THE USE OF ENZYMES IN STRUCTURAL STUDIES ON POLYSACCHARIDES

Transcription

1 THE USE OF ENZYMES IN STRUCTURAL STUDIES ON POLYSACCHARIDES I. THE MODE OF ATTACK OF A 6-D-(1 -+ 3)-GLUCANASE ON LAMINARIN' T. E. NELSON, J. V. SCALETTI, F. SMITH, AND S. KIRKWOOD Departments of Agricultural Biochemistry and Animal Husbandry, University of Minnesota, St. Paul, Minnesota, U.S.A. Received October 19, 1962 ABSTRACT A 0-D-(1 -+ 3)-glucanase from the Basidiomycete sp. QM 806 of Reese and Mandels has been purified and shown to hydrolyze insoluble laminarin by removing glucose residues one at a time from the non-reducing end of the polysaccharide. The enzyme will also hydrolyze simple oligosaccharides and its mode of attack on these is similar to its action on laminarin. The products ~roduced by the action of this preparation on laminarin are consistent with recent information on the structure of this polysaccharide. Reese and R'Iandels (I), in a survey of the occurrence of P-D-(1 -+ 3)-glucanases in fungi, observed that an unidentified species of basidiomycete produced a very high level of P-D-(1 -+ 3)-glucanase activity when grown on a starch medium. The organism was designated Basidiomycete sp. QR;I 806. The criterion for activity was the extent of attack on the seaweed polysaccharide laminarin. The enzyme brought about the complete hydrolysis of laminarin, as judged by reducing value calculated as glucose. The hydrolysis products produced by the action of this enzyme were investigated (paper chromatography) an-d glucose was the only product observed. Reese and i\llandels point out that this observation, coupled with the fact that the enzyme had a very slow rate of attack on a mixture of oligosaccharides produced from laminarin by the action of a glucanase from Rhizopz~s arrhizus, leads to the conclusion that the basidiomycete enzyme attacks laminarin in an exo or endwise splitting fashion. Insoluble laminarin, a cold water insoluble polysaccharide from the brown algae Laminaria cloustoni, has been shown to contain rnannitol (2) to which are attached chains composed of glucose residues. Furthermore, the glucose chains in laminarin are linked by both P-(1 -+ G )and P-(1 -+ 3) bonds (3, 4). Recent investigations have shown that laminarin is heterogeneous and is composed of at least two components. One is a reducing substance, termed laminarose, which consists of glucose residues linked by both P-(1 -+ 6) and P-(1 -f 3) bonds. The other component is a non-reducing substance, termed laminaritol, which appears to be two chains similar to laminarose, both terminating in the same mannitol residue (5). In view of these structural findings it is surprising that the basidiomycete enzyme produces only glucose from laminarin and one must assume that either the preparation is a mixture of enzymes or a single enzyme with a very low order of specificity. In either case the preparation should produce either mannitol or some fragment containing mannitol froin that portion of the laminarin structure containing this substance. In order to investigate these matters further, we have undertaken a study of the action pattern of a purified preparation of this enzyme with the view of using it in structural studies on laminarin and other polysaccharides of similar structure. 'Paper No. 4964, Scientijc Journal Series, Minnesota Agricultural Experiment Station. This work was supported by a grant from the National Institutes of Health. Canadian Journal of Chemistry. Volume 41 (1963) 1671

2 1672 CANADIAN JOURNAL OF CHEMISTRY VOL 41, 1963 Action of the Purz$ed Enzyme on Laminarin The action of our purified preparation on laminarin2 was investigated and compared with the results reported by Reese and Mandels. The enzyme was incubated with laminarin and the increase in reducing power with time followed by the Nelson-Somogyi method (6). Companion determinations of the glucose liberated were carried out by a procedure involving the use of glucose oxidase (7). The results of the experiment (Fig. 1) FIG. 1. Action of the enzyme at high concentration on laminarin, on periodate-oxidized and reduced laminarin. The concentration of eilzyme used was fivefold that used in the laminarin experiment, the results of which are shown in Table I. The release of reducing power, as glucose and glyceritol, was negligible in the absence of enzyme. (b release of glucose equivalent from laminarin as determined by the Nelson- Somogyi procedure; A release of glucose from laminarin as determined by glucose oxidase; 0 release of glucose equivalent from periodate-oxidized and reduced laminarin as determined by the Nelson-Somogyi procedure; A release of glucose from periodate-oxidized and reduced laminarin as determined by glucose oxidase; X release of glyceritol from periodate-oxidized and reduced laminarin. essentially confirm the observations of Reese and Mandels that the increase in reducing power upon enzymatic hydrolysis is due largely to the production of glucose. A further implication of these data is that contamination of this preparation with an endo-type glucanase must be small since the presence of such an enzyme would result in a large difference in the values obtained by the two methods. It should be pointed out also that the fact that the two determinations do not coincide indicates that the enzyme is producing significant amounts of reducing substances other than glucose. Such a result is to be expected on the basis of what is known of the structure of laminarin. In order to make a more definite estimation of the extent of contamination by an endotype enzyme, the preparation was incubated with purified undergraded samples of oat and barley gums at five times the enzyme level used in the laminarin experiment, the results of which are given in Table I. No increase in reducing capacity was observed (Table I). Since both oat and barley gums are known to contain both P-(1 -+ 3) and P-(1 -+ 4) linkages (8-11), the 0-( ) linkages occurring both singly and together (12), it is reasonable to assume that an endo-type enzyme would attack these polysaccharides. In fact recent work by Parrish, Perlin, and Reese (13) has shown that the endo-glucanase from R, arrhzzz~s does indeed produce extensive degradation of both of these polysaccharides. The conclusion may therefore be drawn that our preparation has little or no contamination with an endo-type enzyme. In addition, since cellulase has 2The term "laminarin" is used throughout this paper to denote a preparation of L. cloustoni laminarin obtained from the Liaerpool Borax Co., St. Paul's Square, Litlerpool 3, England.

3 NELSON ET AL.: STRUCTURAL STUDIES ON POLI'SACCHARIDES TABLE I Comparison of the rate of attack on laminarin with that on derivatives of laminarin and on oat and barley gum Substrate Enzyme units added Increase in reducing capacity AODa~n/l5 min Larninarin Periodate-oxidized and reduced laminarin Periodate-oxidized, reduced, and mild acid hydrolyzed laminarin Air-oxidized laminarin Reduced laminarin Oat poly-p-d-glucan Barley poly-0-d-glucan NOTE: All final substrate concentrations were at 0.05%. Determinations were made by adding to 0.50 ml substrate in l.I acetate, ph 4.8, 0.50 ml enzyme solution in the same buffer and incubating for 15 minutes at 37'. The hydrolysis was stopped by the addition of 1.0 ml of Nelson-Somogyi alkaline copper reagent. been shown to degrade oat and barley gums (13), it can be concluded that the cellulase contamination is negligible. Action of the Enzyme on Chemically il.fod$ied Laminarins In order to establish the mode of attack of the glucanase on laminarin, various derivatives of this polysaccharide were prepared in which the reducing and/or non-reducing ends had been altered. By comparing the rate of action of the enzyme on each of these with its rate of attack on unaltered laminarin, it has been possible to reach certain conclusions concerning its mode of attack. Samples of laminarin in which the reducing end of the laminarose component had been altered were-prepared by air oxidation in the presence of dilute alkali and by sodium borohydride reduction. The former procedure converts the reducing ends to acid residues, the latter procedure converts them to glucitol residues. In addition, a sample of laminarin that had been partially purified by dialysis and reprecipitation from water was oxidized with dilute sodium metaperiodate for several days in the dark at 4' and then reduced with sodium borohydride. This procedure alters both the reducing and non-reducing ends of laminarose (see Fig. 2) and laminaritol. The remaining linked residues are, however, left intact (12, 14). A portion of this periodate-oxidized and reduced material was subjected to mild acid hydrolysis at room temperature. The hydrolysis regenerates the non-reducing ends by removing the altered residues and freeing the adjacent intact glucose moieties. It should be noted that this regenerated laminarin resembles the original material only at the non-reducing terminals, the other end of the molecule is probably terminated by an arabitol residue in the case of laminarose and a glyceritol residue in the case of laminaritol. The action of the purified enzyme on these substrates is shown in Table I. An enzyme concentration was used such that the rate of attack on unaltered laminarin was at the maximum value that could be measured with any accuracy. This was done so that very low rates of attack on the modified substrates could be detected and compared with the rate on the unaltered material. As can be seen in Table I, laminarin modified only at the reducing end of the molecule by air oxidation or sodium borohydride reduction is attacked at a rate only slightly less than that characteristic of untreated laminarin. Periodate-oxidized and reduced laminarin, in which both the reducing and non-reducing ends have been altered, is attacked at 1/100

4 CANADIAN JOURNAL OF CHEMISTRY. VOL 41, 1963 CHH C H r [ 4 Ho CH2:H cl CHO r O HOH o periodate CH20H '"gh H,, O OH ~ ~ < ~ ~ O 1, OH 1 - FIG. 2. Structural representation of periodate oxidation, reduction, and mild acid hydrolyses on the laminarose component of laminarin. The branching known to occur in this molecule is ignored for purposes of illustration. The ends of all branches will react in the same fashion as the non-reducing terminal illustrated in the drawing. or less of this rate. The increase in reducing value was so low that it borders on experimental error. The rate of enzyme attack on the periodate-oxidized polysaccharide (tested prior to reduction) was also very low. However, In this case the high reducing value given by the substrate makes the rate of attack-measurement much less accurate. Mild acid hydrolysis of the periodate-oxidized and reduced laminarin, which removes the blockage at the non-reducing end, resulted in a product that was almost as susceptible to enzymatic attack as the original laminarin. This is highly significant since it rules out the possibility that the failure of the enzyme to attack the periodate-oxidized and reduced molecule was due to a structural modification at a point other than the non-reducing terminal. Thus it can be stated conclusively that this glucanase preparation from Basidiomycete sp. QM 806 acts upon laminarin from the non-reducing end of the molecule, removing one residue at a time. Modified laminarin is slowly degraded by the enzyme (Fig. 1). This may be attributed to a limited ability of the glucanase to attack the modified non-reducing end unit, to a slight endo-glucanase contamination, or to spontaneous hydrolysis of the non-reducing ends, allowing enzymatic attack on the exposed glucose residues. These possibilities can be differentiated readily by the fact that hydrolytic cleavage of the altered end unit liberates glyceritol, whereas this polyol would not be formed by attack along the interior of the chain. The data (Fig. 1) show a steady release of glyceritol as well as glucose, and since the modified polysaccharide does not decompose spontaneously at the ph of the enzymatic reaction, the glucanase preparation must have a limited ability to attack the altered non-reducing end unit.

5 NELSON ET AL.: STRUCTURAL STUDIES ON POLYSACCHARIDES 1675 Products of the Action of the Enzyme on Laminarin Aliquots removed at various periods during the hydrolysis of laminarin by the glucanase were examined by paper chromatography. Glucose was the major observable product in all hydrolyses, and in those which were virtually complete it accounted for about 70y0 of the carbohydrate as measured by the phenol - sulphuric acid method (15). In addition, laminaribiose, gentiobiose, laminaritriose, and several higher oligosaccharides appeared. It was shown by elution of the material from the chromatogram, followed by acid hydrolysis and paper electrophoresis in borate, that at least two of the higher oligosaccharides contained mannitol. As the course of the hydrolysis proceeded, laminaritriose and most of the higher oligosaccharides gradually disappeared while gentiobiose remained constant and laminaribiose increased. This pattern of the appearance and disappearance of products during the course of hydrolysis is consistent with recent work on the structure of laminarin (3, 16, 17, 18). The origin of the laminaribiose and laminaritriose is explicable on the basis that they result from the reducing ends of the laminarose component of laminarin. The enzyme apparently has the capacity to attack laminaritriose, since this substance gradually disappears, but it does not attack laminaribiose, which persists to the end of the hydrolysis. Gentiobiose (the,b-(1 -+ 6)-linked disaccharide) arises from the linkages that have been shown to occur in both laminarose and laminaritol. The enzyme is apparently incapable of hydrolyzing a linkage but can bypass it, thus leaving gentiobiose from this structural feature. The mannitol-containing oligosaccharides result from the enzyme failing to totally hydrolyze the laminaritol chains. The action of the enzyme apparently ceases at some distance from the terminal mannitol unit. Action of the Glucanase on Simple Oligosaccharides of Known Structure The pattern of products produced by the action of the enzyme on laminarin suggests that it is incapable of attacking disaccharides but is capable of attacking a trisaccharide of the proper structure at a rate somewhat less than that characteristic of a longer-chain material. In order to confirm this, the action of the enzyme on laminaribiose, gentiobiose, and laminaritriose was investigated using the same conditions as those for laminarin (Table I). It was found that the two disaccharides were not attacked at all, while laminaritriose was acted on at about 1/10 the rate observed with laminarin. These results reinforce the conclusion that the glucanase attacks a 6-(1 + 3)-linked chain of D-glucose residues from the non-reducing terminal and rapidly removes glucose residues one at a time, bypassing linkages, until it approaches the reducing terminal. When the residue from the chain reaches the trisaccharide level, the action of the enzyme is slowed and then ceases after one more residue is removed. The final stages of this process are not established by the data since an attack on either glycoside bond in laminaritriose will yield the same products, namely glucose and laminaribiose. However, the accumulation of mannitol-containing oligosaccharides would indicate that the attack is stepwise, ceasing as the terminal is approached, even if the last residue is not glucose. To establish the terminal process with more certainty, the action of the enzyme on laminaribiosyl-erythritol and laminaritriito13 was determined. If the attack is as predicted, 3Common names have been used throughout to describe the oligosaccharides mentioned in this paper; their more formal designations are: laminaribiosyl-erythritol, O-P-D-~~UCO~~YU~OS~~-(~ --t 3)-0-P-D-glucopyranos$- (1-i 2)-D-erythritol; lanzinaritriitol, O-~-~-~lucop~ranosyl-(1 -+ 3)-0-P-D-glucopyranosyl-(1 -+ 8)-D-glucitol; lanzinaritriose, 0-P-D-glucopyranosyl-(1 -+ 3)-0-P-D-glucopyranosyl-(1 --t 3)-D-glz~copyranose; laminaribiose, 8-0-P-D-glucopyranosyl-D-glucopyranose; gentiobiose, 6-0-~-~-g~~copy~a~0~y~-~-gl~C0pyran0Se; lanzinaribiitol, 8-O-~-~-g~~Copyran0~yl-~-gl~&t0~; glucosyl-erythritol, 2-0-P-D-~lucopyra?zosyl-D-erythrztol.

6 1676 CANADIAN JOURNAL OF CHEMISTRY. VOL namely a single cleavage of the bond connecting the non-reducing terminal in the trisaccharide, then the former should give only glucose and glucosyl-erythritol, the latter only glucose and laminaribiitol. Both substrates were incubated with the enzyme and it was found, by paper chromatography and paper electrophoresis, that only the expected products were produced (Figs. 3 and 4). No erythritol or glucitol were produced even with very high concentrations of enzyme and long periods of incubation. These would be the products produced by any action of the enzyme on the "reducing" end of the chain. FIG. 3. Products of the action of the glucanase on laminaribiosyl-erythritol as shown by paper chromatography.,411 spots are named in order proceeding from the origin. Lane 1 : laminaribiose, glucosyl-erythritol, and glucose standards; lane 2: the enzymatic hydrolyzate; lane 3: laminaribiosyl-erythritol standard; lane 4: laminaribiose, glucose, and erythritol standards. The enzyme concentration used in this experiment was 350-fold that used in the laminarin experiment reported in Table I, the substrate concentration was 0.2y0 and the incubation time 6 hours. FIG. 4. Products of the action of the glucanase on laminaribiosyl-gl~lcitol as show11 by paper electrophoresis. All spots are named in order proceeding from the origin. Lane 1: glucose standard; lane 2: glucitol standard; lane 3: la~ninaritriitol standard; lane 4: enzynratic hydrolyzate; lane 5: laminaribiitol standard; lane 6: laminaribiose standard. The enzyme concentration used in this experiment was 350-fold that used in the laminarin experiment reported in Table I, the substrate concentration was 0.5y0 and the time of incubation was 18 hours. Preparation and Purification of the Enzyme Basidiomycete sp. QM806 was grown on the 0.5% starch medium of Reese and Mandels (1). The crude culture filtrate was evaporated in oacuo at 37", dialyzed, and then fractionated with ammonium sulphate. The ammonium sulphate precipitate was taken up in buffer, dialyzed, and refractionated on a diethylaminoethylcellulose column. The resulting preparation had a specific activity of 163 units per milligram of protein, which represents a 100-fold increase in specific activity over that of the crude culture filtrate. The full details of this and the further purification of this enzyme will be the subject of a separate publication. Enzyme Assay and Unitage To 0.5 ml of substrate solutio11 (O.lyo laminarin in 0.05 A1 acetate buffer, ph 4.8) was added 0.5 ml of an appropriate dilution of the enzyme preparation in the same acetate buffer. The mixture was incubated for 15 minutes at 37" and the reaction stopped by the addition of 1.0 ml of the alkaline copper reagent used in the Nelson-Somogyi reducing sugar estimation (6). The rate of reaction under these conditions is first order with respect to enzyme if the extent of hydrolysis does not proceed beyond 60%. One unit of enzyme is defined as that amount which will liberate 1 pmole of glucose equivalent per minute under the above conditions of incubation. Other Incz~bation Procedures Incubations carried out for purposes other than enzyme assay, such as investigation of the nature of products of hydrolysis etc., were done undcr thc same general conditions as used for assay purposes. If the glucose

7 NELSON ET AL.: STRUCTURAL STUDIES ON POLYSACCHARIDES 1677 was to be estimated by the glucose oxidase procedure, the reaction was stopped by immersing the mixture in a boiling water bath for 5 minutes. In all cases where quantitative determinations were carried out, standards were included in the procedure and both reagent and substrate blanks used. In experiments involving the action of the enzyme on substrates other than larninarin, the latter was always run as a check on enzyme activity. In the case of laminaribiosyl-erythritol and laminaritriitol, a micromethod of incubation was used. Ten microliters of buffered substrate and 5 of enzyme were incubated in a sealed capillary tube, heat-denatured in a boiling-water bath, and then spotted directly onto paper. This method permitted extreme economy in the use of valuable substrates. Glyceritol Deternzination The usual method (19, 20) was modified by carrying out the periodate oxidation in 0.05 M acetate buffer at ph 4.8 rather than in 1 N H2SO4. Under these conditions the altered non-reducing ends of the periodateoxidized and reduced laminarin were not affected and only free glyceritol was determined. The excess periodate was destroyed by arsenious oxide. The glycerol values were corrected for the formaldehyde released by the action of periodate on the glycollic aldehyde produced during enzyme hydrolysis. Paper Chromatography and Electrophoresis All chromatography was done on Whatman No. 1 paper in the solvent system 1-propanol, ethyl acetate, water in the proportions 7:1:2 (21). The usual irrigation period was 36 hours. Paper electrophoresis was carried out with sodium borate in a potential gradient of 50 v/cm. The paper was dried and dipped through 10% HF in acetone to remove borate prior to developing the spots (22). The carbohydrate spots were developed by the Trevelyan modification of the Tollen's method (23). Substrates The "laminarin" used in these experiments was a preparation from L. cloustoni obtained from the Liverpool Borax Co., St. Paul's Square, Liverpool 3, England. Air-oxidized 1anzinarin.-Air-oxidized laminarin was obtained by passing a stream of air through a 0.5% solution of laminarin in 1.0% NaOH at reflux temperature. The reaction was discontinued when the reducing power of the solution had fallen to a negligible value. The solution was then neutralized with HCI, dialyzed in the cold against 0.05 M, ph 4.8 acetate buffer to remove salts, and used as the acetate buffer solution. Reduced 1aminarin.-Reduced laminarin was prepared by reducing an aqueous solution of laminarin with excess sodium borohydride overnight at room temperature. The excess borohydride was destroyed by acidifying with acetic acid, the solution dialyzed in the cold against buffer as described above and used as the substrate solution. It should be noted that laminarin is partially dialyzable so that all dialyses were carried out in the cold, on a rocking dialyzer, for 110 more than 6 hours with two changes of dialyzing medium. All substrates that had been dialyzed were used on the basis of their total carbohydrate content as determined by the phenol - sulphuric acid method (15). Periodate-oxidized and reduced 1aminarin.-Periodate-oxidized and reduced laminarin was prepared by Dr. 13. Lewis of this laboratory and was dialyzed prior to-use. The purified u~ndegraded oat gum and the laminaribiosyl-erythritol used in this work were prepared by Dr. Lewis. The purified barley gum was prepared by Dr. G. Hay... Laminaritriito1.-Laminaritriitol-was prepared from chromatographically pure laminaritriose by sodium borohydride reduction. In this case the borate was removed by deionization with resin followed by evaporation to dryness several times in the presence of anhydrous methanol. Laminaritriose, lanzinaribiose, and gentiobiose.-the first two substances were available in this laboratory, the last was obtained from the Pfanstiehl Co. Other materials.-the glucose oxidase used was the "pure" grade supplied by Nutritional Biochemicals Corp. All glucose oxidase preparations that we have examined have been contaminated with a small amount of 6-(1 -+ 3)-glucosidase activity. All determinations involving glucose oxidase reported in this paper were carried out with control procedures that eliminated this source of error. The peroxidase was a horseradish preparation from the Worthington Biochemical Corp. ACKNOIVLEDGMENTS The authors would like to express their gratitude to Dr. B. Lewis and Dr. G. Hay of this laboratory for their generous gifts of substrates and to Dr. E. T. Reese of the Quartermaster Research Laboratories, Natick, Mass., for supplying us with the culture of Basidiomycete sp. QM REFERENCES 1. E. T. REESE and M. MANDELS. Can. J. Rlicrobiol. 5, 173 (1959). 2. S. PEAT, LV. J. LVHELAN, and H. G. LATVLEY. Chem. Ind. (London), 35 (1955). 3. S. PEAT, W. J. IVHELAN, and H. G. LAWLEY. J. Chem. Soc. 729 (1958).

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