THE BIOLOGICAL SYNTHESIS OF DEXTRAN FROM DEXTRINS*

BY EDWARD J. HEHRE

WITH THE ASSISTANCE OF DORIS M. HAMILTON

(From the Department of Bacteriology and Immunology, Cornell University Medical College, New York, New York)

(Received for publication, March 5, 1951)

The present paper deals with the formation of dextran from certain dextrins by the action of cultures and enzymes of an acetic acid bacte- rium, Acetobacter capsdatum. Hitherto, polysaccharides of the dextran class have been known to be produced only from sucrose, either by living cultures of Leuconostoc mesenteroides and certain related Lactobacteriaceae or, as shown in our laboratory some years ago (l-3), by the in vitro action of soluble Leuconostoc enzymes. Recognition of an alternative pathway of dextran biosynthesis has a close parallel in earlier st.udies of Hehre and Hamilton (4, 5), which showed that amylaceous polysaccharides likewise are not invariably formed from glucose-l-phosphate by phosphorylase systems, but may also arise from sucrose by the act.ion of enzymes of Neis- s&a per$ava. Both past and present observations illustrate the broad validity of the rule that natural products may be formed by diverse meta- bolic pathways, which might not have been anticipated to hold in the case of macromolecular substances.

The conversion of dextrins to dextran by Acetobacter systems brings polysaccharides of the starch-glycogen and dextran classes into the closest biological relationship known to exist between any two polysaccharide classes. The chemical basis for this kinship has of course long been ap- parent. Both amylopolysaccharides and dextrans are polymers of n-glu- copyranose units; the former consist of chains of cr-1,4-linked units, with a-l,6 branching points occurring in amylopectin and glycogen type com- pounds (6), while dextrans comprise chains of a-l ,6-linked units with a-l,4 branching points (7). Both dextrans and glycogen type polysac- charides, moreover, can be synthesized from the same substrate (sucrose) by appropriate enzymes (l-5).

Amylosucrase glycogen type 7 polysaccharide + fructose

I Sucrose

Dextransucrase + dextran + fructose

* Aided by a grant from the Corn Industries Research Foundation. 161

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162 SYNTHESIS OF DEXTRAN FROM DEXTRINS

Indeed, the first indication of biological interconvertibility came as the result of experiments in which a product with the immunological prop- erties of dextran was formed, in small amount, from mixtures of starch or glycogen plus fructose that had been incubated successively with amylo- sucrase and dextransucrase preparations. Search for a biological system capable of converting amylaceous materials to dextran in higher yield led us to investigate a report of Shimwell (8) that certain acetic acid bacteria from “ropy” beer, Acetobacter viscosum and A. capsulatum, become grossly viscous when grown in the presence of sufficient dextrin (a natural con- stituent of beer), but not when grown with glucose, fruct’ose, sucrose, or maltose. We (9) were able to show that the slimy materials elaborated in the dextrin cultures have serological properties like those described earlier (10) for the sucrose-derived dextrans of L. mesenteroides. Moreover, ma- terial recognizable serologically as dextran was produced when cell-free enzymes of A. capsulatum were allowed to act upon certain dext.rins.

Chemical evidence has now been obtained that the product formed from dextrin by cultures of A. capsulatum is indeed a dextran. In addition, dat,a will be given on the preparation and general mode of action of soluble enzymes capable of converting dext,rins to dextran.

EXPERIMENTAL

Isolation and Analysis of Product Formed from Dextrin by Living Cultures of A. capsulatum

Strain NCTC 4943 of A. capsulatum (8, 9) was inoculated into 2 liters of broth containing 10 gm. of Difco yeast extract and 66 gm. (dry weight basis) of Pfanstiehl alcohol-precipitated, soluble “bacteriological” dextrin made from corn-starch. After incubation at 25” for 10 days, the culture was grossly viscous, contained innumerable small jelly-like particles (8), and gave a positive serological test. for dextran (9) when tested in dilutions as high as 1: 100,000. Estimates of the amount of reducing sugars (as maltose) liberated on treatment with salivary amylase indicated that ap- proximately 14.5 gm. (22 per cent) of the dextrin originally present in the culture medium had been utilized.

The gelatinous culture product was isolated without recourse to the destruction of the residual dextrin by amylase in order to preserve any “amylase-sensitive” linkages that it might contain. The separation was guided by serological tests and by tests of iodine coloring capacity; and a product was obtained that accounted for at least 99 per cent of the sero- logically reactive material produced by the culture and contained less than 1 per cent of the dextrin used in the medium.

The culture was first adjusted to pH 6.0, warmed to 70” and shaken for 10 minutes to disperse the jelly-like particles, and then treated with 0.8

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E. J. HEHRE 163

volume of ethanol. The mixture was centrifuged after storage overnight in the cold, and the precipitated gelat,inous product, was dissolved in 2 liters of distilled water and freed of bacterial cells by centrifugation. The solution of crude A. capsulatum gum, following addition of 20 gm. of so- dium acetate, was treated with 800 ml. (0.4 volume) of ethanol; after 15 minutes standing at room temperature, the mixture was cent.rifuged and the supernatant fluid separated from a soft, voluminous precipitate which, upon serological exammation, proved to contain only traces of dextran. To the supernatant fluid, a further 400 ml. (0.2 volume) of ethanol were then added. The massive precipitate that formed was separated by cen- trifugation, dissolved in 1 liter of water, reprecipitated by 0.6 volume of methanol, and finally ground under methanol and dried in vacua at 25” over CaC12. The yield was 14.96 gm. (anhydrous basis), which corre- sponds closely to the amount of dextrin utilized by the culture and to the conversion of approximately 22 per cent of the dextrin originally present in the culture medium.

Properties of A. capsulatum Product-The isolated material was a snow- white, amorphous powder that dissolved slowly in water, after swelling, to give highly opalescent solutions. The ash and nitrogen (11) contents were negligible, i.e. less than 0.05 per cent, and the reducing power (12) was less than l/2000 that of glucose. The optical rotation was [c&& = +190” (c = 0.25 in 0.5 N NaOH). After heating for 6 hours with 1 N

HZ‘S04 in a sealed glass tube immersed in boiling water, the hydrolysate (neutralized) had [cx$&,~ = $53.7” (c = 2.5) and 94 per cent reducing sugar, as glucose (12) ; these values take into account the entry of water during hydrolysis. A 1.00 gm. sample of the product, after similar hy- drolysis, removal of sulfate as BaSO+ and evaporation from alcoholic solu- tion yielded 0.98 gm. (anhydrous basis) of crystalline n-glucose. These data indicate that the A. capsulatum product is a high molecular weight polysaccharide composed chiefly of a-linked n-glucopyranose units.

Further information on the constitution of the Acetobacter polyglucoside was obtained by direct comparison with four reference polysaccharides; i.e., dextran isolated from sucrose broth culture of L. mesenteroides B (lo), oyster glycogen, corn amylopectin, and the “bacteriological” dextrin used as the substrate in the culture medium. All were simultaneously sub- jected to the following procedures.

The limits of conversion to maltose by o(- and ,&amylase were deter- mined on 5 mg. samples of each polysaccharide, dissolved with the aid of heat in 3 ml. of 0.15 M NaCl. Individual samples were incubated for 22 hours at 30” with (a) 3 ml. of a clarified 1:25 solution of saliva, pH 5.8, and (5) with 3 ml. of a solut,ion containing 1 mg. of wheat P-amylnse in 0.1 M acetate buffer, pH 4.6 (13). The reducing sugars liberated were cal-

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164 SYNTHESIS OF DEXTRAN FROM DEXTRINS

culated as per cent of apparent maltose (12, 14) obtained after correction for the entry of water.

Observations on coloration with iodine were made on 5 ml. of 0.2 per cent solutions of the polysaccharide (at pH 5.8) treated with 0.2 ml. of 1 per cent 12 and 2 per cent KI. Rates of hydrolysis by 0.5 N HCI were determined on 0.05 per cent solutions of the polysaccharides. The mix- tures were heated in sealed tubes at 100” for 15, 30, and 45 minutes, then cooled, neutralized, and tested for glucose content (12). The rate is ex- pressed as a multiple of the first order velocity constant, i.e. K1 X lo3 (5, 15).

Capacity to give serological precipitation with dextran-reactive anti- sera was examined with varying dilut,ions of all the polysaccharides. A detailed comparison of the serological properties of the purified Acetobacter and Leuconostoc polysaccharides has already been report,ed (9).

Oxidation by sodium metaperiodate was carried out by a slight modifi- cation of the technique of Jeanes and Wilham (lS).l Between 105 and 110 mg. (dry weight) of polysaccharide were dissolved in freshly boiled water and treated with 6 ml. of 0.275 M sodium metaperiodate (i.e., ap- proximately 2.5 moles of NaI04 per mole of glucose anhydride) and suffi- cient water to make 250 ml. The mixture was stored in the dark at 25” and analyzed after 24, 48, and 72 hours. For determination of the periodate consumption, 50 ml. of the mixture were treated with 6 ml. of 10 per cent NaHC03, 10 ml. of a solution of 0.1 N NazHAsOB in 2 N

NaHCOs, and 0.4 ml. of 20 per cent KI; after storage in the dark for 10 minutes, the excess NazHAsOo was estimated by titration with 0.1 N

iodine. Formic acid liberation was determined on a 25 ml. sample of the polysaccharide-periodate mixture by titration to phenolphthalein with 0.01 N NaOH after preliminary incubation in the dark for 1 hour at 25” with 0.1 ml. of ethylene glycol. Table I lists the periodate consumption and formic acid liberation at 48 hours as moles per mole of the basic unit (glu- case-HTO) of the polysaccharides.

From the data summarized in Table I it is evident that the major fea- tures of the polyglucoside produced by A. capsulatum resemble those of Leuconostoc dextran and differ from those of glycogen, amylopectin, and the dextrin from which the Acetobacter product was formed. Those fea- tures include nearly complete resistance to (Y- and /3-amylase, lack of ap- preciable color with iodine, relatively low rate of hydrolysis by acid, serological activity, and behavior on periodate oxidation. The slight re-

1 The author’s thanks are due to Dr. Allene Jeanes for her kindness in making available, in advance of publication, the technique of periodate oxidation used at the Northern Regional Research Laboratory, as well as the results obtained with the method on various Leuconostoc dextrans.

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E. J. HEHRE 165

lease of reducing sugar on treatment with amylase and the slight capacity of the Acetobacter product to be colored by iodine may be attributed, at least in part, to traces of amylaceous material accompanying the bacterial polysaccharide in its isolation from the dextrin broth culture.2

The data obtained by periodate oxidation furnish good evidence that the Acetobacter product, like the Leuconostoc dextran, is a predominantly 1,6-linked polyglucopyranoside. The Acetobacter polyglucoside (Table I) reduced 1.89 moles of periodate and liberated 0.82 mole of formic acid per

TABLE I Properties of A. capsulatum Polysaccharide Compared with Those of Leuconostoc

Dextran and of Polysaccharides of Starch Class

con;y;;;~ to Rate of

Polysaccharide Color with II, acid hy-

Salivary Wheat 1500 solution drolysis,

a-amyl- /3-amyl- KI x 10’

ase ase -- ger cent per cent min.-=

Acetobacter polyglu- coside.. 1.0 0.2 Nonet 11

Leuconostoc B dex- tran. . . . . . . . . . . . . . 0.0 0.0 “ 10

Oyster glycogen.. 71 33 Red-brown 41 Corn amylopectin. 93 61 Dark purple 54 “Bacteriological”

dextrin.. 86 64 Deep maroon 52

.-

-

I Periodate oxidation

moles

+++ 1.89

+++ 1.83 0 1.02 0 1.06

0 1.22

“%-j liberated

mk

0.82

0.84 0.08 0.04

0.22

* +++, precipitation beyond 1:l million dilution of the polysaccharide with type 2 and type 20 pneumococcus antisera, and from l:lO,OOO to 1:50,000 dilution with type 12 pneumococcus antiserum (9). 0, no precipitation with l:lO,OOO or higher dilutions of the polysaccharide in tests with type 2, 20, or 12 pneumococcus antisera.

t A greenish color was produced, however, when a stronger solution (1:50) of the polysaccharide was treated with iodine.

mole of glucose anhydride during a 48 hour oxidation period, a result closely similar to that obtained with the dextran of L. mesenteroides. B and comparable to results obtained with Leuconostoc dextrans by others (16, 17). An unbranched polysaccharide composed entirely of 1,6-linked

2 It is noteworthy, however, that treatment with saliva (though not with wheat p-amylase) causes some diminution in the opalescence of solutions of the Acetobacter polysaccharide as well as some loss of capacity of the polysaccharide to be precipi- tated by 35 per cent (aqueous) alcohol. These observations suggest that the Aceto- batter polysaccharide molecule? may contain one or more internally located, (Y- amylase-sensitive linkages; and they raise the question of whether such linkages may not arise as the result of the growth of multiple chains of 1,6-linked units from a single dextrin molecule acting as a “primer.”

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166 SYNTHESIS OF DEXTRAN FROM DEXTRINS

n-glucopyranose units theoretically would consume 2 moles of periodate and liberate 1 mole of formic acid per mole of the glucose unit, whereas high molecular weight polymers of glucose other than 1,6-linked poly- glucopyranosides would be expected neither to yield appreciable formic acid nor to reduce appreciably more than 1 mole of periodate per mole of glucose, since their basic glucose units would not have unsubstituted hy- droxyl groups on 3 adjacent carbon atoms, as in the case of the basic glucose units of dextrans.

From the amount of formic acid liberated, Jeanes and Wilham (16)’ have computed that the ratio of 1,6 linkages to linkages other than 1,6 for six specimens of Leuconostoc dextrans ranged from 24: 1 to 3 : 1. In the case of the Acetobacter and Leuconostoc B polysaccharides, the ratio of 1,6 linkages to linkages other than 1,6 is approximately 5: 1.3 The data of course do not give any information as to the nature of the linkages other than 1,6, nor do they indicate whether or not the occurrence of such link- ages causes the molecule to be branched.

Conversion of Dextrins to Dextran by Cell-Free Enzymes from A. capsulatum

The vigor of dextran formation by cultures of A. capsdatum grown with dextrin, the complete lack of dextran formation by cultures grown with other carbohydrates, and 4he approximate equivalence between dex- tran formed and dextrin lost all reflect the operation of an enzyme system capable of converting the oc-1,4-linked n-glucopyranose units of dextrin into the 1 ,6-linked units of dextran.

Solutions of this enzyme system (9), free from bacterial cells and cell fragments, were prepared for the experiments of the present paper from cultures of A. capsulatum 4943 grown at 25’ for 3 or 4 days in broth com- prising 0.5 per cent Difco powdered yeast extract and 0.2 per cent glucose. 4 liter lots bf culture (usually with a final pH about 4.5) were treated with ammonium sulfate (1.5 kilos), centrifuged for 1 hour at 1500 r.p.m., and the resulting-sediments taken up in 75 ml. of distilled water. After sepa- ration of most of the bacterial cells by preliminary centrifugation, the enzyme solutions were cleared in a large angle head centrifuge and stored at -70”. Mixtures with solutions of “bacteriological” dextrin show pro- gressive diminution both in the intensity of the coloration given with iodine and in the amount of reducing sugar liberated by salivary amylase, suggesting utilization of dextrin; at the same time, they acquire in in- creasing degree a new property which may be regarded as presumptive evidence of dextran synthesis (9) ; namely, the capacity to give serological precipitation with types 2 and 20 pneumococcus antisera. These changes

3Since these computations were made, Kent (18) has reported a 5:l ratio for the Leuconostoc mesenteroides dextran of Stacey and Swift (19).

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E. J. HEHRE 167

are especially prominent in enzyme-dextrin mixtures kept in the region of pH 4 to 5.

Substrate SpeciJicity-To gain some understanding of the mechanism of its action, the enzyme was tested for its abi1it.y to synthesize material with the serological properties of dextran from a variety of carbohydrates. Equal volumes of enzyme solution and of substrate (as a 2 per cent or a 0.1 per cent solution in 0.5 M acetate buffer, pH 4.6) were kept for 20 hours at 25”. The incubated mixtures were neutralized, then tested for capacity to give serological precipitation versus a type 2 pneumococcus antiserum capable of detecting Acetobacter or Leuconostoc dextrans in ,con- centrations as low as 1 part in 4 million. Parallel tests were made with control sera as a guard against “false positive” reactions.

It would appear from the data in Table II that the Acetobacter enzyme catalyzes the synthesis of dextran from certain dextrins only. The forma- tion of dextran was not detected, for example, when the enzyme was in- cubated with unhydrolyzed amylose or amylopectin fractions of corn- or potato starch, or with native oyster or rabbit liver glycogens. Hydrol- ysates of amylose, amylopectin, and glycogen produced by acid or sali- vary (a-) amylase did cont.ain products convertible into material with the serological properties of dextran; this was not true, however, of partial hydrolysates of the same substances produced by wheat @-) amylase. Similarly, Schardinger’s fi-dextrin (cycloheptaamylose) was not converted to dextran by the Acetobacter enzyme, while the seven-membered linear dextrin (amyloheptaose) derived from it by opening the cyclic ring by acid (22), as well as the corresponding dextrinic acid, was converted. None of the potential ar-D-glucose donating sugars test.ed, including maltose, sucrose, and glucose-l-phosphate, were converted to dextran. In the case of maltose, samples of the commercial reagent did yield small, variable amounts of dext.ran on incubation with the enzyme, even after they had been treated with carbon and repeatedly recrystallized; however, we be- lieve this activity is attributable to traces of difficultly separable accom- panying dextrins in these reagents, since entirely negative results were obtained with samples of maltose prepared in the laboratory by a method4 which precluded the formation of low mblecular weight dextrins in the digests from which the maltose was crystallized.

One of the most important points shown by the data in Table II is that dextran synthesis can take place from linear fragments composed entirely

4 The maltose was prepared according to the technique of Haworth et al. (25)) with the important difference that a 15 minute digest of potato amylopectin repre- sented the starting material rather than a long term digest of solubilized whole starch. One lot of maltose was made by the use of commercial (Wallerstein) p- amylase from ungerminated barley, a second by the use of p-amylase prepared from wheat flour (21).

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TABLE II Capacity of Soluble A. capsulatum Enzyme to Convert Various Carbohydrates

to Dextran

Carbohydrate substrate

- “Natural” polysaccharidest

HCl hydrolysatess

Salivary (a-) amylase hy- drolysatesn

Wheat (p-) amylase hydroly- sates11

Commercial dextrins

Unbranched dextrins7

Maltose

Other sugars

Recrystallized potato amylose. . “ corn amyloseS. ....

Potato amylopectin .............. Corn amylopectin ................ Rabbit liver glycogen Oyster glycogen. ................ Potato amylose ..................

“ amylopectin .............. Oyster glycogen .................. Potato amylose ..................

“ amylopectin. Oyster glycogen. ................ Potato amylose .................

“ amylopectin .............. Oyster glycogen. ................ “Bacteriological” corn dextrin. .. Soluble potato starch (Lintner) Crystalline cycloheptaamylose . Amyloheptaose .................. Sodium amyloheptaoate .......... Commercial c.p. Reagent 1.. .....

‘I ‘I “ 2. ...... Laboratory Preparation 1. .......

“ “ 2. ....... Glucose ......................... Sucrose**. ....................... Trehalose ........................ Glucose-l-phosphate. ............ Glucose-6-phosphatett . .......... o-Methyl glucoside. .......... Raffinose ..................... Melezitose .....................

-

D&ran after incu- bation with enzyme’

1.0 per cent sub-

strate

0 0 0 0 0 0

4000+ 4000 4000 4000f 4000+ 4006

0 0 0

4000 1000

0 4000+ 4000+

20 100+

0 0 0 0 0 0 0 0 0 0

i

0.05 per cent sub-

strate

0 0 0 0 0 0

400 200 200 400f 400 400

0 0 0

lOOf 0 0

1000+ 1000

0 0 0 0 0 0 0 0 0 0 0 0

* Highest dilution of enzyme-substrate mixture that gave precipitation versus the dextran-reactive antiserum. 0, no precipitation with 1:20 or higher dilution.

t Pentasol fractions (20) from corn-starch were kindly furnished by Dr. T. J. Schoch; those from potato starch were prepared by Mr. A. S. Carlson. The amyloses were dissolved in 0.5 N NaOH at 25’ and adjusted to pH 4.6.

t Extensive retrogradation occurred during the period of incubation with the enzyme.

8 Solutions in 1 N HCl were heated at 80” for 15 minutes, cooled, and adjusted to pH 4.6. The extent of hydrolysis (as glucose) was 14, 11, and 8 per cent for the amylose, amylopectin, and glycogen.

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E. J. HEHRE 169

TABLE II-Concluded

)I Solutions of the polysaccharides digested with diluted saliva at pH 5.5 or wheat P-amylase (21) at pH 4.6 for 15 minutes at 25”, heated at 100” for 10 minutes, and brought to pH 4.6. Hydrolysis of amylose, amylopectin, and glycogen (as “mal- tose”) was 70,59, and 35 per cent in the case of salivary amylase digests and 75,47, and 21 per cent in the case of the wheat amylase digests.

7 Cycloheptaamylose and amyloheptaose (22) were kindly furnished by Dr. Dexter French. Oxidation of amyloheptaose (23) yielded sodium amyloheptaoate with one-fiftieth the reducing power, and Na content of 2.09 per cent.

** Beet sugar, free from the traces of serologically active material found in most reagent sucrose (24).

it Kindly supplied by Dr. W. W. Wainio; Na salt adjusted to pH 4.6 with acetic acid.

of a-l ,4-linked n-glucopyranose units; i.e., from amyloheptaose and from acid and salivary amylase hydrolysates of crystalline potato and corn amyloses. That fact gives strong support to the idea that the essential action of the Acetobacter enzyme is to convert polymerized 1,4-linked glu- cose units into polymerized 1,6-linked units. Moreover, since sodium amyloheptaoate shows activity as a substrate comparable to that of amy- loheptaose, while cycloheptaamylose is inert, the Acetobacter enzyme would appear to require a chain of 1,4-linked units with a free, non-reducing terminal. A single “propagative” step in the enzymatically catalyzed growth of a dextran macromolecule, occurring at the expense of a non- reducing terminal glucose unit of a linear dextrin molecule, might be pic- tured as in Fig. 1. “Branch points” and “reducing terminals” evidently

jz2&!/$*~*~Qoee FIG. 1. Possible basic action step (transfer of glucosyl group from dextrin to

dextran) of the Acetobacter enzyme.

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170 SYNTHESIS OF DEXTRAN FROM DEXTRINS

are not required of a dextrin for its conversion to dextran, though further experiments are needed to determine what influence is exerted on the re- action by glucose units in such special structural positions.

The failure of the Acetobacter enzyme to convert unhydrolyzed starch components or glycogens to serologically detectable dextran is worthy of special comment, since the literature records but few examples of true dextrinases; i.e., enzymes capable of attacking amylaceous split-products, though not st.arch or glycogen. According to current concepts of starch structure, chains of (Y-1,4-linked n-glucose units with non-reducing ter- minals should have been present in all the test mixtures with glycogen, amylopectin, or amylose; they should have been present., indeed, in con- siderably higher concentrations in the inactive mixture containing 1 per cent glycogen or amylopectin than, for example, in the mixture containing 0.05 per cent amyloheptaose, which gave a positive test for dextran when diluted 1000 times. Evidently the arrangement of terminated glucose chains as part of a macromolecule makes them unsuited for conversion to dextran by the Acetobacter enzyme.5

A similar limitation may also account for the failure of the p-amylase digests of glycogen, amylopectin, and amylose to serve as substrates. The glycogen and amylopectin hydrolysates doubtless contained an abundance of terminated chains of glucose units, but presumably all were in the form of short outer branches of high molecular dextrins intermediate between the native polysaccharides and the limit dextrins. The potato amylose hydrolysate, which showed 75 per cent conversion to maltose, likewise may have contained no linear dextrin molecules smaller than several hun- dred glucose units in length.

Action on Amyloheptaose-The most active and most well defined sub- strate found for the Acetobacter enzyme was the seven-membered linear dextrin (amyloheptaose) of French, Levine, and Pazur (22). The action of Acetobacter enzyme on this subst,ance was examined from a quantitative aspect in order to det,ermine the relationship between dextrin breakdown and dextran synthesis as well as to obtain information on the extent of dextrin utilization.

A mixture containing Acetobacter enzyme, amyloheptaose (molecular weight 1152) in 1.00 rniM final concentration, and 0.05 M acetate buffer, pH 4.6, was kept at 10” and analyzed at various intervals for both dextrin and dextran contents. For the analyses, a 1.0 ml. sample of the mixture

5 It is noteworthy that the presence of relatively large amounts of amylose, amy- lopectin, or glycogen was found neither to accelerate nor to retard the enzymatic conversion of dextrin to dextran. Thus, the failure of amylose, amylopectin, and glycogen to serve as substrates would not appear to be due to enzyme inactivation by these macromolecules or to be caused merely by the lack of a suitable “primer” or “acceptor” function.

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E. J. HEHRE 171

was heated at 70” for 10 minutes to destroy the enzyme activity, then treated with 2.0 ml. of 95 per cent ethanol and stored overnight at 10”. The alcoholic mixture was centrifuged, and the supernatant separated from the sediment by careful decantation and drainage into a clean test- tube. Both fractions were brought to near dryness over a gently boiling water bath, then treated with 2.0 ml. of 1 N HCl and hydrolyzed. The tube containing the supernatant (dextrin) fraction was fitted with an air condenser and immersed in boiling water for 23 hours; the tube with the

p 2.0

1s z 1.0

0.01

-, ‘W / / 1 /’ /]3.0 fE .-

HEPTA- - SACCHARIDE -

LOWER AMYLO- SACCHARIDES

\ I\ ,\ ,\ ,\ 1\ I\

0 2 4 6 8 IO I2 14 DAYS

FIG. 2. Action of Acetobacter enzyme on 1.0 mM amyloheptaose

sediment (dextran) fraction was sealed and kept in boiling water for 6 hours. The hydrolysates were then neutralized and analyzed for glucose.

It is evident (Fig. 2) that at each period of analysis the amount of dex- tran that appeared in the mixture corresponded to the amount of dextrin that had been utilized. Moreover, in the course of the reaction, somewhat more than half of the amyloheptaose substrate was converted to macro- molecules of dextran. 6 That is, a concentration of 7.06 mM of glucose, originally derived from 1.00 InM of the heptasaccharide, finally was ac- coumed for by 3.67 mM of glucose from dextran and 3.39 mM of glucose from residual amylosaccharides. This result can be taken as evidence

6 Although not shown in Fig. 2, the incubated enzyme-amyloheptaose mixture was very opalescent and possessed a high degree of serological activity versus type 2 pneumococcus antiserum.

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172 SYNTHESIS OF DEXTRAN FROM DEXTRINS

that the Acetobacter enzyme is able to cause dextran synthesis from dex- trin molecules as small as the tetrasaccharide. Whether the tetrasac- charide or the trisaccharide represents the lowest active homologue, however, cannot be decided from the experiment, since it is quite possible that true equilibrium may not have been reached. Maltose and glucose do not support dextran synthesis (Table II).

DISCUSSION

For the unique enzyme system of A. capsulatum that is able to convert chains of 1,4-linked glucose units into new chains of 1,6-linked units, the name dextran-dextrinase would seem most appropriate. The mode of action of this enzyme system, though incompletely understood, appears to be fundamentally like that of the other polysaccharide synthesizing enzymes (26). That is, dextran-dextrinase would seem to catalyze, as a

7-J. ~o~~o>&3L . . . . . . . ,JchOR--+ 72 DEXTRIN x RESIDUE

+ CIYbVtibb . . DEXTRAN

?-lx

FIG. 3. Concept of the over-all action of Acetobacter dextran-dextrinase

basic reaction step, the transfer of an a-n-glucopyranosyl radical from a terminal position in an appropriate dextrin molecule to a terminal position in a growing dextran molecule, as illustrated in Fig. 1. Repetition of this basic step could then lead to the simultaneous degradation of a dextrin molecule and growth of a dextran molecule; i.e.

Dextrin, + dextran, ---f dextrin, _ I+ dextran, + 1 -+ dextrin, - 2 + dextran, + 2, etc.

where the subscripts represent degrees of polymerization. For such a re- petitive process to occur, the catalytic agent must have aflinity for a range of dextrins as glucosyl group donors as well as for a range of dextrans as glucosyl group acceptors, and we believe the Acetobacter enzyme has such affinity.

The range of suitable dextrin substrates extends from materials that give some color with iodine to 3 or 4 unit amylosaccharides, and thus evi- dently is restricted to relatively small molecules, many of which would be required for the formation of each individual macromolecule of dextran. Whether the enzyme transfers successively all possible glucosyl units from a single dextrin molecule before transferring units from another, or trans- fers a glucosyl unit first from 1 dextrin moledule, t,hen from another, etc.,

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E. J. HEHRE 173

is not known. In either case, the over-all action of the Acetobacter en- zyme may be provisionally expressed as in Fig. 3; that is, n molecules of a particular dextrin with z transferable glucose units (or n molecules of various sized dextrins with an average of x transferable units) would be converted into a dextran polymer of nx units, plus n molecules of a residual saccharide, which, in the case of conversion of linear dextrins, may pos- sibly be maltose or maltotriose.

Further study is needed to determine whether initiation of the reaction requires a trace of preformed dextran (analogous t’o phosphorylase-cata- lyzed reactions which need an amylaceous “primer”) or whether certain dextrins can subserve this function.2

SUMMARY

1. Chemical analyses of the gummy product formed by cultures of Aceto- batter capsulatum growing with dextrin showed it to be a high molecular weight polymer of n-glucopyranose units linked principally in a-l,6 posi- tions. The original serological identification of the product as a dextran similar to the sucrose-derived Leuconostoc dextrans is thereby confirmed.

2. The synthesis of serologically active dextran from dextrins was, more- over, achieved in vitro by the action of a soluble enzyme system, dextran- dextrinase, obtained from A. capsulatum cultures. Products of partial hy- drolysis of amylose, amylopectin, and glycogen by acid or salivary amylase proved to be suitable substrates, but dextran formation could not be de- tected from unhydrolyzed amylose, amylopectin, or glycogen, or from prod- ucts of their partial hydrolysis by P-amylase. Cycloheptaamylose likewise was inert, though bot.h amyloheptaose and sodium amyloheptaoate de- rived from it were active as substrates. Commercial maltose yielded small amounts of dextran on incubation with the enzyme, presumably because of the presence of traces of accompanying dextrins, whereas maltose, pre- pared in the laboratory from P-amylase digests of amylopectin, gave a clearly negative result.

3. Quantitative study of the course of the action of the enzyme on the amyloheptaose of French et al., which was the most active and well defined substrate available, showed that an exact correspondence exists between “dextrin utilized” and ‘(dextran formed,” and also revealed that the en- zyme is able to cause dextran synthesis from molecules at least as small as amylotetraose.

4. The general mode of action of the Acetobacter enzyme system, whereby chains of 1,4-linked glucose units are converted to new chains of 1,6- linked units, would appear to be fundamentally like that of other poly- saccharide-synthesizing enzymes. The mechanism may be pictured as the repetition of a single basic step; namely, the transfer of a glucosyl group

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174 SYNTHESIS OF DEXTRAN FROM DEXTRINS

from a terminal position in a dextrin molecule to a terminal position in a growing dextran molecule. The reaction represents the first known in- stance of the conversion of one polymeric material into another, in which essentially every linkage is affected.

BIBLIOGRAPHY

1. Hehre, E. J., Science, 93, 237 (1941). Hehre, E. J., and Sugg, J. Y., J. Exp. Med., 76, 339 (1942).

2. Hehre, E. J., PTOC. Sot. Exp. Biol. and Med., 64, 240 (1943). 3. Hehre, E. J., J. Biol. Chem., 163, 221 (1946). 4. Hehre, E. J., and Hamilton, D. M., J. Biol. Chem., 166, 777 (1946). 5. Hehre, E. J., J. Biol. Chem., 177, 267 (1949). 6. Schoch, T. J., in Gortner, R. A., Gortner, R. A., Jr., and Gortner, W. A., Out-

lines of biochemistry, 3rd edition, New York, 627 (1949). 7. Evans, T. H., and Hibbert, H., Advances in Carbohydrate Chem., 2, 293 (1946). 8. Shimwell, J. L., J. Inst. Brewing, 63, 280 (1947). 9. Hehre, E. J., and Hamilton, D. M., Proc. Sot. Exp. Biol. and Med., 71,336 (1949).

10. Sugg, J. Y., and Hehre, E. J., J. Immunol., 43, 119 (1942). 11. Koch, F. C., and McMeekin, T. L., J. Am. Chem. Sot., 46, 2066 (1924). 12. Hagedorn, H. C., and Jensen, B. N., Biochem. Z., 135, 46 (1923). 13. Carlson, A. S., and Hehre, E. J., .I. Biol. Chem., 177, 281 (1949). 14. Weise, W., and Brand, T., Biochem. Z., 264, 357 (1933). 15. Sahyun, M., and Alsberg, C. L., J. Biol. Chem., 93, 235 (1931). 16. Jeanes, A., and Wilham, C. A., J. Am. Chem. Sot., 72, 2655 (1950). 17. Carlqvist, B., Acta them. &and., 2, 759 (1948). 18. Kent, P. W., Science, 110, 689 (1949). 19. Stacey, M., and Swift, G., .I. Chem. Sot., 1555 (1948). 20. Schoch, T. J., Advances in Carbohydrate Chem., 1, 247 (1945). 21. Ballou, G. A., and Luck, J. M., .I. Biol. Chem., 139, 233 (1941). 22. French, D., Levine, M. L., and Pazur, J. H., J. Am. Chem. SOL, 71, 356 (1949). 23. Kline, G. M., and Acree, S. F., Ind. and Eng. Chem., Anal. Ed., 2, 413 (1930). 24. Neill, J. M., Hehre, E. J., Sugg, J. Y., and Jaffe, E., J. Exp. Med., 70,427 (1939). 25. Haworth, W. N., Hirst, E. L., Kitchen, H., and Peat, S., .I. Chem. SOL, 791

(1937). 26. Hehre, E. J., Advances in Enzymol., 11, 297 (1951).

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Doris M. HamiltonEdward J. Hehre and With the assistance of

DEXTRAN FROM DEXTRINSTHE BIOLOGICAL SYNTHESIS OF

1951, 192:161-174.J. Biol. Chem. 

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