymes, was purchased from Endo Laboratories, Garden City, NY. Substrates were prepared or obtained as reported in earlier publications (1, 7-10). Laminarin polyalcohol was prepared as by Nelson et al. (11); nonreducing chain ends were regenerated by treatment of this modified polysaccharide with 0.01 N sulfuric acid for 15 h at room temperature.
166
MARSHALL
Glucose oxidase (type II), peroxidase (type l), o-dianisidine dihydrochloride, human serum albumin (Cohn fraction V), cysteine hydrochloride, oxidized glutathione, N-bromosuccinimide, gluconic acid blactone, p-chloromercuribenzoic acid, and 2-hydroxy 5nitrobenzylbromide were purchased from the Sigma Chemical Company. Microgranular DEAE-cellulose (DE-X?) was a product of Whatman Biochemicals; DEAE-Sephadex A-50 and Sephadex G-200 were from Pharmacia Fine Chemicals; Bio-Gel P-60 was from Bio-Rad Laboratories. Analytical methods. Protein concentrations of enzyme solutions were determined by the method of Lowry et al. (12). using bovine serum albumin as standard. In column fractions, protein was detected by measurement of absorbance at 280 nm. Reducing sugars released by enzyme action were measured by reduction of an alkaline copper reagent (13). Glucose was determined specifically by using glucose oxidase (14). Total carbohydrate was determined by the phenol-sulfuric acid method (15), calibrated against glucose. Enzyme assays. During purification of the enzyme, (1 + 3)-@-glucanase activity was determined by measurement of the reducing sugars produced in 1.0 ml digests containing substrate (laminarin, 2.5 mg), acetate buffer (100 mM, pH 5.3, 0.25 ml), calcium chloride (1.25 mg), and a suitable amount of enzyme, incubated at 37°C. One unit of enzyme activity is the amount which releases 1 pmole of glucose equivalents/ min under these conditions. Specific activities are the number of units of activity/mg of protein. Since the purified enzyme was found to exhibit maximum activity at about pH 4.5, studies on the properties of the enzyme were carried out at this lower pH value, unless otherwise indicated. Gel filtration. Gel filtration was performed at 2°C on a column (100 x 2.5 cm) of Bio-Gel P-60 (100-200 mesh), eluted with 25 mM citrate-phosphate buffer pH 8.0, containing 1% sodium chloride, 5.0-ml fractions being collected automatically. Chromatography on Sephadex G-200 was performed using a column of dimensions 90 x 1.6 cm eluted with the same buffer, 2.6-ml fractions being collected. Both types of gel filtration media were prepared as recommended by their respective manufacturers (16-17). Ion-exchange chromatography. Chromatography on microgranular DEAE-cellulose, pretreated as recommended (la), and equilibrated with 25 rnru citrate-phosphate buffer pH 8.0, was performed at 2°C in a column of dimensions 10 x 3.0 cm. Protein was eluted with a pH gradient formed by running 250 ml 25 mM citric acid into 250 ml 25 mM citrate-phosphate buffer pH 8.0, 10 ml fractions being collected. DEAE-Sephadex was prepared for use by the recommended method (19), and equilibrated with 25 mM citrate-phosphate buffer pH 8.0. Chromatogra-
AND GRAND phy on this medium was carried out at 2°C in a 5 x 1.0 cm column, the protein being eluted with a linear gradient (0 - 1.0 M) of sodium chloride in the same buffer. Fractions of volume 10 ml were collected. Polyacrylamide gel electrophoresis. Electrophoresis in polyacrylamide gels was performed according to the method of Ornstein and Davis (20, 21). Molecular weights were determined by gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol (22). Ultrafiltration. Protein solutions were concentrated by ultrafiltration at 2°C in a Diaflo cell (Amicon Corp., Lexington, MA) fitted with a UM 10 membrane. Polarimetry. Optical rotations were measured using a 1-dm quartz cell, thermostatted at 43”C, in a Perkin-Elmer 141 polarimeter. Paper chromatography. Decending paper chromatograms were developed in the solvent system ethyl acetate:pyridine:water (10:4:3, by volume). Sugar spots were vizualized using an alkaline silver nitrate reagent (23). RESULTS
Enzyme Purification Step 1: Chromatography on Bio-Gel P-60. Snail digestive juice (2.5 ml) was chromatographed on Bio-Gel P-60 and the column fractions were assayed for activity towards laminarin. Two peaks of (l--+3)@-glucanase activity were found, the first eluted at the void volume of the column, and a second at a much higher elution volume (Fig. 1). Chromatographic examination of the products of action of fractions in the first peak on laminarin showed glucose to be the main product. The fractions in the second peak produced, during action on the same substrate, glucose and a series of oligosaccharides with the chromatographic mobility of fi-( l&3)linked oligosaccharides. These findings indicated that the first peak contained a p-glucosidase or exo-fi-glucanase, while the second was endo-( 1+3)-P-glucanase. Fractions of the first peak emerging from the column (loo-160 ml) were combined. Step 2: Chromatography on Sephadex G-200. The enzyme preparation from BioGel P-60 was concentrated, dialyzed and chromatographed on Sephadex G-200. Assay of the fractions for (1 + 3)+glucanase activity showed that this procedure separated the activity into two fractions
SNAIL
(Fig. 2). The fractions comprizing the first peak of activity (material eluting at 44-75 ml) were combined. Step
3: Chromatography
on DEAE-
Cellulose. The combined, concentrated and dialyzed enzyme preparation from the Sephadex G-200 column was chromatographed on DEAE-cellulose, using a pH gradient for elution. The majority of the (l-3)-@-glucanase activity was eluted at a late stage in the gradient, between pH
ElUtlOn
167
EXO-B-GLUCANASE
Volume
4.5-2.8 (Fig. 3). The material eluted between 790 and 950 ml was combined. Step 4: Chromatography Sephadex. The combined,
on DEAE-
concentrated and dialyzed (1 --+3)-/3-glucanase preparation from DEAE-cellulose was applied to DEAE-Sephadex and eluted with a gradient of sodium chloride. A single peak of activity towards laminarin was found, eluted at 0.1-0.35 M sodium chloride concentration (Fig. 4). The material eluted
lmll
FIG. 1. Chromatography of crude snail juice on Bio-Gel P-60. --- -, Distribution of protein; C-0, activity towards laminarin. The fractions under the heavy bar were combined and concentrated.
FIG. 2. Chromatography of partly purified (1-3)~@-glucanase on Sephadex G-200. ----, Distribution of protein; CO-O, activity towards laminarin. The fractions under the heavy bar were combined and dialyzed.
168
MARSHALL
ElUtlOn
AND GRAND
Volume
ImU
FIG. 3. Chromatography of partly purified (1 -+ 3)-B-glucanase on DEAE-cellulose, eked with a pH gradient as described in Materials and Methods. ----, Distribution of protein; @-O--O, activity towards laminarin. The fractions under the heavy bars were combined and dialyzed. No significant amount of protein or enzyme activity was present in the portion of the column effluent (approximately 200-500 ml) which has been omitted.
apparent yield of the purified enzyme is misleading, this being largely due to removal of other (l-+3)-@-glucanases at Steps 1 and 2 in the purification scheme. Properties of Purified (1 -+ 3)-p-Glucanase Molecular weight. The minimum molec-
ular weight of the purified enzyme was determined by polyacrylamide gel electrophoresis under denaturing conditions, and was found to be 82,000. This value is in agreement with the approximate value of the molecular weight of the enzyme, judged from its elution characteristics on FIG. 4. Chromatography of partly purified (l-3)Sephadex G-200 (Fig. 2) and is therefore P-glucanase on DEAE-Sephadex, eluted with a graconsidered to be the true molecular weight, dient of sodium chloride. ----, Distribution of protein, rather than the subunit molecular weight, O-0-0, activity towards laminarin. The fracof the enzyme. tions under the heavy bar were combined. Enzyme stability. Tenfold dilution of the concentrated, purified enzyme preparation between 130 and 295 ml was combined, in the presence or absence of human serum concentrated and dialyzed against water. albumin (250 pg/ml) did not result in loss Polyacrylamide gel electrophoresis of the of activity, even after the resulting solupurified enzyme at both pH 4.3 and 8.5, tions were allowed to stand at room temand under denaturing conditions (in the perature for several hours. Neither did presence of sodium dodecyl sulfate and incorporation of human serum albumin 2-mercaptoethanol) showed a single pro- into enzyme digests lead to increased entein band in each case, indicating that the zyme activities. The enzyme did not lose enzyme prepared as above was homogene- activity during freezing and thawing or during freeze-drying and redissolving. ous. The results of a typical purification Effect of pH on enzyme activity and procedure are shown in Table I. The low
SNAIL
Enzyme activity was determined at different pH values using digests containing laminarin (2.5 mg), citrate buffer (25 mM, pH 3.0-6.6) and enzyme solution (10 ~1) in a total volume of 1.0 ml. The pH-activity curve (Fig. 5) which showed maximum activity at pH 4.4-4.5, was the same in the presence and absence of human serum albumin (250 pg/ml) (cf. 10). When the activity was determined after preincubation of the enzyme in the buffers of different pH for 2 h at 37”C, followed by initiation of enzyme action by addition of substrate, a pH-activity curve was obtained which was closely similar on the neutral side of the optimum pH, but showing decreases in activity at pH 4.0 and 3.5 of 10% and 35%, respectively, suggesting that the purified enzyme is labile under acid conditions.
stability.
Optimum temperature and heat stability. Enzyme activity was determined
at different temperatures in digests of composition: laminarin (2.5 mg), acetate buffer (pH 4.5, final concentration 25 mM) and enzyme solution (10 ~1) in a total volume of 1.0 ml, incubated at the chosen temperature for 30 min. Under these conditions, the activity of the enzyme was maximal at 53°C (Fig. 6). The thermal stability of the enzyme was determined by preincubation of all constituents of the above digests, except substrate, at the chosen temperature for 30 min, followed by addition of substrate and measurement of the activity remaining by incubation at 37°C. The enzyme was found to be stable up to 50°C and rapidly inactivated at higher temperatures (Fig. 7). Substrate
TABLE PURIFICATION
Step
1 2 3 4
169
EXO-B-GLUCANASE
SCHEME FOR Helix
Crude snail juice Bio-Gel P-60 chromatography Sephadex G-100 chromatography DEAE-cellulose chromatography DEAE-Sephadex chromatography
of purified
(l-3)-
I
pomatia Protein (mg)
Stage
specificity
Exo-(1
4
3b@-GLUCANASE
(14 3)-PGlucanase activity (U)
370 261 78 7.2 1.0
436 108 44 33.3 24.2
Specific activity Wmd
Yield” (o/o)
1.2 0.4 0.6 4.6 24.2
100 24.6 10.1 7.6 5.4
’ The low overall yield is misleading, being partly due to the removal of other (1 + 3)-B-glucanases at Steps 1 and 2. The presence of a multiplicity of (1 + 3)-@-glucanases in the starting material makes it difficult to calculate accurately the overall extent of purification of the exo-(I + 3)-B-glucanase.
01
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
PH
FIG. 5. Dependence
the text.
of activity
of Helix pomatia
(1 + 3)-P-glucanase
on pH. For conditions
see
170
MARSHALL
Temperature
(“Cl
FIG. 6. Dependence of Helix pomatia canase activity on temperature.
(l-3)-,%glu-
fi-glucanase. To determine the relative rates of action of the purified enzyme on oligo- and polysaccharides, digests of volume 7.0 ml containing substrate (approximate concentration 0.5 mM), acetate buffer (pH 4.5, final concentration 25 mM) and enzyme (20 ~1) were incubated at 37°C. Samples were removed at intervals and analyzed for reducing sugars. In the case of laminarin, laminarin polyalcohol and acidhydrolyzed laminarin polyalcohol, liberated glucose was also determined specifically by using glucose oxidase. The total extent of hydrolysis achieved in 24 h (and in the case of laminarin and lichenin, also 70 h) was determined. The nature of the products produced by action of the enzyme on the various substrates was ascertained by chromatography of samples removed from the digests after incubation for 24 h. The results are shown in Table II and Fig. 8 (a and b). The kinetic constants for the (1 --t 3)-pglucanase acting on laminarin and lichenin were determined by measuring initial reaction rates in digests (1.0 ml) containing substrate (0.1-5.0 mg/ml), acetate buffer (pH 4.5, final concentration 25 mM) and enzyme solution (10 ~1) incubated at 37°C. Values for K, from Lineweaver-Burk double reciprocal plots (Fig. 9), were 1.22 mg/ml and 2.22 mg/ml for laminarin and lichenin respectively. The maximum velocities of enzyme action on laminarin and lichenin were 2.70 and 1.49 pmoles glucose/ml enzymelmin, respectively. A control experiment in which the en-
AND GRAND
zyme was incubated with glucose (5%) for 24 h showed the absence of any detectable oligosaccharide formation indicating that the enzyme does not catalyze reversion reaction. Neither was any evidence found from paper chromatographic examination of the products of enzyme action on its various substrates, to suggest that the enzyme possesses transglycosylase activity. Anomeric form of liberated glucose. To determine whether action, of the enzyme proceeded with retention or inversion of anomeric configuration, the hydrolysis of laminarin in a digest containing substrate (5.0 mg), acetate buffer (pH 5.6, final concentration 5 mM) and enzyme solution (400 ~1) was followed polarimetrically. An increase in optical rotation during incubation, decreasing to the equilibrium value of a mutarotated glucose solution on addition of a drop of ammonium hydroxide solution indicated that the enzyme released glucose with the cu-configuration. Inhibition of purified (1+3)-/3-glucanase. The effect of a number of metal ions and compounds known to inhibit other figlucanases was determined by measuring the activity of the enzyme in digests of composition: laminarin (2.5 mg), acetate buffer (final concentration 100 mM) and enzyme (10 ~1) in a total volume of 1.0 ml,
1
80 s ->I z ; 4 -@l--l
60
40
f zL-!d 10
20
Temperature
40
30
of
50
60
Prelncubatlon
(“0
70
FIG. 7. Heat stability of Helix pomatia (14)-j3glucanase. Enzyme activities were determined at 37°C after heat treatment at the temperatures indicated for 30 min, and are expressed relative to the activity in a control digest incubated at 37°C.
SNAIL
SUBSTRATE
Substrate
SPECIFICITY
171
EXO-P-GLUCANASE TABLE II pomatia
OF Helix
Exo-(1
+ 3)-8-GLUCANASE'
Predominant linkages present (all 8-W=)
Initial rate of hydrolysis (pg glucose/min)
Average degree of polymerization of products after 24 h
Product(s)
Laminaribiose Laminaritriose Laminaritetraose Laminaripentaose Laminarin
l-3 l-3 l-3 l-3 l-3
465 471 502 485 320
1.0 1.1 1.0 1.0 1.8 (1.1 after 70 h)
Pachyman Yeast glucan Lichenin Barley glucan Oat glucan Gentiobiose Luteose CM-Cellulose Cellodextrin
l-3 l-3
very low very low 163 15 N.D. N.D. N.D. very low N.D.
N.D.b N.D. 4.2 (1.7 after 70 h) 6.7 4.0 N.D. N.D. 44 N.D.
Glucose Glucose Glucose Glucose Glucose, gentiobiose and traces of higher oligosaccharides Glucose (trace) Glucose Glucose Glucose Glucose Glucose Glucose Glucose (trace) Glucose
l-3; l-4 1+3;1-4 l-3;1+4 l-6 1-6 144 144
0 The following were not attacked: b N.D. indicates not determined.
(1 + 4)-&mannan,
enzyme action being initiated by addition of substrate, after preincubation of all other constituents of the digests for 15 min. Appropriate control digests were included to correct for any effect of the species being tested on the reducing power measurements. The results are shown in Table III. DISCUSSION
Snail digestive juice provides an alternative to microbial enzyme systems for use in studying the mechanism of enzymic degradation of cellulose, and the enzymic lysis of the cell walls of living yeasts. Crude snail juice contains an extremely complex mixture of glycosidases (2, 3) and it has therefore been necessary to develop methods for separation of these closely related enzymes, prior to examination of their properties. This work has been simplified by the development of affinity binding techniques for the purification of ,&glucanases (7, 8, 10, 24, 25), but the enzyme which is the subject of the present paper could not be purified by such methods. However a combination of conventional ion-exchange and molecular-sieve chromatography techniques, summarized in Table I, yielded a
(1 --t 4)-b- and (1 + 3)-b-xylans,
cellobiose.
homogeneous preparation of the enzyme. It has not been possible to calculate an overall purification factor for the enzyme, because of the multiplicity of (1 -+ 3)+glucanases present in the starting material. Removal of the other (1 --+3)-/3-glucanases is a factor contributing to the apparently low yield of the enzyme; it is estimated that the recovery corresponds to approximately 50% of the amount of the enzyme in the starting material. Action of the enzyme on all substrates results in production of glucose as the sole or major chromatographically-mobile product (Table II). Measurement of the degree of polymerization of the products after action on laminarisaccharide substrates shows that complete degradation into glucose takes place (Table II). The degree of polymerization of the products resulting from action on laminarin also approaches unity after extended incubation times. Analysis of the products during the hydrolysis of laminarin, by using both a copper reducing method and the glucose oxidase method (Fig. 8b), showed glucose to be the primary product of enzyme action. These observations suggest a exo
172
MARSHALL
20
40 Dllrat10n
60
so of
100
lncubatlon
120
AND GRAND
Id0
,m,n,
D”rarlOn
Of
lncubatlon
(mln
)
FIG. 8. (a) Time course of hydrolysis of laminarin (O), lichenin (0) and barley glucan (x) by purified Helix pomatia (1+3)-&glucanase (b) Time course of hydrolysis of laminarin (0, O), oxidized laminarin (m, 0) and oxidized laminarin treated with mild acid (‘I, V) by purified Helix pomatia (l-3)-@-glucanase. The filled symbols show the results obtained using the glucose oxidase method and the open symbols using the Nelson-Somogyi method for glucose determination. TABLE
III
EFFECT OF VARIOUS REAGENTS ON Acrrvrr~ pomatia Exo-(1 + 3)-fl-GLUCANAsF
Reagent* Mercuric chloride Silver nitrate Ferric chloride Copper sulfate Ammonium molybdate Potassium cyanide Phenyl mercuric nitrate Gluconic acid d-lactone N-Bromosuccinimide
‘/[s]
Cm0-YmI )
FIG. 9. Lineweaver-Burk plot of reciprocal of initial reaction rate (V) against reciprocal of substrate concentration ([Sl) for Helix pomatia exo-( l&+3)-& glucanase acting on laminarin (0) and lichenin (A).
action pattern for the enzyme. Action of the enzyme on laminarin is blocked by periodate oxidation of the substrate, a method first used by Nelson et al. (11) to demonstrate the exo nature of a microbial
OF Helix
Inhibition
(%)
47 41 39 41 28 9 23 96 96
“The following were without effect: lead acetate, EDTA, cobalt chloride, zinc sulfate, manganese sulfate, sodium chloride, calcium chloride, cysteine hydrochloride, oxidized glutathione, p-chloromercuribenzoate, 2-hydroxy-5-nitrobenzylbromide, iodoacetic acid, &mercaptoethanol, sodium fluoride, sodium azide.
(1 -+ 3)-/3-glucanase, and substantially restored after mild acid treatment which regenerates intact glucose units at the nonreducing chain ends. These latter ob-
SNAIL
EXO-B-GLUCANASE
servations confirm that the enzyme we have purified is an exo-( l&3)-/3-glucanase. The traces of oligosaccharides produced during action on laminarin are presumed to arise from reducing, or mannitol-terminated, chain. ends. Helix pomatia exo-( 1 + 3)-/3-glucanase is rather unspecific, having the ability to cleave (1 -+ 4)-p- and (1 + 6)-&glucosidic linkages in addition to (1 -+ 3)-P-linkages. Thus the mixed linkage (l-3; l&4)-/3glucan, lichenin, is degraded at half the rate of laminarin, and to an extent approaching completion. Comparison of the kinetic constants of the purified exo(1+3)-/3-glucanase for laminarin and lichenin (K, values 1.22 mg/ml and 2.22 mg/ml; V values 2.70 pmoles glucose released/ml enzyme/min and 1.49 pmoles glucose released/ml enzyme/min, for laminarin and lichenin, respectively) shows the mixed-linkage glucan to be only a slightly poorer substrate for the enzyme than is laminarin. Barley and oat glucans, although somewhat more slowly hydrolyzed than laminarin and lichenin, are nevertheless also extensively degraded. The lower rate of action on the cereal glucans than on lichenin is difficult to explain in view of the indication that they contain a rather smaller proportion (cu. 50%) of (l-4)linkages than does lichenin (cu. 70%), although it is possible that this may be explicable on the basis of differences in the arrangement of the various types of glucosidic linkages in these polysaccharides (5). The less than complete conversion of the “mixed-linkage” substrates into glucose, as apparent from the degrees of polymerization of the products of action on these substrates, indicates the presence in these substrates of some structural feature which resists enzyrne action. As yet, however, we do not have any information to suggest what the nature of such a structural feature might be. The specificity of the Helix pomatia exo-P-glucanase we have purified appears to be less rigid than that of all the other exo-/3-glucanases which have been reported previously. Thus the exo-( l&3)-gglucanase from .Basidiomycetes QM806 (26), and those from various yeasts (27-29) do
173
not degrade the mixed linkage (l-3; l-4) glucans; that from Euglena grucilis (30) releases only small amounts of glucose from these substrates. These enzymes degrade (1 + 3; 1 -+ 6)-glucans by bypassing the (1 + 6)-linkages which are released in the form of gentiobiose. The presence of gentiobiose as a product after action of the enzyme on laminarin suggests that the Helix pomatia also acts in this way, although subsequent hydrolysis of the gentiobiose may take place. While the Helix pomatia enzyme differs from the yeast exo-P-glucanases in being able to degrade lichenin and cereal glucans, it bears certain resemblances to these enzymes in its ability to degrade ,f3-(1+6)-linked glucans. There are indications that one of the yeast enzymes, that produced by Hansenula wingei (29), can also degrade b-(1+4)linked glucose polymers so that the Helix pomatia enzyme bears, qualitatively at least, more similarity to this enzyme than any of the others. It is clear that a number of exo-( 1+3)-P-glucanases exist, each differing in substrate specificity, the Helix pomatia enzyme having the broadest specificity yet found. An analogous situation exists in the case of exo-acting (1 -+ 4)-aglucanases, enzymes of this type differing in their ability to act on (1 + 3) - and (1 + 6)-cY-glucosidic linkages (31). In addition to specificity differences, the Helix pomatia enzyme differs in molecular weight from other enzymes of this type for which values have been reported (82,000 as compared with 51,000 for the Basidiomycetes enzyme (32) and 20,000-40,000 for the yeast enzymes (29)). The molecular weight of Helix pomatia exo-(l-3)-/3glucanase is considerably higher than that of most endo-( 1+3)-P-glucanases which have been studied, including that from Helix pomatia, which lie in the range IO-30,000. This situation, namely exoacting enzymes having molecular weights considerably higher than the corresponding exo-acting enzymes, is very common (33, 34). Unlike a number of endo-acting (l&3)/3-glucanases (10, 35), the Helix porn&u (l&3)-/3-glucanase does not show any tendency to lose activity in dilute solution, and is not stabilized by human serum
174
MARSHALL
albumin. The temperature stability (stable up to 50°C) and optimum temperature (55°C) and susceptibility to acid inactivation suggest that the enzyme does not possess any unusual stability properties. While certain microbial exo-( l-+3)-@glucanases are reportedly activated by manganese, cobalt and ferric ions (36, 37), the Helix pomatia enzyme is unaffected by manganese or cobalt, and markedly inhibited by ferric ions. A number of other workers have failed to demonstrate d:.o(l&3)-P-glucanase activation by manganese and cobalt ions (32, 3%40), and observed Fe3+ inhibition of the enzymes (39). The lack of inhibition of the Helix pomatia enzyme by cysteine, mercaptoethanol, oxidized glutathione and p-chloromercuribenzoate makes it seem unlikely that disulfide or thiol groups are involved in enzyme activity; the effect of phenylmercuric nitrate may not be meaningful since organic mercurial compounds may inhibit other enzymes which do not depend on thiol groups for activity (41-42). The inhibition by N-bromosuccinimide might indicate the participation of tyrosine, tryptophan or histidine residues in the activity of the enzyme. The involvement of tryptophan is, however, unlikely in view of the ineffectiveness of 2-hydroxy 5-nitrobenzylbromide as an inhibitor. The inhibition by mercuric, cupric, ferric and silver ions may support the involvement of histidine (43). Like many other glycosidases (10, 44-46) the Helix pomatia exo-(1 -+ 3)-P-glucanase is strongly inhibited by ammonium molybdate but the mechanism of inhibition by this reagent remains to be determined. The classification of enzymes releasing glucose from oligosaccharide and polysaccharide substrates has been discussed by Reese and co-workers (47). It is not possible to classify the Helix pomatia enzyme unambiguously on the basis of the relative rates of action on (l-3)-P-linked substrates of different chain lengths. The inversion of anomeric configuration which takes place during hydrolysis indicates that the enzyme is an exo-P-glucanase. However, the inhibition by low concentrations of gluconic acid Slactone, which has been considered an indication of ,&glucosi-
AND GRAND
dase activity argues against this conclusion. Thus it is not possible to classify the enzyme we have examined unambiguously by using the criteria of Reese and co-workers. This is a further difference between the Helix pomatia enzyme and other enzymes of this general type. We have preferred to consider the enzyme as an exo-P-glucanase because of its action on a wide range of P-glucan substrates, compared with its limited action on disaccharide substrates. REFERENCES 1. GRAND, R. J. A., AND MARSHALL, J. J. (1975) Comp. Biochem. Physiol. B, in press. 2. HOLDEN, M., AND TRACEY, M. V. (1950) Biochem. J. 47, 407-414. 3. MYERS, F. L., AND NORTHCOTE, D. H. (1958) J. Exp. Biol. 35, 639-648. 4. ANDERSON, F. B., AND MILLBANK, J. W. (1966) Biochem. J. 99, 682-687. 5. MARSHALL, J. J. (1974) Aduan. Carbohyd. Chem. Biochem. 30, 257-370. 6. MARSHALL, J. J. (1975) Advan. Carbohyd. Chem. Biochem. 32, in press. 7. MARSHALL, J. J. (1973) Comp. Biochem. Physiol. 44B, 981-988. 8. MARSHALL, J. J. (1973) Carbohyd. Res. 26, 274-277. 9. MARSHALL, J. J. (1973) Anal. Biochem. 53, 191-198. 10. MARSHALL, J. J. (1974) Carbohyd. Res., 34, 289-305. 11. NELSON, T. E., SCALE~I, J. V., SMITH, F., AND KIRKWOOD, S. (1963) Can. J. Chem. 41, 1671-1678. 12. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. ROBYT, J. F., AND WHELAN, W. J. (1972) Anal. Biochem. 45, 510-516. 14. LLOYD, J. B., AND WHELAN, W. J. (1969) Anal. Biochem. 30, 467-470. 15. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 16. “Gel Chromatography,” Bio-Rad Laboratories, Richmond, California (1971). 17. “Sephadex Gel Filtration in Theory and Practice,” Pharmacia Fine Chemicals, Uppsala, Sweden. 18. “Whatman Advanced Ion-Exchange Celluloses. Laboratory Manual,” W. & L. Balston, Maidstone, Kent, England. 19. “Sephadex Ion Exchangers. An Outstanding Aid in Biochemistry,” Pharmacia Fine Chemicals, Uppsala, Sweden.
SNAIL
EXO-,%GLUCANASE
20. ORNSTEIN, L. (1964) Ann. N.Y. Acad. Sci. 121, 321-349. 21. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 22. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chew 244, 4406-4412. 23. TREVELYAN, W. E., PROCTER,D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444-445. 24. MARSHALL, J. J. (1973) Biochem. Sot. Z’rans. 1, 143-145. 25. MARSHALL, J. J. (1973) J. Chromatogr. 76, 257-260. 26. NELSON, T. E., JOHNSON, J., JANTZEN, J., AND KIRKWOOD, S. (1969) J. Biol. Chem. 244, 5972-5980. 27. TINGLE, M. A., AND HALVORSON, H. 0. (1971) Biochim. Biophys. Acta 250, 165-171. 28. BROCK, T. D. (1965) Biochem. Biophys. Res. Commun. 19, 623-629. 29. ABD-EL-AL, A. T. H., AND PHAFF, H. J. (1968) Biochem. J. 109, 347-360. 30. BARRAS, D. R., AND STONE, B. A. (1969) Biochim. Biophys. Acta. 191, 342-360. 31. MARSHALL, J. J. (1974) Cereal Sci. Today 19,389. 32. HOUTARI, F. I., NELSON, T. E., SMITH, F., AND KIRKWOOD, S. (1968) J. Biol. Chem. 243, 952-956. 33. AHLGREN, E., ERIKSSON, K-E., AND VESTERBERG,0. (1967) Acta Chem. Stand. 21, 937-944.
175
34. MANNERS, D. J., AND MARSHALL, J. J. (1973) Phytochemistry 12, 547-553. 35. MOORE, A. E., AND STONE, B. A. (1972) Biochim. Biophys. Acta 258, 238-247. 36. FELLIG, J. (1960) Science 131, 832. 37. CHESTERS, C. G. C., AND BULL, A. T. (1963) Biochem. J. 86, 38-46. 38. VOGEL, K., AND BARBER, A. A. (1968) J. Protozool. 15, 657-662. 39. KAJI, A., OHSAKI, T., AND YOSHIHARA, 0. J. Agr. Chem. Sot. Jap. 45, 278-283. 40. MOORE, A. E., AND STONE, B. A. (1972) Biochim. Biophys. Acta 258, 248-264. 41. FISCHER, E. H., AND HASELBACH, C. H. (19.51) Helu. Chim. Acta 34, 325-334. 42. SOHLFX, M. R., SEIBERT, M. A., KREKE, C. W., AND COOK, E. S. (1952) J. Biol. Chem. 198, 281-291. 43. GURD, F. R. N., AND WILCOX, P. E. (1956) Aduan. Protein Chem. 11, 311-427. 44. MACWILLIAM, I. C., AND HARRIS, G. (1959) Arch. Biochem. Biophys. 84, 442-454. 45. MANNERS, D. J., AND SPARRA, K. L. (1966) J. Inst. Brew. 72, 360-365. 46. MANNERS, D. J., AND ROWE, K. L. (1969) Carbohyd. Res. 9, 107-121. 47. REESE, E. T., MAGUIRE, A. H., AND PARRISH, F. W. (1968) Can. J. Biochem. 46, 25-34.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Comparative Isolation
and Characterization
167, 165-175 (1975)
Studies
on P-Glucan
Hydrolases
of an Exo(l-+3)-/3-Glucanase Helix pomatia
J. J. MARSHALL3
AND
from the Snail,
‘* ’
R. J. A. GRAND’
Department of Chemistry, Royal Holloway College (University of London), Englefield Green, Surrey TW20 OEX, England., Laboratories for Biochemical Research, Howard Hughes Medical Institute, and Department of Biochemistry, University of Miami School of Medicine, Miami, Florida 33152 Received September
13, 1974
An exo-fi-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-ZOO, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades p-(1 -3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1+3; 1+4)-j3-glucans are also extensively degraded and j3-(l-6)- and &(l-4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-@-glucanases, reported previously, in the broadness of its substrate specificity. The K, values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50-55°C.
The highly complex nature of the /3glucanase mixture present in snail digestive juice has long been recognized (2, 3). Use has been made of the unpurified mixture of glucanases for the formation of yeast protoplasts and related purposes (4), but the nature and role of the enzymes functional in the degradation of microbial cell walls has not been established. In view of the importance of purified &glucanases, r Supported m part by a grant from the Science Research Council. ’ This paper is the third in the series “Comparative Studies on B-Glucan Hydrolases.” Paper II appeared in Comp. Biochem. Ph&iol. B., in press (1). - 3 Investigator of the Howard Hughes Medical Institute. Address enquiries and reprint requests to J. J. Marshall, Department of Biochemistry, University of Miami School of Medicine, P.O. Box 520875, Biscayne Annex, Miami, FL 33152. ’ Present address: Department of Biochemistry, University of Birmingham, Birmingham, Warwicks, England.
with rigidly established specificities, for the examination of the molecular architecture of microbial cell walls, and the mechanism of their enzymolysis, as well as for the examination of the molecular structures of isolated polysaccharides (5, 6), we have undertaken the separation and purification of the constituent ,f3-glucanases of snail juice. We now report on the purification and properties of a new type of exo(l&3)-P-giucanase [( l-3)+glucan glucohydrolase, EC 3.2.1.581 from this source. MATERIALS
165 Copyright 0 1975by Academic Press, Inc. All rights of reproduction in any form reserved
AND
METHODS
Glusulase, a crude mixture of snail digestive enzymes, was purchased from Endo Laboratories, Garden City, NY. Substrates were prepared or obtained as reported in earlier publications (1, 7-10). Laminarin polyalcohol was prepared as by Nelson et al. (11); nonreducing chain ends were regenerated by treatment of this modified polysaccharide with 0.01 N sulfuric acid for 15 h at room temperature.
166
MARSHALL
Glucose oxidase (type II), peroxidase (type l), o-dianisidine dihydrochloride, human serum albumin (Cohn fraction V), cysteine hydrochloride, oxidized glutathione, N-bromosuccinimide, gluconic acid blactone, p-chloromercuribenzoic acid, and 2-hydroxy 5nitrobenzylbromide were purchased from the Sigma Chemical Company. Microgranular DEAE-cellulose (DE-X?) was a product of Whatman Biochemicals; DEAE-Sephadex A-50 and Sephadex G-200 were from Pharmacia Fine Chemicals; Bio-Gel P-60 was from Bio-Rad Laboratories. Analytical methods. Protein concentrations of enzyme solutions were determined by the method of Lowry et al. (12). using bovine serum albumin as standard. In column fractions, protein was detected by measurement of absorbance at 280 nm. Reducing sugars released by enzyme action were measured by reduction of an alkaline copper reagent (13). Glucose was determined specifically by using glucose oxidase (14). Total carbohydrate was determined by the phenol-sulfuric acid method (15), calibrated against glucose. Enzyme assays. During purification of the enzyme, (1 + 3)-@-glucanase activity was determined by measurement of the reducing sugars produced in 1.0 ml digests containing substrate (laminarin, 2.5 mg), acetate buffer (100 mM, pH 5.3, 0.25 ml), calcium chloride (1.25 mg), and a suitable amount of enzyme, incubated at 37°C. One unit of enzyme activity is the amount which releases 1 pmole of glucose equivalents/ min under these conditions. Specific activities are the number of units of activity/mg of protein. Since the purified enzyme was found to exhibit maximum activity at about pH 4.5, studies on the properties of the enzyme were carried out at this lower pH value, unless otherwise indicated. Gel filtration. Gel filtration was performed at 2°C on a column (100 x 2.5 cm) of Bio-Gel P-60 (100-200 mesh), eluted with 25 mM citrate-phosphate buffer pH 8.0, containing 1% sodium chloride, 5.0-ml fractions being collected automatically. Chromatography on Sephadex G-200 was performed using a column of dimensions 90 x 1.6 cm eluted with the same buffer, 2.6-ml fractions being collected. Both types of gel filtration media were prepared as recommended by their respective manufacturers (16-17). Ion-exchange chromatography. Chromatography on microgranular DEAE-cellulose, pretreated as recommended (la), and equilibrated with 25 rnru citrate-phosphate buffer pH 8.0, was performed at 2°C in a column of dimensions 10 x 3.0 cm. Protein was eluted with a pH gradient formed by running 250 ml 25 mM citric acid into 250 ml 25 mM citrate-phosphate buffer pH 8.0, 10 ml fractions being collected. DEAE-Sephadex was prepared for use by the recommended method (19), and equilibrated with 25 mM citrate-phosphate buffer pH 8.0. Chromatogra-
AND GRAND phy on this medium was carried out at 2°C in a 5 x 1.0 cm column, the protein being eluted with a linear gradient (0 - 1.0 M) of sodium chloride in the same buffer. Fractions of volume 10 ml were collected. Polyacrylamide gel electrophoresis. Electrophoresis in polyacrylamide gels was performed according to the method of Ornstein and Davis (20, 21). Molecular weights were determined by gel electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol (22). Ultrafiltration. Protein solutions were concentrated by ultrafiltration at 2°C in a Diaflo cell (Amicon Corp., Lexington, MA) fitted with a UM 10 membrane. Polarimetry. Optical rotations were measured using a 1-dm quartz cell, thermostatted at 43”C, in a Perkin-Elmer 141 polarimeter. Paper chromatography. Decending paper chromatograms were developed in the solvent system ethyl acetate:pyridine:water (10:4:3, by volume). Sugar spots were vizualized using an alkaline silver nitrate reagent (23). RESULTS
Enzyme Purification Step 1: Chromatography on Bio-Gel P-60. Snail digestive juice (2.5 ml) was chromatographed on Bio-Gel P-60 and the column fractions were assayed for activity towards laminarin. Two peaks of (l--+3)@-glucanase activity were found, the first eluted at the void volume of the column, and a second at a much higher elution volume (Fig. 1). Chromatographic examination of the products of action of fractions in the first peak on laminarin showed glucose to be the main product. The fractions in the second peak produced, during action on the same substrate, glucose and a series of oligosaccharides with the chromatographic mobility of fi-( l&3)linked oligosaccharides. These findings indicated that the first peak contained a p-glucosidase or exo-fi-glucanase, while the second was endo-( 1+3)-P-glucanase. Fractions of the first peak emerging from the column (loo-160 ml) were combined. Step 2: Chromatography on Sephadex G-200. The enzyme preparation from BioGel P-60 was concentrated, dialyzed and chromatographed on Sephadex G-200. Assay of the fractions for (1 + 3)+glucanase activity showed that this procedure separated the activity into two fractions
SNAIL
(Fig. 2). The fractions comprizing the first peak of activity (material eluting at 44-75 ml) were combined. Step
3: Chromatography
on DEAE-
Cellulose. The combined, concentrated and dialyzed enzyme preparation from the Sephadex G-200 column was chromatographed on DEAE-cellulose, using a pH gradient for elution. The majority of the (l-3)-@-glucanase activity was eluted at a late stage in the gradient, between pH
ElUtlOn
167
EXO-B-GLUCANASE
Volume
4.5-2.8 (Fig. 3). The material eluted between 790 and 950 ml was combined. Step 4: Chromatography Sephadex. The combined,
on DEAE-
concentrated and dialyzed (1 --+3)-/3-glucanase preparation from DEAE-cellulose was applied to DEAE-Sephadex and eluted with a gradient of sodium chloride. A single peak of activity towards laminarin was found, eluted at 0.1-0.35 M sodium chloride concentration (Fig. 4). The material eluted
lmll
FIG. 1. Chromatography of crude snail juice on Bio-Gel P-60. --- -, Distribution of protein; C-0, activity towards laminarin. The fractions under the heavy bar were combined and concentrated.
FIG. 2. Chromatography of partly purified (1-3)~@-glucanase on Sephadex G-200. ----, Distribution of protein; CO-O, activity towards laminarin. The fractions under the heavy bar were combined and dialyzed.
168
MARSHALL
ElUtlOn
AND GRAND
Volume
ImU
FIG. 3. Chromatography of partly purified (1 -+ 3)-B-glucanase on DEAE-cellulose, eked with a pH gradient as described in Materials and Methods. ----, Distribution of protein; @-O--O, activity towards laminarin. The fractions under the heavy bars were combined and dialyzed. No significant amount of protein or enzyme activity was present in the portion of the column effluent (approximately 200-500 ml) which has been omitted.
apparent yield of the purified enzyme is misleading, this being largely due to removal of other (l-+3)-@-glucanases at Steps 1 and 2 in the purification scheme. Properties of Purified (1 -+ 3)-p-Glucanase Molecular weight. The minimum molec-
ular weight of the purified enzyme was determined by polyacrylamide gel electrophoresis under denaturing conditions, and was found to be 82,000. This value is in agreement with the approximate value of the molecular weight of the enzyme, judged from its elution characteristics on FIG. 4. Chromatography of partly purified (l-3)Sephadex G-200 (Fig. 2) and is therefore P-glucanase on DEAE-Sephadex, eluted with a graconsidered to be the true molecular weight, dient of sodium chloride. ----, Distribution of protein, rather than the subunit molecular weight, O-0-0, activity towards laminarin. The fracof the enzyme. tions under the heavy bar were combined. Enzyme stability. Tenfold dilution of the concentrated, purified enzyme preparation between 130 and 295 ml was combined, in the presence or absence of human serum concentrated and dialyzed against water. albumin (250 pg/ml) did not result in loss Polyacrylamide gel electrophoresis of the of activity, even after the resulting solupurified enzyme at both pH 4.3 and 8.5, tions were allowed to stand at room temand under denaturing conditions (in the perature for several hours. Neither did presence of sodium dodecyl sulfate and incorporation of human serum albumin 2-mercaptoethanol) showed a single pro- into enzyme digests lead to increased entein band in each case, indicating that the zyme activities. The enzyme did not lose enzyme prepared as above was homogene- activity during freezing and thawing or during freeze-drying and redissolving. ous. The results of a typical purification Effect of pH on enzyme activity and procedure are shown in Table I. The low
SNAIL
Enzyme activity was determined at different pH values using digests containing laminarin (2.5 mg), citrate buffer (25 mM, pH 3.0-6.6) and enzyme solution (10 ~1) in a total volume of 1.0 ml. The pH-activity curve (Fig. 5) which showed maximum activity at pH 4.4-4.5, was the same in the presence and absence of human serum albumin (250 pg/ml) (cf. 10). When the activity was determined after preincubation of the enzyme in the buffers of different pH for 2 h at 37”C, followed by initiation of enzyme action by addition of substrate, a pH-activity curve was obtained which was closely similar on the neutral side of the optimum pH, but showing decreases in activity at pH 4.0 and 3.5 of 10% and 35%, respectively, suggesting that the purified enzyme is labile under acid conditions.
stability.
Optimum temperature and heat stability. Enzyme activity was determined
at different temperatures in digests of composition: laminarin (2.5 mg), acetate buffer (pH 4.5, final concentration 25 mM) and enzyme solution (10 ~1) in a total volume of 1.0 ml, incubated at the chosen temperature for 30 min. Under these conditions, the activity of the enzyme was maximal at 53°C (Fig. 6). The thermal stability of the enzyme was determined by preincubation of all constituents of the above digests, except substrate, at the chosen temperature for 30 min, followed by addition of substrate and measurement of the activity remaining by incubation at 37°C. The enzyme was found to be stable up to 50°C and rapidly inactivated at higher temperatures (Fig. 7). Substrate
TABLE PURIFICATION
Step
1 2 3 4
169
EXO-B-GLUCANASE
SCHEME FOR Helix
Crude snail juice Bio-Gel P-60 chromatography Sephadex G-100 chromatography DEAE-cellulose chromatography DEAE-Sephadex chromatography
of purified
(l-3)-
I
pomatia Protein (mg)
Stage
specificity
Exo-(1
4
3b@-GLUCANASE
(14 3)-PGlucanase activity (U)
370 261 78 7.2 1.0
436 108 44 33.3 24.2
Specific activity Wmd
Yield” (o/o)
1.2 0.4 0.6 4.6 24.2
100 24.6 10.1 7.6 5.4
’ The low overall yield is misleading, being partly due to the removal of other (1 + 3)-B-glucanases at Steps 1 and 2. The presence of a multiplicity of (1 + 3)-@-glucanases in the starting material makes it difficult to calculate accurately the overall extent of purification of the exo-(I + 3)-B-glucanase.
01
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
PH
FIG. 5. Dependence
the text.
of activity
of Helix pomatia
(1 + 3)-P-glucanase
on pH. For conditions
see
170
MARSHALL
Temperature
(“Cl
FIG. 6. Dependence of Helix pomatia canase activity on temperature.
(l-3)-,%glu-
fi-glucanase. To determine the relative rates of action of the purified enzyme on oligo- and polysaccharides, digests of volume 7.0 ml containing substrate (approximate concentration 0.5 mM), acetate buffer (pH 4.5, final concentration 25 mM) and enzyme (20 ~1) were incubated at 37°C. Samples were removed at intervals and analyzed for reducing sugars. In the case of laminarin, laminarin polyalcohol and acidhydrolyzed laminarin polyalcohol, liberated glucose was also determined specifically by using glucose oxidase. The total extent of hydrolysis achieved in 24 h (and in the case of laminarin and lichenin, also 70 h) was determined. The nature of the products produced by action of the enzyme on the various substrates was ascertained by chromatography of samples removed from the digests after incubation for 24 h. The results are shown in Table II and Fig. 8 (a and b). The kinetic constants for the (1 --t 3)-pglucanase acting on laminarin and lichenin were determined by measuring initial reaction rates in digests (1.0 ml) containing substrate (0.1-5.0 mg/ml), acetate buffer (pH 4.5, final concentration 25 mM) and enzyme solution (10 ~1) incubated at 37°C. Values for K, from Lineweaver-Burk double reciprocal plots (Fig. 9), were 1.22 mg/ml and 2.22 mg/ml for laminarin and lichenin respectively. The maximum velocities of enzyme action on laminarin and lichenin were 2.70 and 1.49 pmoles glucose/ml enzymelmin, respectively. A control experiment in which the en-
AND GRAND
zyme was incubated with glucose (5%) for 24 h showed the absence of any detectable oligosaccharide formation indicating that the enzyme does not catalyze reversion reaction. Neither was any evidence found from paper chromatographic examination of the products of enzyme action on its various substrates, to suggest that the enzyme possesses transglycosylase activity. Anomeric form of liberated glucose. To determine whether action, of the enzyme proceeded with retention or inversion of anomeric configuration, the hydrolysis of laminarin in a digest containing substrate (5.0 mg), acetate buffer (pH 5.6, final concentration 5 mM) and enzyme solution (400 ~1) was followed polarimetrically. An increase in optical rotation during incubation, decreasing to the equilibrium value of a mutarotated glucose solution on addition of a drop of ammonium hydroxide solution indicated that the enzyme released glucose with the cu-configuration. Inhibition of purified (1+3)-/3-glucanase. The effect of a number of metal ions and compounds known to inhibit other figlucanases was determined by measuring the activity of the enzyme in digests of composition: laminarin (2.5 mg), acetate buffer (final concentration 100 mM) and enzyme (10 ~1) in a total volume of 1.0 ml,
1
80 s ->I z ; 4 -@l--l
60
40
f zL-!d 10
20
Temperature
40
30
of
50
60
Prelncubatlon
(“0
70
FIG. 7. Heat stability of Helix pomatia (14)-j3glucanase. Enzyme activities were determined at 37°C after heat treatment at the temperatures indicated for 30 min, and are expressed relative to the activity in a control digest incubated at 37°C.
SNAIL
SUBSTRATE
Substrate
SPECIFICITY
171
EXO-P-GLUCANASE TABLE II pomatia
OF Helix
Exo-(1
+ 3)-8-GLUCANASE'
Predominant linkages present (all 8-W=)
Initial rate of hydrolysis (pg glucose/min)
Average degree of polymerization of products after 24 h
Product(s)
Laminaribiose Laminaritriose Laminaritetraose Laminaripentaose Laminarin
l-3 l-3 l-3 l-3 l-3
465 471 502 485 320
1.0 1.1 1.0 1.0 1.8 (1.1 after 70 h)
Pachyman Yeast glucan Lichenin Barley glucan Oat glucan Gentiobiose Luteose CM-Cellulose Cellodextrin
l-3 l-3
very low very low 163 15 N.D. N.D. N.D. very low N.D.
N.D.b N.D. 4.2 (1.7 after 70 h) 6.7 4.0 N.D. N.D. 44 N.D.
Glucose Glucose Glucose Glucose Glucose, gentiobiose and traces of higher oligosaccharides Glucose (trace) Glucose Glucose Glucose Glucose Glucose Glucose Glucose (trace) Glucose
l-3; l-4 1+3;1-4 l-3;1+4 l-6 1-6 144 144
0 The following were not attacked: b N.D. indicates not determined.
(1 + 4)-&mannan,
enzyme action being initiated by addition of substrate, after preincubation of all other constituents of the digests for 15 min. Appropriate control digests were included to correct for any effect of the species being tested on the reducing power measurements. The results are shown in Table III. DISCUSSION
Snail digestive juice provides an alternative to microbial enzyme systems for use in studying the mechanism of enzymic degradation of cellulose, and the enzymic lysis of the cell walls of living yeasts. Crude snail juice contains an extremely complex mixture of glycosidases (2, 3) and it has therefore been necessary to develop methods for separation of these closely related enzymes, prior to examination of their properties. This work has been simplified by the development of affinity binding techniques for the purification of ,&glucanases (7, 8, 10, 24, 25), but the enzyme which is the subject of the present paper could not be purified by such methods. However a combination of conventional ion-exchange and molecular-sieve chromatography techniques, summarized in Table I, yielded a
(1 --t 4)-b- and (1 + 3)-b-xylans,
cellobiose.
homogeneous preparation of the enzyme. It has not been possible to calculate an overall purification factor for the enzyme, because of the multiplicity of (1 -+ 3)+glucanases present in the starting material. Removal of the other (1 --+3)-/3-glucanases is a factor contributing to the apparently low yield of the enzyme; it is estimated that the recovery corresponds to approximately 50% of the amount of the enzyme in the starting material. Action of the enzyme on all substrates results in production of glucose as the sole or major chromatographically-mobile product (Table II). Measurement of the degree of polymerization of the products after action on laminarisaccharide substrates shows that complete degradation into glucose takes place (Table II). The degree of polymerization of the products resulting from action on laminarin also approaches unity after extended incubation times. Analysis of the products during the hydrolysis of laminarin, by using both a copper reducing method and the glucose oxidase method (Fig. 8b), showed glucose to be the primary product of enzyme action. These observations suggest a exo
172
MARSHALL
20
40 Dllrat10n
60
so of
100
lncubatlon
120
AND GRAND
Id0
,m,n,
D”rarlOn
Of
lncubatlon
(mln
)
FIG. 8. (a) Time course of hydrolysis of laminarin (O), lichenin (0) and barley glucan (x) by purified Helix pomatia (1+3)-&glucanase (b) Time course of hydrolysis of laminarin (0, O), oxidized laminarin (m, 0) and oxidized laminarin treated with mild acid (‘I, V) by purified Helix pomatia (l-3)-@-glucanase. The filled symbols show the results obtained using the glucose oxidase method and the open symbols using the Nelson-Somogyi method for glucose determination. TABLE
III
EFFECT OF VARIOUS REAGENTS ON Acrrvrr~ pomatia Exo-(1 + 3)-fl-GLUCANAsF
Reagent* Mercuric chloride Silver nitrate Ferric chloride Copper sulfate Ammonium molybdate Potassium cyanide Phenyl mercuric nitrate Gluconic acid d-lactone N-Bromosuccinimide
‘/[s]
Cm0-YmI )
FIG. 9. Lineweaver-Burk plot of reciprocal of initial reaction rate (V) against reciprocal of substrate concentration ([Sl) for Helix pomatia exo-( l&+3)-& glucanase acting on laminarin (0) and lichenin (A).
action pattern for the enzyme. Action of the enzyme on laminarin is blocked by periodate oxidation of the substrate, a method first used by Nelson et al. (11) to demonstrate the exo nature of a microbial
OF Helix
Inhibition
(%)
47 41 39 41 28 9 23 96 96
“The following were without effect: lead acetate, EDTA, cobalt chloride, zinc sulfate, manganese sulfate, sodium chloride, calcium chloride, cysteine hydrochloride, oxidized glutathione, p-chloromercuribenzoate, 2-hydroxy-5-nitrobenzylbromide, iodoacetic acid, &mercaptoethanol, sodium fluoride, sodium azide.
(1 -+ 3)-/3-glucanase, and substantially restored after mild acid treatment which regenerates intact glucose units at the nonreducing chain ends. These latter ob-
SNAIL
EXO-B-GLUCANASE
servations confirm that the enzyme we have purified is an exo-( l&3)-/3-glucanase. The traces of oligosaccharides produced during action on laminarin are presumed to arise from reducing, or mannitol-terminated, chain. ends. Helix pomatia exo-( 1 + 3)-/3-glucanase is rather unspecific, having the ability to cleave (1 -+ 4)-p- and (1 + 6)-&glucosidic linkages in addition to (1 -+ 3)-P-linkages. Thus the mixed linkage (l-3; l&4)-/3glucan, lichenin, is degraded at half the rate of laminarin, and to an extent approaching completion. Comparison of the kinetic constants of the purified exo(1+3)-/3-glucanase for laminarin and lichenin (K, values 1.22 mg/ml and 2.22 mg/ml; V values 2.70 pmoles glucose released/ml enzyme/min and 1.49 pmoles glucose released/ml enzyme/min, for laminarin and lichenin, respectively) shows the mixed-linkage glucan to be only a slightly poorer substrate for the enzyme than is laminarin. Barley and oat glucans, although somewhat more slowly hydrolyzed than laminarin and lichenin, are nevertheless also extensively degraded. The lower rate of action on the cereal glucans than on lichenin is difficult to explain in view of the indication that they contain a rather smaller proportion (cu. 50%) of (l-4)linkages than does lichenin (cu. 70%), although it is possible that this may be explicable on the basis of differences in the arrangement of the various types of glucosidic linkages in these polysaccharides (5). The less than complete conversion of the “mixed-linkage” substrates into glucose, as apparent from the degrees of polymerization of the products of action on these substrates, indicates the presence in these substrates of some structural feature which resists enzyrne action. As yet, however, we do not have any information to suggest what the nature of such a structural feature might be. The specificity of the Helix pomatia exo-P-glucanase we have purified appears to be less rigid than that of all the other exo-/3-glucanases which have been reported previously. Thus the exo-( l&3)-gglucanase from .Basidiomycetes QM806 (26), and those from various yeasts (27-29) do
173
not degrade the mixed linkage (l-3; l-4) glucans; that from Euglena grucilis (30) releases only small amounts of glucose from these substrates. These enzymes degrade (1 + 3; 1 -+ 6)-glucans by bypassing the (1 + 6)-linkages which are released in the form of gentiobiose. The presence of gentiobiose as a product after action of the enzyme on laminarin suggests that the Helix pomatia also acts in this way, although subsequent hydrolysis of the gentiobiose may take place. While the Helix pomatia enzyme differs from the yeast exo-P-glucanases in being able to degrade lichenin and cereal glucans, it bears certain resemblances to these enzymes in its ability to degrade ,f3-(1+6)-linked glucans. There are indications that one of the yeast enzymes, that produced by Hansenula wingei (29), can also degrade b-(1+4)linked glucose polymers so that the Helix pomatia enzyme bears, qualitatively at least, more similarity to this enzyme than any of the others. It is clear that a number of exo-( 1+3)-P-glucanases exist, each differing in substrate specificity, the Helix pomatia enzyme having the broadest specificity yet found. An analogous situation exists in the case of exo-acting (1 -+ 4)-aglucanases, enzymes of this type differing in their ability to act on (1 + 3) - and (1 + 6)-cY-glucosidic linkages (31). In addition to specificity differences, the Helix pomatia enzyme differs in molecular weight from other enzymes of this type for which values have been reported (82,000 as compared with 51,000 for the Basidiomycetes enzyme (32) and 20,000-40,000 for the yeast enzymes (29)). The molecular weight of Helix pomatia exo-(l-3)-/3glucanase is considerably higher than that of most endo-( 1+3)-P-glucanases which have been studied, including that from Helix pomatia, which lie in the range IO-30,000. This situation, namely exoacting enzymes having molecular weights considerably higher than the corresponding exo-acting enzymes, is very common (33, 34). Unlike a number of endo-acting (l&3)/3-glucanases (10, 35), the Helix porn&u (l&3)-/3-glucanase does not show any tendency to lose activity in dilute solution, and is not stabilized by human serum
174
MARSHALL
albumin. The temperature stability (stable up to 50°C) and optimum temperature (55°C) and susceptibility to acid inactivation suggest that the enzyme does not possess any unusual stability properties. While certain microbial exo-( l-+3)-@glucanases are reportedly activated by manganese, cobalt and ferric ions (36, 37), the Helix pomatia enzyme is unaffected by manganese or cobalt, and markedly inhibited by ferric ions. A number of other workers have failed to demonstrate d:.o(l&3)-P-glucanase activation by manganese and cobalt ions (32, 3%40), and observed Fe3+ inhibition of the enzymes (39). The lack of inhibition of the Helix pomatia enzyme by cysteine, mercaptoethanol, oxidized glutathione and p-chloromercuribenzoate makes it seem unlikely that disulfide or thiol groups are involved in enzyme activity; the effect of phenylmercuric nitrate may not be meaningful since organic mercurial compounds may inhibit other enzymes which do not depend on thiol groups for activity (41-42). The inhibition by N-bromosuccinimide might indicate the participation of tyrosine, tryptophan or histidine residues in the activity of the enzyme. The involvement of tryptophan is, however, unlikely in view of the ineffectiveness of 2-hydroxy 5-nitrobenzylbromide as an inhibitor. The inhibition by mercuric, cupric, ferric and silver ions may support the involvement of histidine (43). Like many other glycosidases (10, 44-46) the Helix pomatia exo-(1 -+ 3)-P-glucanase is strongly inhibited by ammonium molybdate but the mechanism of inhibition by this reagent remains to be determined. The classification of enzymes releasing glucose from oligosaccharide and polysaccharide substrates has been discussed by Reese and co-workers (47). It is not possible to classify the Helix pomatia enzyme unambiguously on the basis of the relative rates of action on (l-3)-P-linked substrates of different chain lengths. The inversion of anomeric configuration which takes place during hydrolysis indicates that the enzyme is an exo-P-glucanase. However, the inhibition by low concentrations of gluconic acid Slactone, which has been considered an indication of ,&glucosi-
AND GRAND
dase activity argues against this conclusion. Thus it is not possible to classify the enzyme we have examined unambiguously by using the criteria of Reese and co-workers. This is a further difference between the Helix pomatia enzyme and other enzymes of this general type. We have preferred to consider the enzyme as an exo-P-glucanase because of its action on a wide range of P-glucan substrates, compared with its limited action on disaccharide substrates. REFERENCES 1. GRAND, R. J. A., AND MARSHALL, J. J. (1975) Comp. Biochem. Physiol. B, in press. 2. HOLDEN, M., AND TRACEY, M. V. (1950) Biochem. J. 47, 407-414. 3. MYERS, F. L., AND NORTHCOTE, D. H. (1958) J. Exp. Biol. 35, 639-648. 4. ANDERSON, F. B., AND MILLBANK, J. W. (1966) Biochem. J. 99, 682-687. 5. MARSHALL, J. J. (1974) Aduan. Carbohyd. Chem. Biochem. 30, 257-370. 6. MARSHALL, J. J. (1975) Advan. Carbohyd. Chem. Biochem. 32, in press. 7. MARSHALL, J. J. (1973) Comp. Biochem. Physiol. 44B, 981-988. 8. MARSHALL, J. J. (1973) Carbohyd. Res. 26, 274-277. 9. MARSHALL, J. J. (1973) Anal. Biochem. 53, 191-198. 10. MARSHALL, J. J. (1974) Carbohyd. Res., 34, 289-305. 11. NELSON, T. E., SCALE~I, J. V., SMITH, F., AND KIRKWOOD, S. (1963) Can. J. Chem. 41, 1671-1678. 12. LOWRY, 0. H., ROSEBROUGH,N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. 13. ROBYT, J. F., AND WHELAN, W. J. (1972) Anal. Biochem. 45, 510-516. 14. LLOYD, J. B., AND WHELAN, W. J. (1969) Anal. Biochem. 30, 467-470. 15. DUBOIS, M., GILLES, K. A., HAMILTON, J. K., REBERS, P. A., AND SMITH, F. (1956) Anal. Chem. 28, 350-356. 16. “Gel Chromatography,” Bio-Rad Laboratories, Richmond, California (1971). 17. “Sephadex Gel Filtration in Theory and Practice,” Pharmacia Fine Chemicals, Uppsala, Sweden. 18. “Whatman Advanced Ion-Exchange Celluloses. Laboratory Manual,” W. & L. Balston, Maidstone, Kent, England. 19. “Sephadex Ion Exchangers. An Outstanding Aid in Biochemistry,” Pharmacia Fine Chemicals, Uppsala, Sweden.
SNAIL
EXO-,%GLUCANASE
20. ORNSTEIN, L. (1964) Ann. N.Y. Acad. Sci. 121, 321-349. 21. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121, 404-427. 22. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chew 244, 4406-4412. 23. TREVELYAN, W. E., PROCTER,D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444-445. 24. MARSHALL, J. J. (1973) Biochem. Sot. Z’rans. 1, 143-145. 25. MARSHALL, J. J. (1973) J. Chromatogr. 76, 257-260. 26. NELSON, T. E., JOHNSON, J., JANTZEN, J., AND KIRKWOOD, S. (1969) J. Biol. Chem. 244, 5972-5980. 27. TINGLE, M. A., AND HALVORSON, H. 0. (1971) Biochim. Biophys. Acta 250, 165-171. 28. BROCK, T. D. (1965) Biochem. Biophys. Res. Commun. 19, 623-629. 29. ABD-EL-AL, A. T. H., AND PHAFF, H. J. (1968) Biochem. J. 109, 347-360. 30. BARRAS, D. R., AND STONE, B. A. (1969) Biochim. Biophys. Acta. 191, 342-360. 31. MARSHALL, J. J. (1974) Cereal Sci. Today 19,389. 32. HOUTARI, F. I., NELSON, T. E., SMITH, F., AND KIRKWOOD, S. (1968) J. Biol. Chem. 243, 952-956. 33. AHLGREN, E., ERIKSSON, K-E., AND VESTERBERG,0. (1967) Acta Chem. Stand. 21, 937-944.
175
34. MANNERS, D. J., AND MARSHALL, J. J. (1973) Phytochemistry 12, 547-553. 35. MOORE, A. E., AND STONE, B. A. (1972) Biochim. Biophys. Acta 258, 238-247. 36. FELLIG, J. (1960) Science 131, 832. 37. CHESTERS, C. G. C., AND BULL, A. T. (1963) Biochem. J. 86, 38-46. 38. VOGEL, K., AND BARBER, A. A. (1968) J. Protozool. 15, 657-662. 39. KAJI, A., OHSAKI, T., AND YOSHIHARA, 0. J. Agr. Chem. Sot. Jap. 45, 278-283. 40. MOORE, A. E., AND STONE, B. A. (1972) Biochim. Biophys. Acta 258, 248-264. 41. FISCHER, E. H., AND HASELBACH, C. H. (19.51) Helu. Chim. Acta 34, 325-334. 42. SOHLFX, M. R., SEIBERT, M. A., KREKE, C. W., AND COOK, E. S. (1952) J. Biol. Chem. 198, 281-291. 43. GURD, F. R. N., AND WILCOX, P. E. (1956) Aduan. Protein Chem. 11, 311-427. 44. MACWILLIAM, I. C., AND HARRIS, G. (1959) Arch. Biochem. Biophys. 84, 442-454. 45. MANNERS, D. J., AND SPARRA, K. L. (1966) J. Inst. Brew. 72, 360-365. 46. MANNERS, D. J., AND ROWE, K. L. (1969) Carbohyd. Res. 9, 107-121. 47. REESE, E. T., MAGUIRE, A. H., AND PARRISH, F. W. (1968) Can. J. Biochem. 46, 25-34.
