Characterization of a β-1,4-glucan hydrolase from the snail, Helix pomatia

Comp. Biochem. Physiol., 1976. Vol, 53B, pp. 231 to 237. Pergamon Press. Printed in Great Britain

CHARACTERIZATION OF A fl-I,4-GLUCAN HYDROLASE FROM THE SNAIL, H E L I X P O M A T I A * J. J. MARSHALL'~AND R. J. A. GRAND~ Department of Chemistry, Royal Holloway College (University of London), Englefield Green, Surrey, England and Laboratories for Biochemical Research, Howard Hughes Medical Institute and Department of Biochemistry University of Miami School of Medicine, Miami, FL 33152, U.S.A. (Received 8 October 1974)

Abstract--l. A fl-l,4-glucan hydrolase (fl-l,4-glucan 4-glucanohydrolase E.C. 3.2.1.4), purified to homogeneity from snail intestinal juice, has been characterized in terms of substrate specificity, kinetics of action and other properties. 2. The purified glucanase degrades polysaccharides and oligosaccharides containing fl-l,4-glucosidic linkages, the smallest substrate being cellotetraose. Glucans of the lichenin type, which contain both fl-l,3- and fl-l,4-glucosidic linkages, are also hydrolyzed. 3. The Michaelis constants of the enzyme for carboxymethylcellulose and lichenin are identical (1.5 mg/ml), but the maximum velocity is higher towards the former substrate. 4. The enzyme is without action on native cellulose and a number of other fl-l,4-glycans, but acts slowly on salep mannan. 5. Helix pomatia fl-l,4-glucanase has maximum activity at pH 5-5-6-0, optimum temperature 45°C, and is stable at temperatures up to 45°C. 6. The molecular weight as determined by polyacrylamide gel electrophoresis is 51,000. 7. The enzyme is inhibited by EDTA and stabilized and activated by calcium ions suggesting that, like fl-glucan hydrolases from plant and microbial sources, it is calcium-dependent.

INTRODUCTION CONSIDERABLE use has been made over the period of many years of the mixture of fl-giucanases present in snail intestinal juice, for the formation of yeast protoplasts and related purposes (Anderson & Millbank, 1966; Phaff, 1963). As yet, however, the constitution of this complex glucanase mixture has not been elucidated. For the most part, snail juice has been used unpurified, so that the nature and r01e of the enzymes functional in the degradation of microbial cell walls can only be guessed. In view of the importance of purified fl-glueanases of rigidly established specificity 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 (Marshall, 1974a, 1975a), we have undertaken the separation of the component glucanases of snail juice. In earlier papers (Marshall, 1973b-e) the development of affinity binding methods for the purification of fl-giucan hydrolases has been described, and the application of such a method to the purification of H e l i x pomatia endo-fl-l,4-glueanase reported (Marshall, 1973a). We now report on the properties, speci-

ficity and other aspects of the activity of this purified enzyme. MATERIALS A N D M E T H O D S

Snail intestinal juice

Glusulase, a crude mixture of snail juice enzymes, was purchased from Endo Laboratories. Garden City, New York, U.S.A. Substrates

Carboxymethyl (CM-)cellulose (Cellofas B) was from Imperial Chemical Industries Ltd. Insoluble laminarin was kindly provided by Dr. E. T. Dewar, Inveresk Research International, Inveresk, Midlothian, Scotland. Lichenin was extracted from Iceland Moss (Cetraria islandica) and purified as by Peat et al. (1957), or purchased from Koch-Light Laboratories, Colnbrook, Bucks., England. Oat and barley fl-glucans were prepared as by Preece & MacKenzie (1952). Cellulose powder (CF 1) was purchased from Whatman Biochemicals; cellotriose, cellotetraose and cellodextrin (average degree of polymerization, 12) were prepared by acid hydrolysis of powdered cellulose followed by charcoal column chromatography (Miller et al., 1960). Cellobiose was from Kerfoot. Esparto xylan was prepared as described by Chanda et al. (1950) and luteose was isolated from Penicillium luteum as by Anderson et al. (1939). Pachyman (Warsi & Whelan, 1957) was obtained from Professor W. J. Whelan and yeast fl-l,3-glucan was prepared as described by Manners & Masson (1969). Ivorynut mannan (Aspinall et al., 1953) was kindly provided by Professor J.-E. Courtois, Paris; salep mannan (Husemann, 1940) was a gift from Dr. John Tampion, London.

* Comparative studies on fl-glucan hydrolases, Part II. For Part I, see Marshall (1973a). t Investigator of the Howard Hughes Medical Institute. To whom enquiries and reprint requests should be directed at Department of Biochemistry, University of Miami School of Medicine, P.O. Box 520875, Biscayne Annex, Chemicals Human serum albumin (Cohn fraction V), cysteine hyMiami, FL 33152, U.S.A. ++Present address: Department of Biochemistry, Univer- drochloride, oxidized glutathione, N-bromosuccinimide, gluconic acid fi-lactone, p-chloromercuribenzoic acid, sity of Birmingham, Birmingham B15 2TT, England. 231

232

J.J. MARSHALLAND R. J. A. GRAND

iodoacetic acid, iodoacetamide and 2-hydroxy 5-nitrobenzylbromide were purchased from the Sigma Chemical Company, London, Ltd.

Enzyme purification

4.8

Snail juice endo-/~-l,4-glucanase was prepared as described previously (Marshall, 1973a). Samples from stock solutions of concentration 10-12 U/ml were used after suitable dilution, if necessary, for all the experiments described.

4.6

Gel electrophoresis This was performed in polyacrylamide gels at pH 8.9, as described by Ornstein (1964) and Davis (1964). The mol. wt of the enzyme was determined by polyacrylamide gel electrophoresis under dissociating conditions as described by Weber & Osborn (1969).

4.4

4.2

Analytical measurements Total carbohydrate contents of substrate solutions were determined by the phenol-sulfuric acid method (Dubois et al., 1956). Other analytical methods have been described previously (Manners & Marshall, 1969, 1973; Marshall, 1973a, c, 1974bl. Enzyme action on various substrates was tested using digests of composition essentially as described previously (Marshall, 1973a), exact compositions being given in the text. RESULTS

Homogeneity and molecular weight of snail endo-~-l,4glucanase Electrophoresis in polyacrylamide gels under nondissociating conditions and in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol showed a single protein band in both cases, confirming the homogeneity of the purified enzyme. The mobility of the protein in the presence of SDS and mercaptoethanol, compared with the mobilities of standard proteins (Fig. 1), corresponded to a polypeptide chain of mol. wt approx 51,000.

Stability properties of purified snail endo-~-l,4-glucanase (a) Effect of dilution. Samples (50 #1) of purified enzyme solution (concentration 5.7 U/ml) were diluted 6-fold by addition to water and to human serum albumin solution (0'25 mg/ml) at 22°C. The activities of the diluted solutions were determined after various lengths of time at this temperature by incorporation of samples (10 #1) into digests containing CM-cellulose (2.5 mg), buffer (500 mM acetate pH 5-3, 0.25 ml) and human serum albumin (0.25 mg) in a total vol of 1-0 ml. The human serum albumin was incorporated into all the digests for activity determinations to prevent any loss of activity which might have taken place during the dilution and incubation period involved (cf. Moore & Stone, 1972; Marshall, 1974b). There was, however, no detectable loss in enzymic activity in the diluted solutions, either in the presence or absence of serum albumin, during .the period of 4 hr at 22°C. (b) Effect of freezing/thawing and freeze drying. The effect of freezing and thawing on the enzyme was studied by addition of samples of enzyme solution (50 #1, concentration 5.7 U/ml) separately to 250 #1 of water and to 250 #1 of human serum albumin solution (0.25 mg/ml), freezing the solutions and storing at - 15°C for 2 days, followed by thawing by standing

4.0 0

I 0.2

I 0.4

I 0.6

I 0.8

Mobility

Fig. 1. Determination of the mol. wt of Helix pomatia fl1,4-glueanase by polyacrylamide gel electrophoresis in the presence of SDS and 2-mercaptoethanol. 1,2 and 3 are standard proteins (ovalbumin, chymotrypsinogcn and ribonuclease). The mobilities are expressed relative to that of tracking dye (bromophenol blue). The arrow shows the position of migration of/~-l,4-glucanase.

at room temperature. Samples of the diluted solutions (with and without albumin) were freeze-dried during the period of about 3 hr, and then re-dissolved in the original vol of water. The activities of the frozen and thawed and the freeze-dried, redissolved enzyme solutions were compared with that of a freshly diluted enzyme solution, by incorporation of samples into digests of similar composition to those used for investigation of the dilution effect above, but with 1.25 mg of calcium chloride present. Freezing and thawing did not result in any loss of activity, either in the presence or absence of albumin. Freeze-drying caused losses in activity which were approx the same in the presence and absence of albumin (30 and 25~o respectively).

Effect of buffer composition and pH on snail endo-fl-l,4glucanase activity Enzymic activity was measured at several pH values using digests containing substrate (CM-cellulose, 2-5 mg), buffer (citrate, final concentration 10 mM) and enzyme solution (5 #1) in a total vol of 1-0 ml, with and without calcium chloride (1.25 mg). The results (Fig. 2) show the enzyme to have optimum activity between pH 5.5 and 6.0, and to be slightly more active in the presence of calcium ions. Substitution of acetate buffers for citrate had no effect on the activity.

Optimum temperature and heat stability The optimum temperature of the enzyme was determined using digests containing substrate (CM-cellulose. 2.5 rag), acetate buffer (500 mM, pH 5.3, 0.25 mll and enzyme (5 #1) with and without calcium chloride (1.25 rag). After incubation of the digests at

Snail "cellulase" 12

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Temperature (°C)

I 7.0

pH

Fig. 2. Dependence of activity of Helix pomatia fl-l,4-glucanasc on pH, determined in the presence (O) and absence (e) of calcium ions. For conditions, see the text.

Fig. 3. Dependence of activity of Helix pomatia /~-l.4-glucanase on temperature, determined in the presence (©) and absence (I) of calcium ions. For conditions, see the text.

various temperatures for 30 min, enzyme action was terminated by addition of alkaline copper reagent and the quantity of reducing sugars liberated determined. The effect of temperature on the stability of the enzyme was investigated by using digests of the same composition, substrate solution being added after preincubation of the other constituents of the digests at various temperatures for 30 min, followed by incubation at 37°C for 30 min. Activities were expressed relative to those determined in control digests incubated at 37°C. The results are shown in Figs. 3 and 4.

Kinetic constants for snail endo-fl-l,4-glucanase

Substrate specificity of snail endo-fl-l,4-glucanase The action of the purified enzyme was tested on a number of fl-linked glucose polymers and other related polysaccharides. Digests (5"0 ml) contained polysaceharide or oligosaccharide (2.5 mg/ml), acetate buffer (100 raM, pH 5.3, 1.25 ml), calcium chloride 0.25 mg.ml) and enzyme (0.2 U) and were incubated at 37°C. Aliquots were analyzed for reducing sugars after various incubation times and samples (25-50 pl) were examined by paper chromatography to identify the products of enzyme action. The results are summarized in Table 1.

Initial rates of enzyme action were determined in digests containing substrate (CM-cellulose or lichenin 0-2-7-5 rag), calcium chloride (1.25 rag), acetate buffer (500 raM, pH 5-3, 0.25 ml) and enzyme solution (5 ld). The kinetic constants for the enzyme acting on the two substrates were determined using Lineweaver-Burk double reciprocal plots (Fig. 6) and gave K m values for both lichenin and CM-cellulose of 1.5 mg/ml and V values for these substrates of respectively 14.5 anga]7.7 pmole reducing groups liberated/ ml enzyme per min.

Inhibition of snail endo-fl-l,4-glucanase The effect of various metal ions and other reagents on the enzymic activity was determined using digests containing acetate buffer (500 mM pH 5.3, 0.25 ml), substrate (CM-cellulose, 2.5 rag), enzyme (5 #1, concentration 3'8 U/ml) and the reagent under test (final concentration 1 mM) in a total vol of 1.0 ml. The substrate was added after preincubation of the other digest constituents for 15 min at 37°C. Appropriate control digests were included to correct for the effect 100 80

Action pattern of snail endo-fl-l,4-glucanase The action pattern of the purified enzyme was examined by following the decrease in viscosity and rate of production of reducing groups during hydrolysis of carboxymethyl cellulose. A digest (5'0 ml) containing substrate (25 mg), acetate buffer (pH 5'3, final concentration 25 mM), calcium chloride (1'25 mg/ml) and enzyme (0-04 U) was incubated at 25°C and samples (0.025 ml) removed at intervals for reducing power determination. The viscosity of the digest was determined at various times by measuring the flow times in a miniature Ostwald viscometer at 25°C. The results are shown in Fig. 5.

~>, 6o ~

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0

20

l0

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80

Temper ature (°C)

Fig. 4. Heat stability of Helix pomatia fl-l,4-glucanase, in the prcscncc 1©1 and absence (0) of calcium ions. For details, see the text.

234

J. J. MARSHALLAND R. J. A. GRAND Table 1. Action of Helix pomatia/~-l,4-glucanase on various glycans Initial rate of hydrolysis (expressed relative to CM-cellulose as 100)

Substrate*

Extent of hydrolysis after 30 hr (%)

Average degree of polymerization of products after 30 hr

CM -cellulose

100

17.3

5"8

Cellodextrin

100

27

2.9

Lichenin

31

Barley glucan Oat glucan Salep mannan

16 8 0.1

Cellotetraose Cellulose

6,5

15'3

[ 1.6 10.7 3.3

8.6 9,4 N.D.

very slow very slow

3. I 0,15

Chromatographicallymobile products of enzyme action+ G( 4- ) G2( + I G3( + ) G4(+) G5(+ + +) G6(+ + +) tr G2(+ + ) G3(+ +) G4(+ +) GS(+ +) tr G(±) G2(+) G3(+) G ~ ± ) G5(+I G6(+ +) G4(+ + +) G4(+ + +) unidentified oligosaccharides G2(+ + +) not examined

N.D. ND.

* The following were not hydrolyzed: cellobiose, cellotriose, yeast glucan, pachyman, esparto xylan, cellulose, fl-l,4mannan, laminarin. No reversion products were formed from glucose. t G, G2-G6 indicate glucose and glucose oligomers of DP 2-6 respcctively. The relative amounts are indicated by + + +, heavy; + +. medium; + light; + trace, tr. indicates the presence of unidentified products presumed to arise by transferase action. In the case of CM-cellulose the oligosaccharides indicated by G5 and G6 may not be cellopentaose and cellohexaose, but rather carboxymethylated oligosaccharides of different D.P. N.D. indicates not determined of the inhibitors on reducing power measurements. The results are given in Table 2.

DISCUSSION

The application of an affinity binding method, based on selective interaction with microgranular DEAE-cellulose has enabled us to isolate a fl-l,4-glucan hydrolase from the complex mixture of enzymes in the intestinal juice of the snail, Helix pomatia (Marshall, 1973a). The enzyme is homogeneous by the criterion that it migrates as a single band during polyacrylamide gel electrophoresis under dissociating and non-dissociating conditions. The mol. wt of the enzyme, as determined by polyacrylamide gel electrophoresis in SDS (Fig. 1) is 51,000, a value considerably higher than the preliminary estimate based on gel filtration measurements (Marshall, 1973a). The reason for the discrepancy is anomalous retardation of the enzyme on polyacrylamide gel columns, a phenomenon which has recently been observed (J. J.

,ooi

Marshall, unpublished work) in the case of several fl-glucan hydrolases, and which will be discussed in detail elsewhere. The value of 51,000 for the mol wt of this animal fl-l,4-glucanase is similar to that of most microbial fl-l,4-glucanases which are in the range of 40-60,000 (summarized by Keilich et al., 1969, 1970) Several microbial and plant fl-glucanases have been shown to be unstable in dilute solution and susceptible to inactivating during freezing and thawing, and to be protected by the presence of serum albumin under such conditions (Miller & Birzgalis, 1961; Moore & Stone, 1972; Marshall, 1974b). In contrast, the purified snail fl-l,4-glucanase shows no evidence of instability in dilute solution or on freezing, and only moderate inactivation takes place during freeze drying. There is no evidence to suggest that serum albumin stabilizes this enzyme. It is not known whether the difference between the stability properties of the fl-glucanases from these different sources is the result of structural differences or whether it is due to other factors such as traces of proteolytic enzymes

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60

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20

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40

60

80

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Duration of Incubation (min)

Fig. 5. Decrease in specific viscosity (q~,,) and production of reducing groups (expressed as ~o hydrolysis) during action of Helix pomatia fl-l,4-glucanase on CM-cellulose. For details, see the text.

J 0

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1/[sl tmo-!mO Fig. 6. Lineweaver-Burk double reciprocal plot of the dependence of initial velocity, V on substrate concentration, IS], for CM-cellulose (Q) and lichenin (A). For details, see the text.

Snail "cellulase"

235

In an effort to identify functional groupings involved in the activity of the enzyme, the effect of Compound* Inhibition ('~,,)t several reagents which modify amino acid side chains in proteins was investigated. Since inhibition was not caused by treatment with cysteine, 2-mercaptoethanol, Mercuric chloride 100 EDTA (15 minutes preincubation) 0 p-chloromercuribenzoate or oxidized glutathione, it is EDTA (overnight preincubation) 35 unlikely that thiol or disulfide groups are involved Silver nitrate 61 in the activity of the enzyme. The considerable inactit:erri¢ ¢hhwidc 63 vation caused by phenylmercuric nitrate may be due Copper sulphate 93 to reaction with some other type of amino acid side Ammonium molybdate 61 chain, since this rbagent has been shown to inhibit N -Bromosuccinimide 80 enzymes which do not depend on sulfhydryl groups Phenylmercuric nitrate 57 for activity (Sohler et al., 1952). The inhibition by 2-Hydroxy 5-nitrobenzylbromide 30 2-hydroxy 5-nitrobenzyl bromide and N-bromosuc* All used at final concentrations of 1 mM. The follow- cinimide suggests the involvement of tryptophan in ing were without effect: lead acetate, cobalt chloride, zinc the activity of the enzyme, although it cannot be sulfate, manganese chloride, sodium chloride, cysteine, oxi- excluded that histidine and tyrosine, which also react dized glutathione, gluconic acid 6-1actone, p-chloromercur- with N-bromosuccinimide, are involved. The lack of ibenzoate, 2-mercaptoethanol, potassium cyanide, sodium inhibition by gluconic acid fi-lactone indicates that azide, sodium fluoride, cellobiose, iodoacetic acid and the enzyme is a fl-glucan endo-hydrolase, rather than iodoacetamide. an exo-hydrolase (Reese et al., 1968). t All activities are expressed relative to the activity deterThe purified Helix pomatia fl-l,4-glucanase has a mined in the presence of calcium chloride. specificity enabling it to split fl-l,4-glucosidic linkages in soluble cellulose derivatives such as CM-cellulose, cellodextrins and cellulose-derived oligosaccharides of degree of polymerization four or more, with formation of cellobiose as the principal chromatographiwhich are frequently present in microbial enzyme cally-mobile product. Action on CM-cellulose and preparations (cf. Marshall, 1974b). cellodextrin yielded some oligosaccharides thought to The rather higher optimum pH of the snail fl-l,4- be the result of transferase action. The enzyme does glucanase (5.5-6-0), compared with that of most other not act on cellobiose or cellotriose, showing that at fl-l,4-glucanases which have been studied, presumably least one glycosidic linkage must be present on each reflects the differences in pH of the environments in side of the linkage being hydrolyzed. The slow rate which these enzymes act in vivo. The enzyme is of hydrolysis of cellotetraose shows that this condineither markedly heat stable, nor heat labile, nor does tion by itself is not, however, sufficient for rapid hyit show any unusual heat stability properties such as drolysis and that the maximum rate is only observed have been observed with a fungal fl-glucanase (Mar- when the glycosidic linkage hydrolyzed is part of a somewhat longer sequence of fl-l,4 glycosidic linkages. shall, 1974b). Helix pomatia fl-l,4-glucanase shows slightly The ability of the enzyme to partly degrade salep greater (10-15~o) activity in the presence of calcium mannan, but not ivory-nut mannan, probably indithan in its absence (Fig. 2), and is stabilized by this cates that action on the former substrate is restricted cation during heat treatment (Fig. 4). Plant fl-glu- to portions of the polysaccharide where the glucose canases have previously been shown to be calcium residues are situated. The large decrease in viscosity dependent (Manners & Marshall, 1969), and the same of CM-cellulose associated with a small extent of is also true of certain microbial fl-glucanases (J. J. hydrolysis (50~ decrease in viscosity being observed Marshall, unpublished work). Although the experi- at an extent of hydrolysis of about 0'5~o, Fig. 5) shows ments reported were performed with CM-cellulose that the enzyme acts in an endo fashion. Like many as substrate, and are thus subject to the possible criti- other fl 1,4-glucan hydrolases, the Helix pomatia cism that the observed effect is due to interaction of enzyme is without action on native cellulose suggestcalcium ions with the charged substrate, rather than ing that, in vivo, as is the case with the corresponding with the enzyme, this is unlikely. A similar extent of microbial enzymes, it requires the prior or concomiactivation was observed when the uncharged sub- tant action of a C1-cellulase (Selby, 1969) to effect strate, lichenin, was used. Furthermore, the inhibition the degradation of this insoluble polysaccharide. In common with most other endo-fl 1,4-glucanases by EDTA confirms this conclusion. Pre-incubation of the enzyme with this chelating agent at a con- (Barras et al., 1969), the Helix pomatia enzyme degrades centration of 1 mM for 15 min had no effect on the mixed-linkage (fl-l,3; fl-l,4) glucans such as lichenin activity; overnight pre-incubation at the same con- and barley glucan, in addition to substrates containcentration resulted in 35~o inhibition, presumably due ing only fl-l,4-glucosidic linkages. Comparison of the to the removal of some very firmly bound calcium kinetic constants for the enzyme acting on lichenin ions present in the enzyme. Helix pomatia fl-l,4-glu- and CM-cellulose show the K,, for both to be the canase was inhibited by a number of heavy metal same, indicating that the fl-l,3-glucosidic linkages do ions (Hg 2+, Ag ÷, Fe 3 +, and Cu 2+) but not by Others not interfere with enzyme-substrate binding. They do, (Pb 2+, Co z+, Zn 2+, and Mn2 +). In this regard it does however, cause a decreased catalytic rate, because the not differ significantly from other plant and micro- maximum velocity of enzyme action is greater on bial fl-glucanases which have been examined pre- CM-cellulose. While the rate of enzyme action on lichenin is relatively fast, the extent of enzyme action viously (Mandels & Reese, 1963). Table 2. Inhibition of Helix pomatia fl-l,4-glucanase

236

J.J. MARSHALLAND R. J. A. GRAND

is small, and the major products of enzyme action are chromatographically immobile. While the rate of action on the cereal glucans is lower, the extent of hydrolysis is greater and paper chromatography showed the presence in the products of major amounts of what appeared to be a tetrasaccharide, together with chromatographically immobile oligosaccharides. Clearly lichenin and the cereal glucans differ markedly in their susceptibility to hydrolysis by Helix pomatia fl-l,4-glucanase, this presumably reflecting differences in the arrangements of the two types of glycosidic linkages in these polysaccharides. The fast initial rate of action on lichenin, and the appearance of some low mol. wt products may indicate the presence of segments of chain containing significant numbers of contiguous /~-l,4-1inked glucose residues which are readily susceptible to the action of the enzyme. Such arrangements are presumably not present in any significant amount in the cereal glucans so that enzyme action takes place at susceptible linkages in short segments of fl-l,4-1inked glucose units, with fl-l,3-1inkages in the immediate vicinity. The action of Helix pomatia fl-l,4-glucanase on the mixed linkage substrates differs from that of the other purified fl-l,4-glucanases whose action on these substrates has been tested. Thus Streptomyces (Parrish et al., 1960; Perlin & Suzuki, 1962) and Aspergillus niger (Clarke & Stone, 1965, 1966) cellulases extensively degraded lichenin and the cereal glucans, giving largely trisaccharide and tetrasaccharide products, together with some glucose and disaccharides. Further work is in progress to examine in more detail the action of Helix pomatia fl-l,4-glucanase on these polysaccharides, including characterization of the major low mol. wt product produced during action on oat and barley glucans. The Helix pomatia enzyme may be of value in the elucidation of the fine structures of the mixed-linkage fl-glucans in view of the apparent difference in its hydrolytic action from that of the other known fl-l,4-glucanases on these polysaccharides (Marshall, 1974a, 1975a).

CLARKE A. E. & STONE B. A. (1965) Properties of a fl-l,4glucan hydrolase from Aspergillus niger. Biochem. J. 96, 802-807. CLARKE A. E. t~ STONE I. A. (1966) Enzymic hydrolysis of barley and other fl-glucans by a fl-(l ~ 4)-glucan hydrolase. Biochem. J. 99, 582-588. DAVIS B. J. (1964) Disc electrophoresis--II. Methods and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427. DUBOIS M., GILLES K. A., HAMILTONJ. K., REBERSP. A. & SMITH F. (1956) Colorimetric method for the determination of sugars. Analyt. Chem. 78, 350-356. HUSEMANN E. ( 1 9 4 0 ) Macromolecular compounds--CCXLVI. The constitution of salep mannan. J. prakt. Chem. 155, 241 260. KEILING G., BAILEYP. J., AFTING E. G. & LIESE W. (1969) Cellulase (fl-l,4-glucan 4-glucanohydrolase) from the wood-degrading fungus Polyporus schweinitzii Fr.--ll. Characterization. Biochim. biophys. Acta 185, 392-401. KEILICH G., BAILEYP. & LIESEW. (1970) Enzymatic degradation of cellulose, cellulose derivatives and hemicelluloses in relation to the fungal decay of wood. Wood Sci. Technol. 4, 273 283. MANNERSD. J. t~ MARSHALLJ. J. (1969) Studies on carbohydrate-metabolizing enzymes--XXII. The fl-glucanase system of malted barley. J. inst. Brew. 75, 550-561. MANNERS D. J. & MARSHALLJ. J. (1973) Some properties of a fl-l,3-glucanase from rye. Phytochem. 12, 547 553. MANNERS D. J. 8z MASSON A. J. (1969) The structures of two glucans from yeast-cell walls. FEBS Lett. 4, 122 124. MARSHALLJ. J. (1973a) Purification of a fl-l,4-glucan hydrolase (cellulase) from the snail, Helix pomatia. Comp. Biochem. Physiol. 44B, 981-988. MARSHALLJ. J. (1973b) Use of ion-exchangers for the purification of fl-glucan hydrolases. Biochem. Soc. Trans. 1, 198-200. MARSHALL J. J. (1973c) Behavior of fl-glucan hydrolases on ion-exchangers. Analyt. Biochem. 53, 191-198. MARSHALL,J. J. (1973d) Nature of the binding of a fl-l,4glucan hydrolase to ion-exchangers. J. Chromat. 76, 257-260. MARSHALL J. J. (1973e) Separation and characterization of the fl-D-glucan hydrolases from a species of Cytophaga. Carbohyd. Res. 26, 24~277. MARSHALL J. J. (1974a) Application of enzymic methods to the structural analysis of polysaccharides Part I. Adv. Carbohyd. Chem. Biochem. 30, 257-370. Acknowledgement--This work was supported, in part, by MARSHALL J. J. (1974b) Studies on the structure and a grant from S.R.C. mechanism of action of glycoside hydrolases. Part 1: Purification and study of some factors affecting the activity of Rhizopus arrhizus (1 ~ 3)-fl-D-glucanase. Carbohyd. Res. 34, 289-305. MARSHALL J. J. (1975a) Application of enzymic methods REFERENCES to the structural analysis of polysaccharides Part II. Adv. Carbohyd. Chem. Biochem. (In preparation). ANDERSON C. G., HAWORTH W. N., RAISTRICK H. & STACEY M. (1939) Polysaccharides synthesized by microor- MARSHALLJ. J. (1975b) Degradation of barley glucan by a purified (1--4)-fl-D-glucanase from the snail, Helix ganisms--IV. The molecular constitution of luteose. Biopomatia. Carbohyd. Res. 42, 203-207. chem. J. 33, 272-279. ANDERSON F. B. & MILLBANK J. W. (1966) Protoplast for- MARSHALL J. J. & GRAND R. J. A. (1975) Comparative studies on fl-glucan hydrolases. Isolation and characterimation and yeast cell-wall structure. The action of the enzymes from the snail, Helix pomatia. Biochem. J. 99, zation of an exo(1 ---, 3)-fl-glucanase from the snail, Helix pomatia. Archs. Biochem. Biophys. 167, 165-175. 682-687. ASPINALL G. O., HIRST E. L., PERCIVAL E. G. V. & W1L- MANDELSM. & REESEE. T. (1963) Inhibition of cellulases LIAMSONI. R. (1953) The mannans of ivory nut (Phyteleand fl-glucosidases. In Advances in Enzymic Hydrolysis phas macrocarpa)--1. The methylation of mannan A and of Cellulose and Related Materials. pp. 115-157. PergaB. J. chem. Soc. 3184-3188. mon Press, Oxford. BARRASD. R., MOOREA. E. & STONEB. A. (1969) Enzyme- MILLER G. L., DEAN J. & BLUM R. (1960) A study of substrate relationships among fl-glucan hydrolases. In methods for preparing oligosaccharides from cellulose. Cellulases and Their Applications. Adv. Chem. Set. No. Archs. Biochem. Biophys. 91, 21-26. 95, pp. 105-138. A.C.S., Washington. MILLER G. L. & BIRZGALISL. (1961) Protective action of CHANDA S. K., HIRST E. L., JONES J. K. N. & PERCIVAL bovine serum albumin in measurements of activity of E. G. V. (1950) Constitution of xylan from esparto grass low concentrations of cellulase. Analyt. Biochem. 2, 393(Stipa tenacissima). J. chem. Soc. 1289-I 297. 395.

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