CARBOHYDRATE METABOLISM OF COLI GROUP BACTERI

IV. OLIGOSACCHARIDE SYNTHESIS FROM SUCROSE BY

ESCH. COLI VAR. COMMUNIOR

NOBUICHI MOROOKA

The National Institute of Health Tokyo, Japan

(Received: May 3rd, 1953)

With reference to the synthesis of various kinds of polysaccharides in a

living cell, their synthetic mechanism has partly been made clear.

In 1946, it was reported that various kinds of polysaccharides were produced

by such enzymes as phosphorylase (1), dextransucrase (2), levansucrase (2),

amylosucrase (3, 4, 5), amylomaltase (1) and Q-enzyme (6) acting on such

substrates as glucose-phosphate, sucrose, rafinose and maltose.

On the other hand, finding the starch like polysaccharides produced in

process of the fermentation of maltose by some strains of E. coli, Monod, Torriani reported in 1948 (7), Doudoroff, Hassid, Putmann, Potter and

Lederberg reported in 1949, in order to prove the mechanism of the direct

fermentation, that the synthesis of the polysaccharides by Esch. coli (mutabile)

was due to amylomaltase (1).

In 1952, Aronson reported on the transgalactosidation at the time of the

hydrolysis of lactose by yeast enzyme (8). Barker and Bourne , also, reported on the resynthesis of such oligosaccharides as maltotriose and maltotetraose

when maltose was metabolized by Esch. coli (Monod coli mutabbile) (9) . Pazur, reported that 4-isomaltosyl-D-glucose (panose), 6-isomaltosyl-D-glucose (dex-

trantriose) and 4-dextrantoriosyl-D-glucose of tetrasaccharides were detected

at the time of the metabolism of maltose by As pergillus oryzae (10). More

recently he gave another report on the resynthesis of such a new type of

oligosaccharides as I-inulobiosyl-D-glucose and I-inulotriosyl-D-glucose at the

time of the fermentation of sucrose by Asp. oryzae enzyme (11) .As described in the previous report, a new type of oligosaccharide was

found at the time of the metabolism of sucrose by Esch. toll var. communior . Following description is the detailed report thereof.

This substance was produced when sucrose was metabolized by the intact

cell of Esch. toll var. communior. Akiya-Takahashi's method (12) was used

for extraction and fractionation of the substance. The substance thus produced

showed negative ninhydrin and Biuret reactions, positive Molisch reaction and

negative ammoniac AgNO3 and iodine reactions. It was found to be a kind

of oligosaccharide with keton radical by the paper-chromatography of new type

oligosaccharide and it was supposed to be a combination of maltose and fructose

on the basis of the findings of the paper-chromatography of acid hydrolysis

1

2 MOROOKA

of this substance and the extinction curve by Beckman's spectro-photometer

(D. U.) according to carbazol method.Moreover, the acetate of this substance was prepared in order to refine

the substance and determine its molecular weight. The melting-point of this

substance (acetart) was at 41•`42•Ž. The molecular weight determined by

Akiya-Barger's method was ca. 1000 (combination of about 3 molecules of

monosaccharide). Judging from the above-mentioned results, it was deter-

mined that the new type of oligosaccharide was a combination of maltose-like

substance and fructose, (G-G-F).

METHOD AND RESULTS

I. Synthesis of oligosaccharide

1. Enzyme solution (N=0.2g/100cc)

For the preparation of enzyme solution, Esch. coli var. communior (received

from the Inst. Infect. Diseases of T•¬ky•¬ Univ.) cultured at 37•Ž for 20•`24

hrs on a plate of neutral agar added with 1 % maltose. These bacterial cells

were suspended in sterile physiological saline solution and centrifuged at 4000

r.p.m. for 15 minutes. The supernatant fluid was decanted. Repeating the

washing three times, the saccharide was removed from the medium.

The enzyme (intact cell) was prepared by suspending the washed cell in

sterile physiological saline solution with the addition of some drops of toluene

and subsequent efficient mixing.

2. Buffer Solution

Buffer solution (pH 4.6) was prepared by mixing 10.2 cc of M/5 CH3COOH

solution with 9.3cc of M/5 CH3COONa solution.

3. Substrate solution

Sucrose solution (12%) was used as substrate solution.

4. Reaction

Mixing enzyme solution (N=0.2 g/100 cc), buffer solution and substrate

solution in a proportion of 2:1:1, 100 cc of reaction mixture was prepared.

After adding toluene, the mixture was kept at 37•Ž for 20•`24 hrs. After

reaction, the cells were removed by Zeiz-filter. Moreover, pH was neutralized

at 7.0 with sodium acetate.

II. The separation and character of oligosaccharide

1. Refinement

Immediately the reaction mixture was electro-dialysed in Pauly apparatus

(bovine and cellophane membranes) in order to remove inorganic substance,

and was dialysis in running water using a cellophane membrane (Nippon

cellophane. Co. LTD, No. 600). (see paper-chromatogram photo. 2). In the

electro-dialysis the temperature was not allowed to rise, and pH value was

kept at 7.0. Water was often changed in the solutions of the positive and the

negative poles at the beginning of the dialysis. Thus, the electro-dialysis was

3CARBOHYDRATE METABOLISM

completed in 72 hrs. (see paper-chromatogram photo . 3) . After dialysis, the

solution was divided into three parts and acetone was added in a proportion

of 30, 45 and 70 %. After each solution was kept in a cooling from overnight,

the appearance of some deposite was examined . On examination it was found

that little deposite apeareed in each solution . This substance gave positive nin-

hydrin, Molisch and modified Seliwanoff reaction (13) . After filtration deposits,

evapolated acetone and water. Thus about 1 g of oligosaccharide was gained.

This oligosaccharide formed irregular plate crystal in alcohol and was very

absorptive. It showed no color on thee paper-chromatogram the stain reagent

of which was ammoniac AgNO3 solution, it gave negative iode reaction and

positive Seliwanoff reaction (see paper-chromatogram photo. 3) .

2. Determination of rotation

Free fructose in an amount of precisely 20 mg was dissolved in 2 cc of

distilled water and the rotation of this solution was determined as the control

for the determination of the rotation of the sample and acid hydrolyzate of

the sample. The rotation of the sample was determined to be ([ƒ¿]14D=+95 .9•‹).

The sample in an amount of 20 mg was mixed with 1 cc of 1 % hydrochloric

acid solution and resolved in a boiling water bath for 1 hr . After resolution,

it was neutralized by sodium hydroxide . The rotation determined by using

2 cc of the solution was [ƒ¿]14D=-4•‹.

3. Determination of reducing power

As reported in the previous paper, substrate solution had no reducing

power, but when it was decomposed by heating (100•‹ 1 hr) in 1 % hydrochloric

acid, its reducing power was found to be 43% of fructose .

4. Acetylation of the oligosacchride

The sample in an amount of 150 mg was added with 3 cc of formamide,

3 cc of pyridine and 3 cc of acetic unhydride and kept under anhydrous condition

at the room temperaturee for 48 hrs . After reaction, the solution was slowly

dropped into a small quantity of ice water . Shaking occasionally to avoid

that the reaction might be limited to one spot . White precipitate formed was

collected by filtration. It was washed with water and dried at 37•‹ in a

vaccum over phosphate oxide . And this substance was recrystalized four times

with absolute alcohol and again dried at 37•‹ in a vaccum over P2O5. Then

its melting point was determined and found to be 41•`42•Ž.

5. Determination of the molecular weight o f acetylated oligosaccharide

The molecular weight of the acetylated oligosaccharide was determined by

Akiya-Barger's method. A solution of 0 .1 M of azobenzene (3.64 mg/2 cc actone)

was prepared, and this was further diluted to obtain solutions with different

concentrations of 0.05, 0.025, 0.016 and 0.001 M. On the other hand acetylated

oligosaccharide 1.6 mg/0 .12 cc acetone was prepared. A capillary with 1 mm

outside diameter and 20•`25 cm in length was made . Both ends of the capillary

were cut perpendicularly with an ampoule cutter . The standard solution (azo-

4 MOROOKA

benzene) and the sample solution were sucked up alternately into it so that

alternate empty spaces might be formed. When the sample solution was

sucked up 5 or 7 times, both ends of the glass capillary were closed over a

little flame. Then, using a microscope 100 magnifications, the changes produced

in the spaces between the standard solution and the sample solution in the

capillary was determined following the elapse of time at a constant temperature.

Judging from the above results of the determination (Table I), the mo-

lecular weight of the acetate of oligosaccharide was supposed to be about 1,000.

The percentage of the acetate of substance was 51.17 as shown in the following

table 2.

Table 1. Determination of the molecular weight of acetylated

oligosaccharide by Akiya-Barger's method

Table 2

6. Extinction curve by car bazol method

Oligosaccharide was removed from approximately 10 mg of acetate substance

with unhydrous liquid ammonia, and 1 hour after the ammonium was evapolated.

Then it was washed with ether three times and the ether was removed by

heating at 30•`40•Ž under low pressure. The substance was dried with P2O5

and recrystalized after washing with absolute alcohol. The acid hydrolysis of

this substance was done by adding hydrochloric acid solution (1% 1 cc) and

boiling at 1 hour. After acid hydrolysis, the substance was removed of its

water by evapolation at 35•`40•Ž under low pressure. This process was re-

peated four times. A small amount of distilled water was added to the dried

sample and any remaining hydrochloric acid is neutralized with barium car-

bonate. Barium carbonate was precipitated as BaCI when acid was dissolved.

At the same time superfluous barium carbonate and ammonium chloride were

removed. The filtrate was kept overnight in a cool room and a small quantity

of its precipitate was removed by means of filtration. After keeping in an

ice-box for additional 48 hrs, its precipitate formed was removed again by

5CARBOHYDRATE METABOLISM

means of filtration. Then this filtrate was concentrated at 35•`40•Ž under

low pressure, and dissolved in water.

Thus some precipitate was produced again with redistilled ethyl alcohol.

This substance formed irregular plate crystal in alcohol and was very absorptive.

This substance was dried with P2O5 by heating at 37•Ž and kept in a vaccum

desiccater for five days. Thus an acid hydrolytic substance of about 5 mg

was gained.

Above substance in an amount of 5 mg was dissolved in 10 cc of distilled

water for the preparation of a 0.1% substrate solution. A part of this solution

was used for the following paper-chromatogram.

The other part was further diluted to 0.01%, and the extinction curve

was determined as follows:

According to F. B. Seibert's method (14), 8 cc of conc. H2SO4 was added

to 1 cc of distilled water in a test tube (diameter; 22 mm, length 200 mm) kept

in ice water. Sample in an amount of 0.5 cc was carefully layered on the sur-

face of the acid, without wetting the wall of the tube. As controll tube, 0.5 cc

of distilled water was added. On the other hand, 7 tubes containing glucose

and fructose of different ratio were prepared for comparison. Next, 0.5

alcohol solution of carbazol in an amount of 0.3 cc was poured into each of the

above tubes, which were shaken and chilled in an ice wated bath. Then, boiled

in the water bath at 100•Ž for 10 minutes and again chilled in an ice water

bath. On completion of the above mentioned treatments, the extinction curve

of each solution was determined with Beckman's spectrophotometer (wave

length.; 400mƒÊ•`600mƒÊ). The results thus obtained are shown in Fig 1.

Table 3. Absorption ratio of D 540/D 440

The curve of the sample was much the same as the absorption curve drown

by maltose mixed with fructose in a proportion of 2/3 to 1/3. The extinction

curve of the sample shown by the ratio of D 540/ D 440 was as follow (see

Table 3):

6 MOROOKA

Fig. I. absorption curves of carbazole reaction with

various mixtures of glucose, fructose, mal-tose and sucrose

(I) (a) pure fructose, (i) (b) pure glucose(II) glucose 1/3: fructose 2/3(III) glucose 1/2: fructose 1/2(IV) glucose 2/3: fructose 1/3(V) sucrose 1/2: fructose 1/2(VI) maltose 2/8: fructose 1/3(VII) sample

7CARBOHYDRATE METABOLISM

7. Paper-chlomatographic investigation o f oligosaccharide

Acetyl was separated from the acetylized substance (see 6) and other hand

we used no acetylized sample. These 20 mg were decomposed by 1 % hydro-

chloric acid at boiling water for 20 minutes.

Each 0.2 cc of approximately 1 % solution of the substance was developed

with the solvent of butanol, acetic acid, and water in the proportion of 4:1:2

mixture through the Toyo filter-paper No. 50 at 20•Ž for 15 hrs, and colour

was developed by the use of hydrochloric acid resorcin and ammoniac AgNO3

reagent. The results showed that the sample was positive by hydrochloric acid

resorcin and was negative by ammoniac AgNO3 reagent (cf. photograph 3. i)

By the test result of the paper-chromatography it found that the substance

decomposed by the hydrochloric acid gave the spots in the place near fructose

which also appeared in the substance in the occasion to stain its colour by

ammoniac AgNO3 reagent.

The substance of the sample decomposed by acid was developed with the

solution of phenol added with 15% distilled water, and was presented for stain-

ing its colour with benzidine-trichloroacetic acid. In consequence, a substance

with Rf value of 0.51 which had the same colour of light yellow of fructose

was observed as shown in photgraph 4, 5. Another substance, in the place

near the spot of maltose, which had a little lighter brown colour than the one

of glucose and showed no colour by hydrochloric acid resorcin, was caught.

This was the substance remained after separation of a substance to contain

ketone from the sample in the way of the acid decomposition of the sample.

In use of benzidine-trichloroacetic acid, the substances, which were unable to

generate the colour or had low sensibility for colour generation with hydro-

chloric acid resorcin and ammoniac AgNO3solution, could be found.

The benzidine-trichloroacetic acid used for the generation of the colour

was composed in the proportion that 0.5 g benzidine was solved into 10 cc of

40% (w/v) trichloroacetic acid mixed with 80 cc of ethanol. (15)

Judging from the absorption curves, the ratios of D540/D440 and the

results of paper-chromatography, the sample was found to be the combination

of glucose and fructose (2/3:1/3). The molecular weight of this substance

was found to be three times as much the weight of the molecular of monosac-

charides when determined by Barger's method. Moreover, it was supposed that

the combination unit of the sample was G-G-F and that G-G was combined

like maltose. (see photo 4. d, 5. b,)

CONSIDERATION

In view of the findings obtained from i) dialysis ii) determination of

molecular weight iii) paper-chromatogram, and iv) extinction curve by car-

bazol method, it is supposed that the intermediate metabolite of sucrose by

Esch. coli var. communior is oligosaccharide of G-G-F combination.

8 CARBOHYDRATE METABOLISM

The production mechanism of this oligosaccharide is G-o-F (sucrose) +

enzymeZ•c•c•¨G. E(E=enzymesystem),•c•c•c•cF (free fructose), when sucrose

is given to Esch. coli var. communior as substrate. It is supposed that G. E

is combined with the active part of G-o-F and G. E as follows:

G-o-F+Enzyme system•¬•¨+

G.E•c•c•c•c•c•¨F (free fructose)+

G-o-F•¨G-G-F•c(free enzyme)

Oligosaccharide is produced the enzyme system (E) being freed in the

above mentioned manner.

It is supposed that the enzyme producing this oligosaccharide is a specific

transferase of its bacteria and, moreover, it is supposed that i) the reversible

action of a-glucosidase system. ii) phospholyrase system and the above men-

tioned transferase take part in sucrose producing G-G-F. Esch. coli var. com-

munior does ferment sucrose, however, it has neither a-glucosidase nor fruc-

tosidase. If Aerobacter transfers oligosaccharide in process of the fermentation

of sucrose, this transferring may be made possible by the reversible action of

a-glucosidase. But since Esch. coli var. communior has neither a-glucosidase

nor fructosidase this transferring can not be made possible by this enzyme.

For this reason it is supposed that a specific transf erase takes part in Esch. coli

var. communior systhesizing oligosaccharide (G-G-F) at the time of the meta-

bolism of sucrose.

CONCLUSION

1) The molecular weight of oligosaccharide synthesized at the time of

the fermentation of sucrose by Esch. coli var. communior was about M.W.

1000 when determined by the acetate of this oligosaccharide using Akiya-

Barger's method.

2) The acetate of this acetyl oligosaccharide is about 50%, and the

molecular weight of this oligosaccharide is about 500. It is also supposed

that the combination order of this oligosaccharide is G-G-F (combination with

three molecules of monosaccharide).

3) It is supposed that the enzyme taking part in the synthesis of this

oligosaccharide in the new transf erase system of Esch. coli var. communior.

ACKNOWLEDGEMENTS

The author wishes to acknowledge his indebtedness to Miss Naoko Ono for her

faithful assistance in experiments. Also to Dr. Y•¬z•¬ T•¬yama, Dr. K. Sahashi and

Dr. T. Miwa who have directed this work, and Dr. H. Takahashi for his thoughtful-

ness to give any convenience to the author in the chemical part of the experiments.

9MOROOKA

REFERENCES

(1) M. Doudoroff, H. A. Barker and W. Z. Hassid, E. W. Putman, A. L. Potter

and J. Lederberg, Direct utilization of maltose by Esch. coli. J. Biol. Chem.,

179, 921. 1949.

(2) Hehre, E. J., and Neil, J. M., Formation of serologically reactive dextrans

by Streptococci from subacute bacterial endocarditis. J. Expel. Med., 83,

147. 1946.

(3) Hehre, E. J., Synthesis of polysaccharide of the starch-glycogen class from

sucrose by a cell-free bacterial enzyme system (amylosucrase). J. Biol.

Chem., 177; 267-279. 1949.

(4) Hehre, E. J., Studies on the enzymatic synthesis on dextran from sucrose. J.

Biol. Chem., 163; 221-233. 1946.

(5) Hehre, E. J., and Hamilton, D. M., The conversion of sucrose to a polysaccharide

of the starch-glycogen class by Neisseria from the pharynx. J. Bact., 55,

197. 1948.

(6) Hehre, E. J., Enzymic synthesis of polysaccharides. Adv. Enzymol., 11: 297-

337. 1951.

(7) Monod, J., and. A. M. Torriani, Synthese d'un polysaccharide du type amidon

aux dð¥pens, du maltose en presence d'un extrait enzymatic d'origine bac-

terienne. C. r. Acad. Sci. Paris, 227: 240-242. 1948.

(8) M. Aronson, Transgalactosidation during lactose hydrolysis. Arch. Biochem.

and Bioph., 37, 370. 1952.

(9) S. A. Barker and E. J. Bourne, The oligosaccharides synthesised from maltase

by Esch. coli. J. Chem. Soc., 209-215. 1952.

(10) J. H. Pazur and D. French. The action of transglucosidase of Asp. oryzae on

maltose. J. Biol. Chem., 196. 265. 1952.

(11) J. H. Pazur, Transfructosidation reactions of an enzyme of Asp. oryzae. J.

Biol. Chem., 199. 217. 1952.

(12) S. Akiya, H. Takahashi, M. Kuriyama and N. Ogawa. Bacterial component of

Haemophilus pertussis VII. Studies on polisaccharides (1) Japan Med. Jour.,

Vol. 4, No. 6. 325-329. 1951.

(13) W. G. C. Forsyth, A method for studing the carbohydrate metabolism of micro-

organisms. Nature 162. 150. 1948.

(14) F. B. Seibert, Jane Atno, Determination of polysaccharide in serum. J. Biol.

Chem., 163. 511. 1946.

(15) J. S. D. Bacon and J. E delman. The carbohydrates of the Jerusalem artichoke

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11MOROOKA

Photograph 1. Paper-chromatogram of new type of oligosaccharide

a. fructose b. glucosec. maltose d. sucrosee. f. new type of oligosaccharide

g. sucrose h. before dialysis

Photograph 2. Phenomenon of oligosaccharide after dialysis by cellophane membrane

a. after 48 hrs (in cellophane membrane)

b. after 24 hrs (out of the cellophane membrane)

c.•¬ (in cellophane membrane)

d. after 48 hrs (out of the cellophane membrane)

e. maltose f. sucrose g. glucose h. fructose

12 CARBOHYDRATE METABOLISM

Photograph 3. Paper-chromatogram of the oligosaccharide

(G-G-F) stained by hydrochloric acid resorcin

Sample: oligosaccharide (G-G-F)

R: raffinose F: fructoseS: sucrose

Sample: after electro-dialysis

HCI: after hydrolysis substance

R: raffinose F: fructose

S: sucrose

13MOROOKA

Photograph 4. Paper-chromatogram of

hydrolysis substance

a. maltose

b. mixture of fructose, glucose and maltose

c. after hydrolysis substance

d. after hydrolysis substance of refined oligo-

saccharide by acetylation

Photograph 5. Paper-chromatogram of

hydrolysis substance

a. fructose and glucose mixtureb. after acid hydrolysis of

oligosaccharide