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[21] Polysaccharide synthesis from disaccharides
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ENZYMES OF CARBOHYDRATE METABOLISM
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that aged solutions of malt retained considerable power to hydrolyze lichenin, whereas the cellobiase activity was lost. The work of Karrer and Lier 18 indicated that lichenase and cellobiase could be separated by fractional absorption on aluminum hydroxide. However, the products of hydrolysis were very complex and a simple product was not identified. Freudenberg and Ploetzl* present similar results. Grassmann et al.~l and later Zechmeister et al. ~° applied the adsorption technique for the solution of a cellobiase solution. The Grassmann procedure is a continuation of step 2 for the purification of cellulase.
Purification Procedure The combined absorbed material (Al~03 plus adsorbed enzyme fraction) was stirred with 20 ml. of 0.20 M sodium bicarbonate solution and 80 ml. of water and centrifuged. This neutralized supernatant solution was employed for the activity determination. The results of this determination are outlined in Table I. Grassmann 11 further indicated that the cellobiase preparation from A s p e r g i l l u s oryzae exhibited some lichenase activity ( ~ cellobiase potency to 1/~ lichenase). The pH optimum of cellobiase is between 4 and 5. Enders and Saji 21 found that the enzyme from malt and barley exhibited optimum activity at pH 4 to 4.5 and a temperature of 37 °, whereas Grassmann et al. 11 and Zechmeister et al. 2° suggest working in the pH range of 4.5 to 5 and a temperature of 30 °. Smith, ~s working in Nord's laboratory, found that the mold M e r u l i u s l a c h r y m a n s hydrolyzed cellobiase initially to glucose at pH 4.28 and a temperature of 22°. Pringsheim 2 reports that cellobiase is deactivated at temperatures above 67 ° . 18 p. Karrer and H. Lier, HelD. Chim. Acta 8, 248 (1925). 19 K. Freudenberg and T. Ploetz, Z. physiol. Chem. 259, 19 (1939). s o L. Zechmeister, G. Tdth, P. Fiirth, and J. B£rsony, Enzymologia 9, 155 (1941). 21 C. Enders and T. Saji, Biochem. Z. 306, 430 (1940). 2~ V. M. Smith, Arch. Bioehem. 28, 446 (1949).
[21] Polysaccharide Synthesis from Disaccharides B y E D W A R D J. H E H R E
Introductory Note This section deals exclusively with enzymes known to catalyze, at least under certain conditions, the synthesis of high molecular weight polysaccharides from disaccharides. No attempt has been made to treat related biological systems that effect syntheses of series of oligosaccharides
[9"1]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
179
from disaccharides, or syntheses of polysaccharides from oligosaccharides other than disaccharides. For simplicity, all reactions have been considered principally from the forward or synthetic aspect. Moreover, all polymer products have been defined in generic terms, since none of the polysaccharide synthesizing enzymes has yet been prepared in a state of high purity, and it is not known to what extent accompanying enzymes operate to introduce branching or other structural irregularities in the polymers considered. I. Dextransucrase n-C12H22011 -~-
Sucrose
H O R --~ H(CeH10Os)~OR + n-CeHl~Oe Acceptor Dextran Fructose
Assay Method Principle. Measurement of fructose liberated under conditions providing a zero-order type reaction is the basis for estimating dextransucrase activity (Hehrel). The technique given below is that adopted by KoepselF and by Tsuchiya et al. 3 for the assay of dextransuerase in culture fluids of Leuconostoc mesenteroides. Reagents
0.04 N N a 0 H solution, containing phenolphthalein indicator (1 drop of indicator per 100 ml. of N a 0 H solution). 3 N acetic acid-NaOH buffer, pH 5.4. Buffered sucrose solution (60 %). 10 ml. of 3 M acetate buffer, pH 5.4, and 60 g. of sucrose are dissolved in distilled water and made up to 100 ml. The final pH should be 5.2. Enzyme. Adjust the cell-free culture fluid to pH 5.0 to 5.2. Prepare an accurate dilution with water, if necessary, to obtain solution for assay containing less than 40 units of enzyme per milliliter. (See definition below.) Procedure. Pipet 10 ml. of the enzyme solution, attempered to 30 °, into a 1 X 8-inch test tube containing 2 ml. of buffered 60 % sucrose solution also at 30 °. Note time of beginning of the pipetting step. Mix thoroughly and incubate at 30 °. At the end of I hour pipet 2 ml. of the mixture into a 100-ml. volumetric flask containing 10 ml. of 0.04 N NaOH solution. 1E. J. Hehre, J. Biol. Chem. 163, 221 (1946). 2 H. J. Koepsell, in U.S. Department of Agriculture Report of Working Conference on Dextran, Peoria, Illinois, p. 13, 1951; H. J. Koepsell and H. M. Tsuchiya, J. Bacteriol. 63, 293 (1952). a H. M. Tsuchiya, H. J. Koepsell, J. Corman, G. Bryant, M. O. Bogard, V. H. Feger, and R. W. Jackson, J. Bacteriol. 64, 521 (1952); additional details were kindly furnished by Dr. Tsuchiya.
180
ESZYMES OF CARBOHYDRATE METABOLISM
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Add more NaOH solution if necessary to make the sample slightly alkaline to phenolphthalein. Make up to volume, mix, and determine the amount of fructose present in the sample by the Somogyi method for reducing sugars 4 (fructose factor = 1.108 × milligrams of glucose equivalent to 1 ml. of 0.005 N Na~S203). Determine similarly the reducing power (as fructose) in a blank of enzyme made slightly alkaline to phenolphthalein with NaOH. Correct the amount of fructose present per milliliter of enzyme in the reaction mixture for the amount of reducing sugar (as fructose) per milliliter of enzyme in the blank. The difference, representing milligrams of fructose released per milliliter of enzyme, is //mol. wt. sucrose~ multiplied by 1.9 or \ ~ ~. ~ ] to give the amount of sucrose converted to dextran per milliliter of enzyme. Definition of Unit. One dextransucrase unit (DSU) is defined as the amount of enzyme which will convert 1 rag. of sucrose to dextran in 1 hour (releasing 0.52 mg. of fructose) under the above conditions. Extent and Limitations of Application. Crude filtrates or fluids from cultures of various bacteria that form dextran from sucrose, as well as solutions of partially purified dextransucrase, may be assayed with a high degree of accuracy by the above method. Two sources of potential interference, however, are recognized. 1. The presence of any other enzyme capable of yielding reducing sugar from sucrose will, of course, render the assay inaccurate. Since certain strains of dextran-forming bacteria are able also to hydrolyze sucrose or convert it to levan plus glucose, it is important to determine that neither invertase nor levansucrase accompanies the dextransucrase under assay. Methods for differentiating various sucrose-derived products in crude bacterial culture fluids have been described) 2. Accuracy of the dextransuerase assay also depends on freedom of the enzyme solution from high concentrations of certain sugars (sometimes employed to modify the synthesis so as to obtain oligosaecharides or low molecular weight dextrans as products) that affect the reaction velocity. The rate of fructose liberation is depressed, for example, by concentrations of sucrose higher than ca. 0.4 M.l Fructose and melibiose in high concentration likewise have a rate-depressing action, whereas isomaltose, maltose, a-methyl glucoside, and glucose have the opposite effect) Hence, enzyme unitage should be determined either prior to the 4 M. Somogyi,J. Biol. Chem. 160, 61 (1945). 6E. J. Hehre and J. M. Neill, J. Exptl. Med. 83, 147 (1946); W. G. C. Forsyth and D. M. Webley, J. Gen. Microbiol. 4, 87 (1950). 6 H. J. KoepseU, H. M. Tsuchiya, N. N. Hellman, A. Kazenko, C. A. Hoffman, E. S. Sharpe, and R. W. Jackson, J. Biol. Chem. 200, 793 (1953).
[21]
POLYSA.CCHARIDE SYNTHESIS FROM DISACCHARIDES
181
addition of any sugar modifier or after separation of the enzyme from such sugar. Supplementary Procedures. For purposes of orientation, dextransucrase activity may be crudely estimated by noting the speed of appearance (in mixtures with sucrose) of opalescence, serological activity, alcohol precipitable material, or other properties referable to dextran itself. 7 Use of the rate of dextran formation for assay of enzyme activity is proposed by Braswell et al., 7~ who find linear relationships between reaction time and (1) the logarithm of the relative viscosity, and (2) the relative turbidity measured at an angle of 90 ° in a light scattering photometer.
Preparation Biological Source. Although dextran is formed from sucrose by a number of different lactic acid bacteria, most dextransucrase preparations so far described have been made from Leuconostoc mesenteroides. Appreciable quantities of the enzyme are found extracellularly in broth cultures of these bacteria, provided that sucrose is present in the culture medium. 7 The higher the concentration of sucrose in the medium (at least up to 5%), the higher is the enzyme yield, s High-Potency Culture Fluids. The following preparative procedure of Tsuchiya et al. (1952) 3 gives notably improved yields of enzyme over the earlier methods. 1,~Slightly modified, it has proved suitable for the production of enzyme-rich fluids on an industrial scale. Inoculate L. mesenteroides N U R B B-512 at a 1 to 2% rate into flasks of culture medium, and incubate these at 25 ° for 24 hours on a reciprocating shaker. A suitable culture medium contains 2 to 3 % sucrose, 2 % corn steep solids or 0.5 % yeast extract, 2 to 3 % K2HP04, 0,02 % MgSO4.7H20, 0.001% NaC1, 0.001% FeSO4"7H20, and 0.001% MnSO4.H20. Bacterial cells are removed from the grown cultures by filtration or centrifugation. The crude, essentially cell-free fluids assay 90 to 120 DSU/ml. Separation of Enzyme from Preformed Dextran. Dextran and usually also sucrose and fructose are undesirable accompanying substances, present in the sucrose broth culture fluids. The following procedure has been used by the author to remove the greater part of these preformed reaction components in preparations from L. mesenteroides, strain B. 1 To each liter of chilled culture fluid add 370 g. of ammonium sulfate, taking care to dissolve the salt completely. Centrifuge the mixture in the cold, and decant the supernatants containing most of the dextran and sugars. Drain the precipitates in the cold, then wash three times with 7 E. J. Hehre and J. Y. Sugg, J. Exptl. Med. 75, 339 (1942). 7o E. Braswell, G. Oster, and K. G. Stern, work reported at Meeting-in-Miniature, New York Section Am. Chem. Soc., Feb. 1954.
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ENZYMES OF CARBOHYDRATE METABOLISM
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250-ml. portions of half-saturated a m m o n i u m sulfate in 0 . 1 % acetic acid. F o r each washing, work the sticky precipitates f r o m the b o t t o m s and sides of the centrifuge tubes into suspension with the aid of a r u b b e r policeman, centrifuge, and decant the wash fluid. E x t r a c t the final washed precipitate with 100 ml. of 0.025 M citrate buffer ( p H 6.3), and clear b y centrifugation in the cold. Properties
Specificity. Sucrose is the only established " d o n o r s u b s t r a t e " for polysaccharide formation b y dextransucrase, with the Michaelis constant (K,) recorded as 19 to 20 raM. 1 a n d 15.8 m M . s for enzymes of different source. N o n e of a long list of other sugars and sugar derivatives has been found to serve in this capacity. 6,~ Isomaltose, dextran, and leucrose have, however, been reported to serve as donor substrates for oligosaccharide form a t i o n b y the enzyme. 9~ T h e notion 1° t h a t Leuconostoc systems form small a m o u n t s of d e x t r a n f r o m glucose in the absence of sucrose stems f r o m the use of a nonspecific (gravimetric) test m e t h o d for dextran. T h e e n z y m e likewise has no detectable action u p o n inorganic p h o s p h a t e or glucose-l-phosphate, 11 or upon glucose-6-phosphate, fructose-6-phosphate, fructose-l,6-diphosphate, or A T P . s With respect to " a c c e p t o r s u b s t r a t e " specificity, the e n z y m e has, first of all, a strong preferential affinity for low molecular weight dextrans ;~2-14 t h a t is, the addition of fractions with average molecular weights of ca. 5000 to ca. 60,000 in small a m o u n t to enzyme-sucrose mixtures causes synthesis to be so modified t h a t dextran molecules of low or intermediate size rather t h a n of large size are formed as m a j o r reaction products. ~5
8 W. W. Carlson, C. L. Rosano, and V. Whiteside-Carlson, J. Bacterial. 65, 136 (1953). 8 E. J. Hehre, Science 93, 237 (1941). Traces of material with the serological properties of dextran were formed in incubated enzyme-raffinose mixtures, suggesting that raffinose may be a donor substrate of low affinity. However, this effect may possibly have been due to enzymic action on traces of sucrose liberated in some way from the trisaccharide. 8~ H. M. Tsuchiya and C. S. Stringer, Bacteriol. Proc., p. 98 (1954). lo H. L. A. Tart and H. Hibbert, Can. J. Research 5, 414 (1931); Staff Report, Chem. Eng. New~ 29, 650 (1951). The lack of formation of any dextran in incubated enzyme-glucose mixtures was shown by the use of serological tests of high specificity and sensitivity2 lz E. J. Hehre, Proc. ,.%c. Exptl. Biol. Med. 54, 240 (1943). 18E. J. Hehre, or. Am. Chem. ,%c. 75, 4866 (1953). 18H. M. Tsuchiya, N. N. Hellman, and H. J. Koepsell, J. Am. Chem. Soc. 75, 757 (1953). 14H. Nadel, C. I. Randles, and S. L. Stahly, Appl. Microbiol. 1, 217 (1953). ~6 Reaction products of suitable molecular weight for use as a blood volume expander have been produced in good yield from mixtures (kept at pH 5.0 and 15°) containing
[21]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
183
The enzyme also utilizes certain sugars (especially isomaltose and maltose, but also a-methyl glucoside and glucose) as acceptors for the glucosyl units from sucrose. ~ Enzymic action on equimolecular concentrations of sucrose and any one of these sugars differs from the action on sucrose alone in t h a t the reaction rate (as measured b y fructose liberation) is increased, oligosaccharides based on the sugar acceptor appear as major reaction products, and less high molecular weight dextran is synthesized. Fructose, leucrose, melibiose, and galactose also appear to act as acceptors, since in their presence (equimolecular with sucrose) some oligosaccharide formation occurs and the reaction rate is slightly depressed; the activity of both pyranose and furanose forms of fructose is indicated b y the formation of two fructose-containing disaccharides, leucrose (5-D-glucopyranosyl-D-fructopyranose) and isomaltulose (6-Dglucopyranosyl-n-fructofuranose), ~s in mixtures containing added fructose. e,~7 Dextransucrase apparently does not utilize as acceptors xylose, D- and L-arabinose, ribose, rhamnose, mannose, sorbose, cellobiose, trehalose, lactose, melezitose, raffinose, inulin, inositol, mannitol, sorbitol, 2-ketogluconate, 5-ketogluconate, or glycerol, e High molecular weight (native) dextrans cannot be excluded as acceptors, but their addition does not affect the rate of dextran synthesis from sucrose. ~ The production of small amounts of glucose in enzyme-sucrose mixtures (Forsyth and Webley 5) suggests t h a t water m a y be an acceptor substrate of low affinity, unless traces of invertase contaminate the available enzyme preparations. E q u i l i b r i u m . The reaction in the synthetic direction proceeds beyond 99.5% completion; ~,~ reversal, although theoretically possible, has not been detected. Activators and Inhibitors. Apart from the sugars noted above, no special activators or inhibitors of dextransucrase are known. The following common enzyme poisons are without detectable effect: 0.05 M fluoride 40 units/ml, dextransucrase, 100 mg./ml, sucrose, and from 2 to 20 mg./ml. (depending on the molecular weight) of low molecular weight dextran "acceptor." H. M. Tsuchiya, N. N. Hellman, H. J. Koepsell, J. Corman, C. S. Stringer, F. R. Senti, and R. W. Jackson, Abstr. 124th Meeting Am. Chem. Soc., 35A (1953). 16F. H. Stodola, H. J. Koepsell, and E. S. Sharpe, J. Am. Chem. Soc. 74, 3202 (1952); F. H. Stodola, E. S. Sharpe, and H. J. Koepsell, Abstr. 126th Meeting Am. Chem. ,Soc. (1954). 17Since fructose accumulates as a product of enzymic action upon sucrose, its effect may be observed even under "normal" circumstances. Leucrose in small amount appears on paper chromatograms in the terminal phases of action of dextransuerase from L. mesenteroides N U R B B-512 upon 0.125 M sucrose; larger amounts appear when the enzyme operates on higher concentrations of sucrose (and larger amounts of fructose are released) or when fructose is added to enzyme-sucrose m i x t u r e s )
184
ENZYMES OF CARBOHYDRATE METABOLISM
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or cyanide, 0.025 M azide or iodoacetate, 0.01 M pyridine or aniline, 0.001 M CuS04, ZnS04, or MnC12, or 0.00001 M AgN03.1 Enzymic activity likewise is unaffected by dialysis or by the presence of sodium ethylenediamine tetraaeetate (versene).S An early suggestion that dextransucrase may be activated by PABA has not been confirmed. Effect of pH and Temperature. The enzyme is most active and stable between pH 4 and 6, ~ with the optimum reported as pH 5.0 to 5.2 ~ or pH 5.6. 8 Activity is rapidly lost on exposure to pH 6.7 or above, even at 25 to 37 °. Moreover, the enzyme catalyzes the formation of dextran over a wide temperature range (3 to 37 °) but is, nevertheless, very heat labile. 7 For example, it shows high activity and stability at 30 °, yet is destroyed in a few minutes at 40 °, even at pH 5.0. ~ Lyophilized preparations, sealed in vacuo and stored at 5 °, retain their potency for many years. Reaction temperature has been observed to influence the nature of the dextran produced when the enzyme operates in the presence of added dextran acceptor. Lowering the reaction temperature of such systems from 30 ° to 15 ° greatly favors the formation of intermediate- as opposed to high-molecular weight dextran. 1~ II. Amylosucrase n-C1uI-I2~O11 -~- H O R --* H(CeHloOb)~OR -I" ~-C6H1206 Sucrose Acceptor Glycogen-type Fructose polysaccharide
Assay Method A detailed procedure for estimating enzymic activity has not been reported, but a suitable basis is measurement of the fructose by-product released per unit of time under conditions yielding zero-order reaction kinetics. Such conditions are approached when enzyme solutions are incubated at 10 ° with an equal volume of 0.2 M sucrose in 0.025 M maleate buffer, pH 6.4. is Rough appraisals of activity may be obtained also from the speed of development of opalescence, color with iodine, aleohol-preeipitable material, or other attribute of the glycogen-type polymer that is synthesized.
Preparation The biological source is Neisseria perflava, a species of bacteria readily isolated from the throat. 19 Crude but cell-free enzyme preparations may be obtained b y the author's method. ~s Cultivate N. perflava (strain 19-34) 18E. J. Hehre, J. Biol. Chem. 177, 267 (1949). 19E. J. Hehre and D. M. Hamilton, Y. Biol. Chem. 166, 777 (1946); J. Bacl~riol. 66, 197 (1948).
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POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
185
for 5 days at 37 ° in unshaken flasks of broth (800 ml.) comprising 0.1% peptone, 0.15% sodium citrate, 0.02% yeast extract, 0.06% KH2PO4, 0.15% Na~HPO4, and 0.05% glucose. (Inoculate each flask with 10 ml. of a 1-day culture in the same medium.) Treat each 800 ml. of grown culture with 300 g. of ammonium sulfate, centrifuge the mixture, and decant the supernatant fluids. Suspend the sediments in 100 ml. of half-saturated ammonium sulfate previously adjusted to pH 6.4 with ammonia, centrifuge, and discard the wash fluid. Extract the washed and drained precipitates with 15 ml. of 0.025 M maleate buffer (pH 6.4), and clarify by centrifugation.
Properties Specificity. The only known "donor substrate" for amylosucrase acting synthetically is sucrose. Neither glucose, maltose, lactose, trehalose, a-methyl glucoside, raffinose, melezitose, nor glucose-l-phosphate serves the enzyme in this capacity. 18.2° Amylosucrase presumably utilizes a series of amylosaeeharides as "aeceptor substrates" in the course of building up polysaccharide molecules, but its affinity for various acceptors has not been systematically examined. Equilibrium. The conversion of sucrose to polysaccharide and fructose proceeds to ca. 98% completion. 21 Reversal to the extent of about 1% can be demonstrated when the enzyme is incubated with fructose plus amylose, amylopectin, or glycogen. Inhibitors. Synthesis of polysaccharide from sucrose is not impaired by the presence of inorganic phosphate in concentrations sufficiently high (8 moles P to 1 of substrate) to suppress all synthesis from glucose-lphosphate by the phosphorylase in N. perflava extracts. 18,1g Traces of salivary amylase, however, inhibit the formation of polysaccharide and fructose from sucrose, possibly by destroying essential acceptor substrates. 21 Effects of pH and Temperature. Satisfactory enzymic action occurs over the temperature range of 10 to 37 ° at pH 6.4. Amylosucrase (pH 6.4) appears stable at 5 ° for many weeks but is almost completely inactivated at 45 ° for 10 minutes (under which conditions the phosphorylase activity of the extracts remains unaffected~8). Information is lacking on the activity and stability in different pH ranges. 2o N . perflava preparations produce an amylaceous polysaccharide from glucose-lphosphate, and also a glycogen-type polysaccharide from amylose, but these actions are attributable to an accompanying phosphorylase and to a branching enzyme of the Q type. I1 E. J. Hehre, Advances in Enzymol. 11, 297 (1951).
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ENZYMES OF CARBOHYDRATE METABOLISM
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HI. Levansucrase
n-ClsH2~Oll ~Sucrose
H0R --~ H(C6HIoOs). -{- n-C6HI~O~ Acceptor Levan Glucose
Assay Method
P r i n c i p l e . The circumstance that a variable but generally large amount of sucrose hydrolysis regularly accompanies levan formation in reactions catalyzed by available enzyme preparations has prevented development of an assay method based on the rate of liberation of glucose. The following procedure of Hestrin and Avineri-Shapiro ~2 measures the rate of formation of highly polymerized levan itself under conditions where the synthesis follows an essentially linear course. (For rough appraisals of enzyme activity, the speed of release of reducing sugars and of development of features referable to levan, e.g., opalescence, viscosity, serological activity, or alcohol-precipitable polyfructoside, may be found useful.) Procedure. The assay mixture comprises 1 ml. of enzyme solution, 1 ml. of 15 % sucrose in distilled water, 1 ml. of SOrensen citrate buffer (pH 5.0), and 1 drop of chloroform containing thymol for maintaining sterility. Incubate at 37 ° until the levan concentration is in the range of 50 to 200 mg./100 ml. (as judged by comparison of the opalescence of the assay mixture with reference solutions containing known concentrations of levan), and then determine the levan content as follows.23 Pipet 1.0 ml. of the assay solution into a centrifuge tube of 15-ml. capacity, and add 3.0 ml. of ethanol. If flocculation is slow, it can be hastened and rendered quantitative by the addition of a drop of 1% CaC12. The suspension is centrifuged, and the sedimented levan freed from reducing sugar and sucrose residues by twice-repeated solution in H20 over a water-bath and precipitation each time with ethanol. Addition of a glass bead to the mixture facilitates these operations. The final sediment is taken up in 3.0 ml. of 0.5% oxalic acid and hydrolyzed in a boiling water bath for 1 hour. Evaporation is restricted during this treatment by placing a glass bulb at the tube aperture. The hydrolyzate is neutralized, cleared with Zn(OH)2, and diluted to a suitable volume. The reducing power of the filtrate is estimated by the method of Somogyi. The amount of levan is calculated from the "glucose value" by multiplying by a factor which allows both for the small difference in reducing power between glucose and fructose and for the entry of HsO during hydrolysis.
~2S. tIestrin and S. Avineri-Shapiro,Biochem. J. $8, 2 (1944); S. Avineri-Shapiroand S. Hestrin, Biochem. J. $9, 167 (1945). 23 S. Hestrin~ S. Avineri-Shapiro,and M. Aschner, Biochem. J. 37, 450 (1943) ; Nature 149, 527 (1942).
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POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
187
Definition of Unit. One levansucrase unit (LU) is defined as the minimal amount of enzyme which induces production of 100 rag. of levan per 100 ml. in 2 hours under the above conditions. This definition is designed to circumvent the variation observed in the specific activity of levansucrase at different enzyme concentrations. Limitations. The presence of levan-splitting enzymes (levanases) in certain enzyme preparations (e.g., from Bacillus subtilis) may lead to an erroneously low levansucrase assay figure. Likewise, the presence of preformed levan in crude sucrose broth culture fluids (unless corrected for) may give an error in the opposite direction. Extensive dextran production, which may occur under levansucrase assay conditions in the case of extracts from certain bacteria (especially certain streptococci), may also lead to unduly high assay values and should in any case be recognized.
Preparation Bacteria of many kinds produce levan from sucrose or raffinose, and crude, cell-free levansucrase preparations have been obtained from varieties in such diverse genera as Bacillus, Streptococcus, and Aerobacter. Hestrin et al. 23 give the following method for preparing enzymes suitable for kinetic studies from Aerobacter levanicum. The bacterium (Jerusalem strain) is cultivated in 1-1. conical flasks at 30 ° in a two-phase medium consisting of a bottom solid layer of nutrient agar devoid of sucrose and a thin top layer of 2 % aqueous sucrose. After 20 hours the cells are harvested, rinsed repeatedly in water to free them from medium constituents and levan, and finally left to autolyze at 37 ° under water containing a little thymol and chloroform. The volume ratio of culture medium to autolyzate fluid is 100:1. After 24 hours of autolysis, the suspension is centrifuged. The cell-free supernates may assay as high as 5 units/ml. Various alternative methods have been used in preparing levansucrase from spore-forming bacilli or streptococci. ~,~3-27 Since a procedure found applicable to one particular bacterial strain may not be suitable for another, the trial of several methods is advised.
Properties Specificity. Levansucrase utilizes sucrose and raffinose (a galactosyl sucrose) as "donor substrates," with the Michaelis constant for sucrose in the range of 0.02 to 0.06 M. The enzyme does not utilize any other common sugar or other substance with a terminal fructofuranose group (e.g., 24 E. J. Hehre, Proc. Soc. Exptl. Biol. Med. 58, 219 (1945). ~5 M. Doudoroff and R. O'Neal, J. Biol. Chem. 159, 585 (1945). 26 F. L. Horsfall, Jr., J. Exptl. Med. 93, 229 (1951). ~v R. Dedonder and Mine. Noblesse~ Ann. Inst. Pasteur 85, 356 (1953).
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ENZYMES OF CARBOHYDRATE METABOLISM
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fructose-l,6-diphosphate, fructose-6-phosphate, methyl fructofuranoside, inulin), in this way. There is also no action upon " f r e e " or " n a s c e n t " fructofuranose or upon inorganic phosphate. ~2,28,~9 Little information is as yet available concerning the "acceptor substrate" specificity of levansucrase. A series of glucose-ended 2,6-1inked polyfructosides is probably used in synthesis of the levan macromolecule. ~9~In addition, B. subtilis preparations seem to have some affinity for glucose (but not fructose) as an acceptor. The action on a mixture of 10 % sucrose and 5% glucose, for example, differs from the action of 10% sucrose alone in that oligosaccharides containing glucose as well as fructose appear as prominent (although transient) reaction products, and high molecular weight levan is less rapidly synthesized.~7 (Cf. Activators and Inhibitors.) Equilibrium. Because of the complication of concomitant hydrolytic breakdown of the substrate (sucrose) by the available enzyme preparations, the exact position of equilibrium in levan synthesis remains undefined. Sufficient data have been obtained, ~2however, to establish that the ratio of levan to sucrose at equilibrium is definitely greater than unity. Yields of levan as high as 62 % of the theoretical maximum have been obtained under certain conditions. Since reversal of the reaction by the action of enzyme on levan and glucose has not been detected by direct means, ~ an equilibrium position far to the side of synthesis seems likely. Activators and Inhibitors. Certain sugars, including D-glucose, L-arabinose, D-xylose, L-sorbose, lactose, maltose, a-methyl glucoside, and D-galactose (but not D-fructose, D-mannose, mannitol, sorbitol, glueosamine, or trehalose), retard the formation of highly polymerized levan from sucrose by A. levanicum preparations. The "inhibitory" effect increases, in the case of D-glucose at least, as its concentration relative to sucrose is increased; and levan formation is completely suppressed, for example, in mixtures containing 1% sucrose and 16% D-glucose. ~2 It would appear most probable that this "inhibition" effect actually represents a modification of the synthetic reaction, and that D-glucose and the other sugars in question are "acceptor substrates." Some of them may actually stimulate the rate of fructosyl group transfer by the enzyme. 28It should be noted, however, that gentianose, verbaseose, and several recently dis-
covered sucrose-ended oligosaeeharidespassing terminal fructofuranosyl units, i.e., erlose, kestose, inulobiosyl glucose, have not been examined and may be able to serve as donor substrates for levansucrase. ~9S. Hestrin, Nature 164, 581 (1944). ~9~Neither levanbiose, levantriose, nor levantetraose, however, has been found to serve as an acceptor substrate (or modifier of levan synthesis) in the case of Aerobacter levansucrase. See S. Hestrin, Instituto Superiore di Sanita' Symposium on Microbial Metabolism, Rome, p. 53, 1953.
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POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
189
Known poisons of respiration or of glycolysis, including 0.002 M cyanide, 0.02 M fluoride, 0.001 M monoiodoacetate, and 0.001 M phloridzin, fail to suppress or markedly inhibit levan formation. Dialysis similarly does not affect the activity of the enzyme, and A. levanicum extracts do not contain free adenylic acid. 22 Effect of pH and Temperature. The pH-activity curve of levansucrase operating upon sucrose at 37 ° is regular and symmetrical, with its peak in the region of pH 5.0 to 5.8. 22 The relationship between activity and temperature has not been systematically studied, but the enzyme operates satisfactorily over the range of 5 to 37 °. Enzyme stability (at pH 5.0) is very high at temperatures up to 30 ° . Storage for several days at this temperature, or for several months at 4 ° , has no deleterious effect. Gradual loss of activity at 37 ° is discernable over a period of days, and there is complete and almost immediate inactivation on exposure at 100°. 23 Preparations frozen and dried in vacuo are suitable for prolonged storage.
IV. Amylomaltase n-Cl~H22011 ~ H O R --) H(C~H10Os),OR ~ n-C6HI~O~ Maltose Acceptor Amylose-type Glucose polysaccharide
Assay Method Principle. The assay procedure (Monod and Torriani 3°) has as a basis determination of the glucose liberated under conditions where the reaction follows a kinetically zero-order course; the glucose is determined (in the presence of the maltose substrate) by measuring manometrically the uptake of 02 during its specific oxidation by notatin. Reagents 0.1 M phosphate buffer, pH 6.8 Notatin. Purified glucose oxidase of Penicillium notatum21 Buffered maltcse solution. The solution used as substrate for the assay contains 100 ~,/ml. notatin, 0.033 M maltose, and 0.02 M sodium azide (for inactivation of catalase) in 0.1 M phosphate buffer of pH 6.8. Enzyme. Prepare a known dilution, if necessary, to obtain an enzyme solution for assay that contains between 20 and 130 amylomaltase units/ml. (See definition below.) 3o j. Monod and A. M. Torriani, Ann. Inst. Pasteur 78, 65 (1950) ; Compt. rend. 227, 240 (1948). 31 C. E. Coulthard, R. Michaelis, W. F. Short, G. Sykes, G. E. H. Skrimshire, A. F. Standfast, J. H. Birkinshaw, and H. Raistrick, Biochem. J. $9, 24 (1945); see Vol. I [45].
190
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
Procedure. Place 3.0 ml. of the buffered maltose solution containing notatin in the main chamber of a Warburg flask, and 0.1 ml. of enzyme in the side arm. After assembly of the manometric apparatus and equilibration at 28 ° , mix the contents of the flask. Measure the oxygen consumption at 10-minute intervals until a steady rate is achieved (usually 20 to 40 minutes). The 02 uptake in microliters per hour divided by 2.26 gives the micromoles of 0~ consumed per hour per milliliter of enzyme and, hence, the micromoles of glucose formed per hour per milliliter of enzyme. Definition of Unit. One amylomaltase unit is defined as the amount of enzyme which will liberate from maltose 1 ~M. of glucose per hour under the above conditions. Specific activity is expressed as units per milligram of Kjeldahl N. a° Extent and Limitations of Application. The procedure has been used for assay of amylomaltase in toluene-treated suspensions of intact bacterial cells of the ML strain of E. coli. s2 For use with cells or with impure soluble preparations, however, the possibility of interference by the presence of enzymes yielding glucose from maltose or from amylosaccharides would, of course, have to be guarded against.
Preparation The presence of amylomaltase has been described in two special mutant strains of E. coli, namely, the maltose- and lactose-positive form of strain ML, 3° and the maltose-positive lactose-negative form of strain K122 s The enzyme evidently is produced only when the bacteria are cultivated in the presence of maltose. Using strain ML, Monod and Torriani 3° prepare the cell-free enzyme as follows. The bacteria are heavily inoculated into 250-ml. amounts of culture medium in 2-1. conical flasks. (The medium is made up of 2.72 % KH2PO,, 0.4% (NH,),SO,, 0.04% MgSO,-7H~O, 0.001% CaCl~, 0.00005% FeSOr7H20, and sufficient NaOH to attain pH 7.5; after sterilization, a filtered solution of maltose is added aseptically to give an 0.8% concentration.) The inoculated flasks are incubated, with agitation, at 34 ° for 14 hours. The bacterial cells are harvested by Sharples centrifugation, washed in the centrifuge with a volume of distilled water equal to that of the original culture medium (2 to 10 1.), then suspended in 0.05 M phosphate buffer, pH 6.8; 200 ml. of buffer is used for each 100 g. of the washed cells. Fine sand, 2 g. for each 1 ml. of suspension, is then added, and the mixture vigorously agitated in a shaking apparatus for 40 minutes. The creamy mass is centrifuged at 12,000 r.p.m, for 15 minutes, and ~2G. Cohen-Bazire and M. Jolet, Ann. inst. Pasteur 84, 937 (1953). " M. Doudoroff, W. Z. Hassid, E. W. Putman, A. L. Potter, and J. Lederberg, J. Biol. Chem. 179, 921 (1949).
[9.1]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
191
the supernatant fluid removed. The precipitate is resuspended in 100 ml. of buffer and again centrifuged. This operation is repeated a second time, and the supernatant fluids combined. Maltose is added to give a 0.05 M solution, followed by solid ammonium sulfate to 75 % saturation (in the cold). After 2 hours, the precipitate is separated by centrifugation and redissolved in 100 ml. of buffer. The precipitation is repeated three times under similar conditions with sufficient solid ammonium sulfate to give 50% saturation. The final precipitate is taken up in about 20 ml. of buffer, and insoluble material eliminated by centrifugation. All operations are at 0 °. To remove accompanying amylase and phosphorylase, the solution is dialyzed in the cold against distilled water. A precipitate should form as a result of this treatment or be induced by careful acidification of the dialyzate with acetic acid to pH 5.2. The precipitate is collected by centrifugation and extracted with 5 ml. of 0.025 M veronal buffer, pH 6.8, containing 0.2 M sodium sulfate. After storage overnight at 0 °, this final extract is cleared by centrifugation. The best preparations contain 5000 amylomaltase units/ml. (660 units/mg. N)2 °
Properties Specificity. The enzyme has affinity for amylosaccharides as well as for maltose as "donor substrates." It is uncertain, however, whether any donor other than maltose is utilized in the forward or synthetic reaction. Lactose, sucrose, melibiose, cellobiose, a-methyl glucoside,/~-methyl glucoside, trehalose, and glucose-l-phosphate definitely do not serve as donor substrates for the enzyme2 ° Amylomaltase is presumed to utilize maltose and its higher homologs as "acceptor substrates" in the course of polysaccharide synthesis. Glucose definitely is utilized in this capacity, since enzymic action on maltose in the presence of glucose leads to the formation of oligosaccharides as major reaction products, whereas action in the absence of glucose (in systems containing notatin) leads to the formation of an amylose-type polysaccharide as the product. 3°.33,34 The presence of glucose also permits the reverse reaction (polysaccharide degradation to maltose) to occur. 3° There is some indication that D-xylose and to some extent D-mannose (but not D-fructose, D-galactose, D-arabinose, or I,-arabinose) can replace D-glucose as the acceptor substrate in this reverse reaction. 3s Equilibrium. The polymerative action of amylomaltase upon maltose proceeds until about 60 % of the substrate has been converted; a similar equilibrium position is reached in the reverse direction, i.e., after enzymic action on equimolecular amounts of polysaccharide and glucose2 ° (When s4 S. A. Barker a n d E. J. Bourne, J. Chem. Soc. 1952, 209.
192
ENZYMES OF CARBOHYDRATE METABOLISM
[22]
the synthesis from maltose is conducted in the presence of notatin, an equilibrium is not observed and conversion to polysaccharide proceeds to completion.) Activators and Inhibitors. Enzymic activity is unaffected by the presence or absence of orthophosphate, and it is retained after dialysis20 E. coli cells that contain amylomaltase are active in converting maltose to higher homologs in the presence of 0.002 M iodoacetate2 T M Effect of pH and Temperature. Little has been recorded concerning the effect of pH and temperature upon enzyme activity and stability. Suitable activity is manifested in mixtures set at pH 6.8 and 28 to 32 °.
[22] Phosphorylases from Plants By W. J. WHELAN CH,0H
H
OH
CH ~OH
H
P0~Ks
0
OH a- D-Glucose-I. phosphate
CH20H
CH ~OH
OH
CH ~0It
0 OH
0 .... OH
Primer
11
CH,OH
CH~0H
CH~0H
K,HPO~ + H
0 OH
0 OH
0 OH
0 .... OH
Amyiose
Assay Method Pr/nc/p/e. Primer and G-1-P are mixed with enzyme, and the orthophosphate set free during amylose synthesis is measured colorimetrically. The following procedure is based on that of Green and Stumpf. 1
Reagent8 G-1-P solution (0.1 M) contains 37.22 g./1. of the dipotassium salt. 2 5 % soluble starch solution. 0.5 M citric a c i d - - N a 0 H buffer, pH 6.0. l D. E. G r e e n a n d P. K. S t u m p f , J. Biol. Chem. 142, 355 (1942).
' Pure, primer-free G-1-P is conveniently prepared by the method of R. M. MeCready and W. Z. Hasald, J. Am. Chem. 8oe. 66, 560 (1944).
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
that aged solutions of malt retained considerable power to hydrolyze lichenin, whereas the cellobiase activity was lost. The work of Karrer and Lier 18 indicated that lichenase and cellobiase could be separated by fractional absorption on aluminum hydroxide. However, the products of hydrolysis were very complex and a simple product was not identified. Freudenberg and Ploetzl* present similar results. Grassmann et al.~l and later Zechmeister et al. ~° applied the adsorption technique for the solution of a cellobiase solution. The Grassmann procedure is a continuation of step 2 for the purification of cellulase.
Purification Procedure The combined absorbed material (Al~03 plus adsorbed enzyme fraction) was stirred with 20 ml. of 0.20 M sodium bicarbonate solution and 80 ml. of water and centrifuged. This neutralized supernatant solution was employed for the activity determination. The results of this determination are outlined in Table I. Grassmann 11 further indicated that the cellobiase preparation from A s p e r g i l l u s oryzae exhibited some lichenase activity ( ~ cellobiase potency to 1/~ lichenase). The pH optimum of cellobiase is between 4 and 5. Enders and Saji 21 found that the enzyme from malt and barley exhibited optimum activity at pH 4 to 4.5 and a temperature of 37 °, whereas Grassmann et al. 11 and Zechmeister et al. 2° suggest working in the pH range of 4.5 to 5 and a temperature of 30 °. Smith, ~s working in Nord's laboratory, found that the mold M e r u l i u s l a c h r y m a n s hydrolyzed cellobiase initially to glucose at pH 4.28 and a temperature of 22°. Pringsheim 2 reports that cellobiase is deactivated at temperatures above 67 ° . 18 p. Karrer and H. Lier, HelD. Chim. Acta 8, 248 (1925). 19 K. Freudenberg and T. Ploetz, Z. physiol. Chem. 259, 19 (1939). s o L. Zechmeister, G. Tdth, P. Fiirth, and J. B£rsony, Enzymologia 9, 155 (1941). 21 C. Enders and T. Saji, Biochem. Z. 306, 430 (1940). 2~ V. M. Smith, Arch. Bioehem. 28, 446 (1949).
[21] Polysaccharide Synthesis from Disaccharides B y E D W A R D J. H E H R E
Introductory Note This section deals exclusively with enzymes known to catalyze, at least under certain conditions, the synthesis of high molecular weight polysaccharides from disaccharides. No attempt has been made to treat related biological systems that effect syntheses of series of oligosaccharides
[9"1]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
179
from disaccharides, or syntheses of polysaccharides from oligosaccharides other than disaccharides. For simplicity, all reactions have been considered principally from the forward or synthetic aspect. Moreover, all polymer products have been defined in generic terms, since none of the polysaccharide synthesizing enzymes has yet been prepared in a state of high purity, and it is not known to what extent accompanying enzymes operate to introduce branching or other structural irregularities in the polymers considered. I. Dextransucrase n-C12H22011 -~-
Sucrose
H O R --~ H(CeH10Os)~OR + n-CeHl~Oe Acceptor Dextran Fructose
Assay Method Principle. Measurement of fructose liberated under conditions providing a zero-order type reaction is the basis for estimating dextransucrase activity (Hehrel). The technique given below is that adopted by KoepselF and by Tsuchiya et al. 3 for the assay of dextransuerase in culture fluids of Leuconostoc mesenteroides. Reagents
0.04 N N a 0 H solution, containing phenolphthalein indicator (1 drop of indicator per 100 ml. of N a 0 H solution). 3 N acetic acid-NaOH buffer, pH 5.4. Buffered sucrose solution (60 %). 10 ml. of 3 M acetate buffer, pH 5.4, and 60 g. of sucrose are dissolved in distilled water and made up to 100 ml. The final pH should be 5.2. Enzyme. Adjust the cell-free culture fluid to pH 5.0 to 5.2. Prepare an accurate dilution with water, if necessary, to obtain solution for assay containing less than 40 units of enzyme per milliliter. (See definition below.) Procedure. Pipet 10 ml. of the enzyme solution, attempered to 30 °, into a 1 X 8-inch test tube containing 2 ml. of buffered 60 % sucrose solution also at 30 °. Note time of beginning of the pipetting step. Mix thoroughly and incubate at 30 °. At the end of I hour pipet 2 ml. of the mixture into a 100-ml. volumetric flask containing 10 ml. of 0.04 N NaOH solution. 1E. J. Hehre, J. Biol. Chem. 163, 221 (1946). 2 H. J. Koepsell, in U.S. Department of Agriculture Report of Working Conference on Dextran, Peoria, Illinois, p. 13, 1951; H. J. Koepsell and H. M. Tsuchiya, J. Bacteriol. 63, 293 (1952). a H. M. Tsuchiya, H. J. Koepsell, J. Corman, G. Bryant, M. O. Bogard, V. H. Feger, and R. W. Jackson, J. Bacteriol. 64, 521 (1952); additional details were kindly furnished by Dr. Tsuchiya.
180
ESZYMES OF CARBOHYDRATE METABOLISM
[21]
Add more NaOH solution if necessary to make the sample slightly alkaline to phenolphthalein. Make up to volume, mix, and determine the amount of fructose present in the sample by the Somogyi method for reducing sugars 4 (fructose factor = 1.108 × milligrams of glucose equivalent to 1 ml. of 0.005 N Na~S203). Determine similarly the reducing power (as fructose) in a blank of enzyme made slightly alkaline to phenolphthalein with NaOH. Correct the amount of fructose present per milliliter of enzyme in the reaction mixture for the amount of reducing sugar (as fructose) per milliliter of enzyme in the blank. The difference, representing milligrams of fructose released per milliliter of enzyme, is //mol. wt. sucrose~ multiplied by 1.9 or \ ~ ~. ~ ] to give the amount of sucrose converted to dextran per milliliter of enzyme. Definition of Unit. One dextransucrase unit (DSU) is defined as the amount of enzyme which will convert 1 rag. of sucrose to dextran in 1 hour (releasing 0.52 mg. of fructose) under the above conditions. Extent and Limitations of Application. Crude filtrates or fluids from cultures of various bacteria that form dextran from sucrose, as well as solutions of partially purified dextransucrase, may be assayed with a high degree of accuracy by the above method. Two sources of potential interference, however, are recognized. 1. The presence of any other enzyme capable of yielding reducing sugar from sucrose will, of course, render the assay inaccurate. Since certain strains of dextran-forming bacteria are able also to hydrolyze sucrose or convert it to levan plus glucose, it is important to determine that neither invertase nor levansucrase accompanies the dextransucrase under assay. Methods for differentiating various sucrose-derived products in crude bacterial culture fluids have been described) 2. Accuracy of the dextransuerase assay also depends on freedom of the enzyme solution from high concentrations of certain sugars (sometimes employed to modify the synthesis so as to obtain oligosaecharides or low molecular weight dextrans as products) that affect the reaction velocity. The rate of fructose liberation is depressed, for example, by concentrations of sucrose higher than ca. 0.4 M.l Fructose and melibiose in high concentration likewise have a rate-depressing action, whereas isomaltose, maltose, a-methyl glucoside, and glucose have the opposite effect) Hence, enzyme unitage should be determined either prior to the 4 M. Somogyi,J. Biol. Chem. 160, 61 (1945). 6E. J. Hehre and J. M. Neill, J. Exptl. Med. 83, 147 (1946); W. G. C. Forsyth and D. M. Webley, J. Gen. Microbiol. 4, 87 (1950). 6 H. J. KoepseU, H. M. Tsuchiya, N. N. Hellman, A. Kazenko, C. A. Hoffman, E. S. Sharpe, and R. W. Jackson, J. Biol. Chem. 200, 793 (1953).
[21]
POLYSA.CCHARIDE SYNTHESIS FROM DISACCHARIDES
181
addition of any sugar modifier or after separation of the enzyme from such sugar. Supplementary Procedures. For purposes of orientation, dextransucrase activity may be crudely estimated by noting the speed of appearance (in mixtures with sucrose) of opalescence, serological activity, alcohol precipitable material, or other properties referable to dextran itself. 7 Use of the rate of dextran formation for assay of enzyme activity is proposed by Braswell et al., 7~ who find linear relationships between reaction time and (1) the logarithm of the relative viscosity, and (2) the relative turbidity measured at an angle of 90 ° in a light scattering photometer.
Preparation Biological Source. Although dextran is formed from sucrose by a number of different lactic acid bacteria, most dextransucrase preparations so far described have been made from Leuconostoc mesenteroides. Appreciable quantities of the enzyme are found extracellularly in broth cultures of these bacteria, provided that sucrose is present in the culture medium. 7 The higher the concentration of sucrose in the medium (at least up to 5%), the higher is the enzyme yield, s High-Potency Culture Fluids. The following preparative procedure of Tsuchiya et al. (1952) 3 gives notably improved yields of enzyme over the earlier methods. 1,~Slightly modified, it has proved suitable for the production of enzyme-rich fluids on an industrial scale. Inoculate L. mesenteroides N U R B B-512 at a 1 to 2% rate into flasks of culture medium, and incubate these at 25 ° for 24 hours on a reciprocating shaker. A suitable culture medium contains 2 to 3 % sucrose, 2 % corn steep solids or 0.5 % yeast extract, 2 to 3 % K2HP04, 0,02 % MgSO4.7H20, 0.001% NaC1, 0.001% FeSO4"7H20, and 0.001% MnSO4.H20. Bacterial cells are removed from the grown cultures by filtration or centrifugation. The crude, essentially cell-free fluids assay 90 to 120 DSU/ml. Separation of Enzyme from Preformed Dextran. Dextran and usually also sucrose and fructose are undesirable accompanying substances, present in the sucrose broth culture fluids. The following procedure has been used by the author to remove the greater part of these preformed reaction components in preparations from L. mesenteroides, strain B. 1 To each liter of chilled culture fluid add 370 g. of ammonium sulfate, taking care to dissolve the salt completely. Centrifuge the mixture in the cold, and decant the supernatants containing most of the dextran and sugars. Drain the precipitates in the cold, then wash three times with 7 E. J. Hehre and J. Y. Sugg, J. Exptl. Med. 75, 339 (1942). 7o E. Braswell, G. Oster, and K. G. Stern, work reported at Meeting-in-Miniature, New York Section Am. Chem. Soc., Feb. 1954.
182
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
250-ml. portions of half-saturated a m m o n i u m sulfate in 0 . 1 % acetic acid. F o r each washing, work the sticky precipitates f r o m the b o t t o m s and sides of the centrifuge tubes into suspension with the aid of a r u b b e r policeman, centrifuge, and decant the wash fluid. E x t r a c t the final washed precipitate with 100 ml. of 0.025 M citrate buffer ( p H 6.3), and clear b y centrifugation in the cold. Properties
Specificity. Sucrose is the only established " d o n o r s u b s t r a t e " for polysaccharide formation b y dextransucrase, with the Michaelis constant (K,) recorded as 19 to 20 raM. 1 a n d 15.8 m M . s for enzymes of different source. N o n e of a long list of other sugars and sugar derivatives has been found to serve in this capacity. 6,~ Isomaltose, dextran, and leucrose have, however, been reported to serve as donor substrates for oligosaccharide form a t i o n b y the enzyme. 9~ T h e notion 1° t h a t Leuconostoc systems form small a m o u n t s of d e x t r a n f r o m glucose in the absence of sucrose stems f r o m the use of a nonspecific (gravimetric) test m e t h o d for dextran. T h e e n z y m e likewise has no detectable action u p o n inorganic p h o s p h a t e or glucose-l-phosphate, 11 or upon glucose-6-phosphate, fructose-6-phosphate, fructose-l,6-diphosphate, or A T P . s With respect to " a c c e p t o r s u b s t r a t e " specificity, the e n z y m e has, first of all, a strong preferential affinity for low molecular weight dextrans ;~2-14 t h a t is, the addition of fractions with average molecular weights of ca. 5000 to ca. 60,000 in small a m o u n t to enzyme-sucrose mixtures causes synthesis to be so modified t h a t dextran molecules of low or intermediate size rather t h a n of large size are formed as m a j o r reaction products. ~5
8 W. W. Carlson, C. L. Rosano, and V. Whiteside-Carlson, J. Bacterial. 65, 136 (1953). 8 E. J. Hehre, Science 93, 237 (1941). Traces of material with the serological properties of dextran were formed in incubated enzyme-raffinose mixtures, suggesting that raffinose may be a donor substrate of low affinity. However, this effect may possibly have been due to enzymic action on traces of sucrose liberated in some way from the trisaccharide. 8~ H. M. Tsuchiya and C. S. Stringer, Bacteriol. Proc., p. 98 (1954). lo H. L. A. Tart and H. Hibbert, Can. J. Research 5, 414 (1931); Staff Report, Chem. Eng. New~ 29, 650 (1951). The lack of formation of any dextran in incubated enzyme-glucose mixtures was shown by the use of serological tests of high specificity and sensitivity2 lz E. J. Hehre, Proc. ,.%c. Exptl. Biol. Med. 54, 240 (1943). 18E. J. Hehre, or. Am. Chem. ,%c. 75, 4866 (1953). 18H. M. Tsuchiya, N. N. Hellman, and H. J. Koepsell, J. Am. Chem. Soc. 75, 757 (1953). 14H. Nadel, C. I. Randles, and S. L. Stahly, Appl. Microbiol. 1, 217 (1953). ~6 Reaction products of suitable molecular weight for use as a blood volume expander have been produced in good yield from mixtures (kept at pH 5.0 and 15°) containing
[21]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
183
The enzyme also utilizes certain sugars (especially isomaltose and maltose, but also a-methyl glucoside and glucose) as acceptors for the glucosyl units from sucrose. ~ Enzymic action on equimolecular concentrations of sucrose and any one of these sugars differs from the action on sucrose alone in t h a t the reaction rate (as measured b y fructose liberation) is increased, oligosaccharides based on the sugar acceptor appear as major reaction products, and less high molecular weight dextran is synthesized. Fructose, leucrose, melibiose, and galactose also appear to act as acceptors, since in their presence (equimolecular with sucrose) some oligosaccharide formation occurs and the reaction rate is slightly depressed; the activity of both pyranose and furanose forms of fructose is indicated b y the formation of two fructose-containing disaccharides, leucrose (5-D-glucopyranosyl-D-fructopyranose) and isomaltulose (6-Dglucopyranosyl-n-fructofuranose), ~s in mixtures containing added fructose. e,~7 Dextransucrase apparently does not utilize as acceptors xylose, D- and L-arabinose, ribose, rhamnose, mannose, sorbose, cellobiose, trehalose, lactose, melezitose, raffinose, inulin, inositol, mannitol, sorbitol, 2-ketogluconate, 5-ketogluconate, or glycerol, e High molecular weight (native) dextrans cannot be excluded as acceptors, but their addition does not affect the rate of dextran synthesis from sucrose. ~ The production of small amounts of glucose in enzyme-sucrose mixtures (Forsyth and Webley 5) suggests t h a t water m a y be an acceptor substrate of low affinity, unless traces of invertase contaminate the available enzyme preparations. E q u i l i b r i u m . The reaction in the synthetic direction proceeds beyond 99.5% completion; ~,~ reversal, although theoretically possible, has not been detected. Activators and Inhibitors. Apart from the sugars noted above, no special activators or inhibitors of dextransucrase are known. The following common enzyme poisons are without detectable effect: 0.05 M fluoride 40 units/ml, dextransucrase, 100 mg./ml, sucrose, and from 2 to 20 mg./ml. (depending on the molecular weight) of low molecular weight dextran "acceptor." H. M. Tsuchiya, N. N. Hellman, H. J. Koepsell, J. Corman, C. S. Stringer, F. R. Senti, and R. W. Jackson, Abstr. 124th Meeting Am. Chem. Soc., 35A (1953). 16F. H. Stodola, H. J. Koepsell, and E. S. Sharpe, J. Am. Chem. Soc. 74, 3202 (1952); F. H. Stodola, E. S. Sharpe, and H. J. Koepsell, Abstr. 126th Meeting Am. Chem. ,Soc. (1954). 17Since fructose accumulates as a product of enzymic action upon sucrose, its effect may be observed even under "normal" circumstances. Leucrose in small amount appears on paper chromatograms in the terminal phases of action of dextransuerase from L. mesenteroides N U R B B-512 upon 0.125 M sucrose; larger amounts appear when the enzyme operates on higher concentrations of sucrose (and larger amounts of fructose are released) or when fructose is added to enzyme-sucrose m i x t u r e s )
184
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
or cyanide, 0.025 M azide or iodoacetate, 0.01 M pyridine or aniline, 0.001 M CuS04, ZnS04, or MnC12, or 0.00001 M AgN03.1 Enzymic activity likewise is unaffected by dialysis or by the presence of sodium ethylenediamine tetraaeetate (versene).S An early suggestion that dextransucrase may be activated by PABA has not been confirmed. Effect of pH and Temperature. The enzyme is most active and stable between pH 4 and 6, ~ with the optimum reported as pH 5.0 to 5.2 ~ or pH 5.6. 8 Activity is rapidly lost on exposure to pH 6.7 or above, even at 25 to 37 °. Moreover, the enzyme catalyzes the formation of dextran over a wide temperature range (3 to 37 °) but is, nevertheless, very heat labile. 7 For example, it shows high activity and stability at 30 °, yet is destroyed in a few minutes at 40 °, even at pH 5.0. ~ Lyophilized preparations, sealed in vacuo and stored at 5 °, retain their potency for many years. Reaction temperature has been observed to influence the nature of the dextran produced when the enzyme operates in the presence of added dextran acceptor. Lowering the reaction temperature of such systems from 30 ° to 15 ° greatly favors the formation of intermediate- as opposed to high-molecular weight dextran. 1~ II. Amylosucrase n-C1uI-I2~O11 -~- H O R --* H(CeHloOb)~OR -I" ~-C6H1206 Sucrose Acceptor Glycogen-type Fructose polysaccharide
Assay Method A detailed procedure for estimating enzymic activity has not been reported, but a suitable basis is measurement of the fructose by-product released per unit of time under conditions yielding zero-order reaction kinetics. Such conditions are approached when enzyme solutions are incubated at 10 ° with an equal volume of 0.2 M sucrose in 0.025 M maleate buffer, pH 6.4. is Rough appraisals of activity may be obtained also from the speed of development of opalescence, color with iodine, aleohol-preeipitable material, or other attribute of the glycogen-type polymer that is synthesized.
Preparation The biological source is Neisseria perflava, a species of bacteria readily isolated from the throat. 19 Crude but cell-free enzyme preparations may be obtained b y the author's method. ~s Cultivate N. perflava (strain 19-34) 18E. J. Hehre, J. Biol. Chem. 177, 267 (1949). 19E. J. Hehre and D. M. Hamilton, Y. Biol. Chem. 166, 777 (1946); J. Bacl~riol. 66, 197 (1948).
[21]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
185
for 5 days at 37 ° in unshaken flasks of broth (800 ml.) comprising 0.1% peptone, 0.15% sodium citrate, 0.02% yeast extract, 0.06% KH2PO4, 0.15% Na~HPO4, and 0.05% glucose. (Inoculate each flask with 10 ml. of a 1-day culture in the same medium.) Treat each 800 ml. of grown culture with 300 g. of ammonium sulfate, centrifuge the mixture, and decant the supernatant fluids. Suspend the sediments in 100 ml. of half-saturated ammonium sulfate previously adjusted to pH 6.4 with ammonia, centrifuge, and discard the wash fluid. Extract the washed and drained precipitates with 15 ml. of 0.025 M maleate buffer (pH 6.4), and clarify by centrifugation.
Properties Specificity. The only known "donor substrate" for amylosucrase acting synthetically is sucrose. Neither glucose, maltose, lactose, trehalose, a-methyl glucoside, raffinose, melezitose, nor glucose-l-phosphate serves the enzyme in this capacity. 18.2° Amylosucrase presumably utilizes a series of amylosaeeharides as "aeceptor substrates" in the course of building up polysaccharide molecules, but its affinity for various acceptors has not been systematically examined. Equilibrium. The conversion of sucrose to polysaccharide and fructose proceeds to ca. 98% completion. 21 Reversal to the extent of about 1% can be demonstrated when the enzyme is incubated with fructose plus amylose, amylopectin, or glycogen. Inhibitors. Synthesis of polysaccharide from sucrose is not impaired by the presence of inorganic phosphate in concentrations sufficiently high (8 moles P to 1 of substrate) to suppress all synthesis from glucose-lphosphate by the phosphorylase in N. perflava extracts. 18,1g Traces of salivary amylase, however, inhibit the formation of polysaccharide and fructose from sucrose, possibly by destroying essential acceptor substrates. 21 Effects of pH and Temperature. Satisfactory enzymic action occurs over the temperature range of 10 to 37 ° at pH 6.4. Amylosucrase (pH 6.4) appears stable at 5 ° for many weeks but is almost completely inactivated at 45 ° for 10 minutes (under which conditions the phosphorylase activity of the extracts remains unaffected~8). Information is lacking on the activity and stability in different pH ranges. 2o N . perflava preparations produce an amylaceous polysaccharide from glucose-lphosphate, and also a glycogen-type polysaccharide from amylose, but these actions are attributable to an accompanying phosphorylase and to a branching enzyme of the Q type. I1 E. J. Hehre, Advances in Enzymol. 11, 297 (1951).
186
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
HI. Levansucrase
n-ClsH2~Oll ~Sucrose
H0R --~ H(C6HIoOs). -{- n-C6HI~O~ Acceptor Levan Glucose
Assay Method
P r i n c i p l e . The circumstance that a variable but generally large amount of sucrose hydrolysis regularly accompanies levan formation in reactions catalyzed by available enzyme preparations has prevented development of an assay method based on the rate of liberation of glucose. The following procedure of Hestrin and Avineri-Shapiro ~2 measures the rate of formation of highly polymerized levan itself under conditions where the synthesis follows an essentially linear course. (For rough appraisals of enzyme activity, the speed of release of reducing sugars and of development of features referable to levan, e.g., opalescence, viscosity, serological activity, or alcohol-precipitable polyfructoside, may be found useful.) Procedure. The assay mixture comprises 1 ml. of enzyme solution, 1 ml. of 15 % sucrose in distilled water, 1 ml. of SOrensen citrate buffer (pH 5.0), and 1 drop of chloroform containing thymol for maintaining sterility. Incubate at 37 ° until the levan concentration is in the range of 50 to 200 mg./100 ml. (as judged by comparison of the opalescence of the assay mixture with reference solutions containing known concentrations of levan), and then determine the levan content as follows.23 Pipet 1.0 ml. of the assay solution into a centrifuge tube of 15-ml. capacity, and add 3.0 ml. of ethanol. If flocculation is slow, it can be hastened and rendered quantitative by the addition of a drop of 1% CaC12. The suspension is centrifuged, and the sedimented levan freed from reducing sugar and sucrose residues by twice-repeated solution in H20 over a water-bath and precipitation each time with ethanol. Addition of a glass bead to the mixture facilitates these operations. The final sediment is taken up in 3.0 ml. of 0.5% oxalic acid and hydrolyzed in a boiling water bath for 1 hour. Evaporation is restricted during this treatment by placing a glass bulb at the tube aperture. The hydrolyzate is neutralized, cleared with Zn(OH)2, and diluted to a suitable volume. The reducing power of the filtrate is estimated by the method of Somogyi. The amount of levan is calculated from the "glucose value" by multiplying by a factor which allows both for the small difference in reducing power between glucose and fructose and for the entry of HsO during hydrolysis.
~2S. tIestrin and S. Avineri-Shapiro,Biochem. J. $8, 2 (1944); S. Avineri-Shapiroand S. Hestrin, Biochem. J. $9, 167 (1945). 23 S. Hestrin~ S. Avineri-Shapiro,and M. Aschner, Biochem. J. 37, 450 (1943) ; Nature 149, 527 (1942).
[21]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
187
Definition of Unit. One levansucrase unit (LU) is defined as the minimal amount of enzyme which induces production of 100 rag. of levan per 100 ml. in 2 hours under the above conditions. This definition is designed to circumvent the variation observed in the specific activity of levansucrase at different enzyme concentrations. Limitations. The presence of levan-splitting enzymes (levanases) in certain enzyme preparations (e.g., from Bacillus subtilis) may lead to an erroneously low levansucrase assay figure. Likewise, the presence of preformed levan in crude sucrose broth culture fluids (unless corrected for) may give an error in the opposite direction. Extensive dextran production, which may occur under levansucrase assay conditions in the case of extracts from certain bacteria (especially certain streptococci), may also lead to unduly high assay values and should in any case be recognized.
Preparation Bacteria of many kinds produce levan from sucrose or raffinose, and crude, cell-free levansucrase preparations have been obtained from varieties in such diverse genera as Bacillus, Streptococcus, and Aerobacter. Hestrin et al. 23 give the following method for preparing enzymes suitable for kinetic studies from Aerobacter levanicum. The bacterium (Jerusalem strain) is cultivated in 1-1. conical flasks at 30 ° in a two-phase medium consisting of a bottom solid layer of nutrient agar devoid of sucrose and a thin top layer of 2 % aqueous sucrose. After 20 hours the cells are harvested, rinsed repeatedly in water to free them from medium constituents and levan, and finally left to autolyze at 37 ° under water containing a little thymol and chloroform. The volume ratio of culture medium to autolyzate fluid is 100:1. After 24 hours of autolysis, the suspension is centrifuged. The cell-free supernates may assay as high as 5 units/ml. Various alternative methods have been used in preparing levansucrase from spore-forming bacilli or streptococci. ~,~3-27 Since a procedure found applicable to one particular bacterial strain may not be suitable for another, the trial of several methods is advised.
Properties Specificity. Levansucrase utilizes sucrose and raffinose (a galactosyl sucrose) as "donor substrates," with the Michaelis constant for sucrose in the range of 0.02 to 0.06 M. The enzyme does not utilize any other common sugar or other substance with a terminal fructofuranose group (e.g., 24 E. J. Hehre, Proc. Soc. Exptl. Biol. Med. 58, 219 (1945). ~5 M. Doudoroff and R. O'Neal, J. Biol. Chem. 159, 585 (1945). 26 F. L. Horsfall, Jr., J. Exptl. Med. 93, 229 (1951). ~v R. Dedonder and Mine. Noblesse~ Ann. Inst. Pasteur 85, 356 (1953).
188
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
fructose-l,6-diphosphate, fructose-6-phosphate, methyl fructofuranoside, inulin), in this way. There is also no action upon " f r e e " or " n a s c e n t " fructofuranose or upon inorganic phosphate. ~2,28,~9 Little information is as yet available concerning the "acceptor substrate" specificity of levansucrase. A series of glucose-ended 2,6-1inked polyfructosides is probably used in synthesis of the levan macromolecule. ~9~In addition, B. subtilis preparations seem to have some affinity for glucose (but not fructose) as an acceptor. The action on a mixture of 10 % sucrose and 5% glucose, for example, differs from the action of 10% sucrose alone in that oligosaccharides containing glucose as well as fructose appear as prominent (although transient) reaction products, and high molecular weight levan is less rapidly synthesized.~7 (Cf. Activators and Inhibitors.) Equilibrium. Because of the complication of concomitant hydrolytic breakdown of the substrate (sucrose) by the available enzyme preparations, the exact position of equilibrium in levan synthesis remains undefined. Sufficient data have been obtained, ~2however, to establish that the ratio of levan to sucrose at equilibrium is definitely greater than unity. Yields of levan as high as 62 % of the theoretical maximum have been obtained under certain conditions. Since reversal of the reaction by the action of enzyme on levan and glucose has not been detected by direct means, ~ an equilibrium position far to the side of synthesis seems likely. Activators and Inhibitors. Certain sugars, including D-glucose, L-arabinose, D-xylose, L-sorbose, lactose, maltose, a-methyl glucoside, and D-galactose (but not D-fructose, D-mannose, mannitol, sorbitol, glueosamine, or trehalose), retard the formation of highly polymerized levan from sucrose by A. levanicum preparations. The "inhibitory" effect increases, in the case of D-glucose at least, as its concentration relative to sucrose is increased; and levan formation is completely suppressed, for example, in mixtures containing 1% sucrose and 16% D-glucose. ~2 It would appear most probable that this "inhibition" effect actually represents a modification of the synthetic reaction, and that D-glucose and the other sugars in question are "acceptor substrates." Some of them may actually stimulate the rate of fructosyl group transfer by the enzyme. 28It should be noted, however, that gentianose, verbaseose, and several recently dis-
covered sucrose-ended oligosaeeharidespassing terminal fructofuranosyl units, i.e., erlose, kestose, inulobiosyl glucose, have not been examined and may be able to serve as donor substrates for levansucrase. ~9S. Hestrin, Nature 164, 581 (1944). ~9~Neither levanbiose, levantriose, nor levantetraose, however, has been found to serve as an acceptor substrate (or modifier of levan synthesis) in the case of Aerobacter levansucrase. See S. Hestrin, Instituto Superiore di Sanita' Symposium on Microbial Metabolism, Rome, p. 53, 1953.
[21]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
189
Known poisons of respiration or of glycolysis, including 0.002 M cyanide, 0.02 M fluoride, 0.001 M monoiodoacetate, and 0.001 M phloridzin, fail to suppress or markedly inhibit levan formation. Dialysis similarly does not affect the activity of the enzyme, and A. levanicum extracts do not contain free adenylic acid. 22 Effect of pH and Temperature. The pH-activity curve of levansucrase operating upon sucrose at 37 ° is regular and symmetrical, with its peak in the region of pH 5.0 to 5.8. 22 The relationship between activity and temperature has not been systematically studied, but the enzyme operates satisfactorily over the range of 5 to 37 °. Enzyme stability (at pH 5.0) is very high at temperatures up to 30 ° . Storage for several days at this temperature, or for several months at 4 ° , has no deleterious effect. Gradual loss of activity at 37 ° is discernable over a period of days, and there is complete and almost immediate inactivation on exposure at 100°. 23 Preparations frozen and dried in vacuo are suitable for prolonged storage.
IV. Amylomaltase n-Cl~H22011 ~ H O R --) H(C~H10Os),OR ~ n-C6HI~O~ Maltose Acceptor Amylose-type Glucose polysaccharide
Assay Method Principle. The assay procedure (Monod and Torriani 3°) has as a basis determination of the glucose liberated under conditions where the reaction follows a kinetically zero-order course; the glucose is determined (in the presence of the maltose substrate) by measuring manometrically the uptake of 02 during its specific oxidation by notatin. Reagents 0.1 M phosphate buffer, pH 6.8 Notatin. Purified glucose oxidase of Penicillium notatum21 Buffered maltcse solution. The solution used as substrate for the assay contains 100 ~,/ml. notatin, 0.033 M maltose, and 0.02 M sodium azide (for inactivation of catalase) in 0.1 M phosphate buffer of pH 6.8. Enzyme. Prepare a known dilution, if necessary, to obtain an enzyme solution for assay that contains between 20 and 130 amylomaltase units/ml. (See definition below.) 3o j. Monod and A. M. Torriani, Ann. Inst. Pasteur 78, 65 (1950) ; Compt. rend. 227, 240 (1948). 31 C. E. Coulthard, R. Michaelis, W. F. Short, G. Sykes, G. E. H. Skrimshire, A. F. Standfast, J. H. Birkinshaw, and H. Raistrick, Biochem. J. $9, 24 (1945); see Vol. I [45].
190
ENZYMES OF CARBOHYDRATE METABOLISM
[21]
Procedure. Place 3.0 ml. of the buffered maltose solution containing notatin in the main chamber of a Warburg flask, and 0.1 ml. of enzyme in the side arm. After assembly of the manometric apparatus and equilibration at 28 ° , mix the contents of the flask. Measure the oxygen consumption at 10-minute intervals until a steady rate is achieved (usually 20 to 40 minutes). The 02 uptake in microliters per hour divided by 2.26 gives the micromoles of 0~ consumed per hour per milliliter of enzyme and, hence, the micromoles of glucose formed per hour per milliliter of enzyme. Definition of Unit. One amylomaltase unit is defined as the amount of enzyme which will liberate from maltose 1 ~M. of glucose per hour under the above conditions. Specific activity is expressed as units per milligram of Kjeldahl N. a° Extent and Limitations of Application. The procedure has been used for assay of amylomaltase in toluene-treated suspensions of intact bacterial cells of the ML strain of E. coli. s2 For use with cells or with impure soluble preparations, however, the possibility of interference by the presence of enzymes yielding glucose from maltose or from amylosaccharides would, of course, have to be guarded against.
Preparation The presence of amylomaltase has been described in two special mutant strains of E. coli, namely, the maltose- and lactose-positive form of strain ML, 3° and the maltose-positive lactose-negative form of strain K122 s The enzyme evidently is produced only when the bacteria are cultivated in the presence of maltose. Using strain ML, Monod and Torriani 3° prepare the cell-free enzyme as follows. The bacteria are heavily inoculated into 250-ml. amounts of culture medium in 2-1. conical flasks. (The medium is made up of 2.72 % KH2PO,, 0.4% (NH,),SO,, 0.04% MgSO,-7H~O, 0.001% CaCl~, 0.00005% FeSOr7H20, and sufficient NaOH to attain pH 7.5; after sterilization, a filtered solution of maltose is added aseptically to give an 0.8% concentration.) The inoculated flasks are incubated, with agitation, at 34 ° for 14 hours. The bacterial cells are harvested by Sharples centrifugation, washed in the centrifuge with a volume of distilled water equal to that of the original culture medium (2 to 10 1.), then suspended in 0.05 M phosphate buffer, pH 6.8; 200 ml. of buffer is used for each 100 g. of the washed cells. Fine sand, 2 g. for each 1 ml. of suspension, is then added, and the mixture vigorously agitated in a shaking apparatus for 40 minutes. The creamy mass is centrifuged at 12,000 r.p.m, for 15 minutes, and ~2G. Cohen-Bazire and M. Jolet, Ann. inst. Pasteur 84, 937 (1953). " M. Doudoroff, W. Z. Hassid, E. W. Putman, A. L. Potter, and J. Lederberg, J. Biol. Chem. 179, 921 (1949).
[9.1]
POLYSACCHARIDE SYNTHESIS FROM DISACCHARIDES
191
the supernatant fluid removed. The precipitate is resuspended in 100 ml. of buffer and again centrifuged. This operation is repeated a second time, and the supernatant fluids combined. Maltose is added to give a 0.05 M solution, followed by solid ammonium sulfate to 75 % saturation (in the cold). After 2 hours, the precipitate is separated by centrifugation and redissolved in 100 ml. of buffer. The precipitation is repeated three times under similar conditions with sufficient solid ammonium sulfate to give 50% saturation. The final precipitate is taken up in about 20 ml. of buffer, and insoluble material eliminated by centrifugation. All operations are at 0 °. To remove accompanying amylase and phosphorylase, the solution is dialyzed in the cold against distilled water. A precipitate should form as a result of this treatment or be induced by careful acidification of the dialyzate with acetic acid to pH 5.2. The precipitate is collected by centrifugation and extracted with 5 ml. of 0.025 M veronal buffer, pH 6.8, containing 0.2 M sodium sulfate. After storage overnight at 0 °, this final extract is cleared by centrifugation. The best preparations contain 5000 amylomaltase units/ml. (660 units/mg. N)2 °
Properties Specificity. The enzyme has affinity for amylosaccharides as well as for maltose as "donor substrates." It is uncertain, however, whether any donor other than maltose is utilized in the forward or synthetic reaction. Lactose, sucrose, melibiose, cellobiose, a-methyl glucoside,/~-methyl glucoside, trehalose, and glucose-l-phosphate definitely do not serve as donor substrates for the enzyme2 ° Amylomaltase is presumed to utilize maltose and its higher homologs as "acceptor substrates" in the course of polysaccharide synthesis. Glucose definitely is utilized in this capacity, since enzymic action on maltose in the presence of glucose leads to the formation of oligosaccharides as major reaction products, whereas action in the absence of glucose (in systems containing notatin) leads to the formation of an amylose-type polysaccharide as the product. 3°.33,34 The presence of glucose also permits the reverse reaction (polysaccharide degradation to maltose) to occur. 3° There is some indication that D-xylose and to some extent D-mannose (but not D-fructose, D-galactose, D-arabinose, or I,-arabinose) can replace D-glucose as the acceptor substrate in this reverse reaction. 3s Equilibrium. The polymerative action of amylomaltase upon maltose proceeds until about 60 % of the substrate has been converted; a similar equilibrium position is reached in the reverse direction, i.e., after enzymic action on equimolecular amounts of polysaccharide and glucose2 ° (When s4 S. A. Barker a n d E. J. Bourne, J. Chem. Soc. 1952, 209.
192
ENZYMES OF CARBOHYDRATE METABOLISM
[22]
the synthesis from maltose is conducted in the presence of notatin, an equilibrium is not observed and conversion to polysaccharide proceeds to completion.) Activators and Inhibitors. Enzymic activity is unaffected by the presence or absence of orthophosphate, and it is retained after dialysis20 E. coli cells that contain amylomaltase are active in converting maltose to higher homologs in the presence of 0.002 M iodoacetate2 T M Effect of pH and Temperature. Little has been recorded concerning the effect of pH and temperature upon enzyme activity and stability. Suitable activity is manifested in mixtures set at pH 6.8 and 28 to 32 °.
[22] Phosphorylases from Plants By W. J. WHELAN CH,0H
H
OH
CH ~OH
H
P0~Ks
0
OH a- D-Glucose-I. phosphate
CH20H
CH ~OH
OH
CH ~0It
0 OH
0 .... OH
Primer
11
CH,OH
CH~0H
CH~0H
K,HPO~ + H
0 OH
0 OH
0 OH
0 .... OH
Amyiose
Assay Method Pr/nc/p/e. Primer and G-1-P are mixed with enzyme, and the orthophosphate set free during amylose synthesis is measured colorimetrically. The following procedure is based on that of Green and Stumpf. 1
Reagent8 G-1-P solution (0.1 M) contains 37.22 g./1. of the dipotassium salt. 2 5 % soluble starch solution. 0.5 M citric a c i d - - N a 0 H buffer, pH 6.0. l D. E. G r e e n a n d P. K. S t u m p f , J. Biol. Chem. 142, 355 (1942).
' Pure, primer-free G-1-P is conveniently prepared by the method of R. M. MeCready and W. Z. Hasald, J. Am. Chem. 8oe. 66, 560 (1944).