SYNTHESIS OF A POLYSACCHARIDE OF THE STARCH- GLYCOGEN CLASS FROM SUCROSE BY A CELL-

FREE, BACTERIAL ENZYME SYSTEM (AMYLOSUCRASE)”

BY EDWARD J. HEHRE

WITH THE ASSISTANCE OF DORIS M. HAMILTON AND ARTHUR S. CARLSON

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

(Received for publication, July 28, 1948)

The present paper deals with the conversion of sucrose to an amylopec- tin- or glycogen-like polysaccharide by means of a cell-free bacterial enzyme system obtained from Neisseria perjava, a Gram-negative coccus that occurs in the throats of healthy people. This reaction is of interest, not only because of its possible bearing on the well known interrelationship between sucrose and starch in the plant world, but also because of its apparent freedom from mediation by glucose-l-phosphate, which is com- monly believed to be the essential substrate for the synthesis of all members of the starch-glycogen class.

We have already reported (1,2) that cultures and resting cell suspensions of Neisseria per$ava convert sucrose into a polysaccharide with the chemical properties of amylopectin; large amounts are formed from sucrose, smaller amounts from glucose-l-phosphate, but none at all from other common sugars. The enzyme system responsible for the conversion of sucrose to polysaccharide is, in addition, obtainable in cell-free form (2). The pres- ent paper gives a method for obtaining the enzyme solutions from the bacterial cultures, and deals with the action of these enzyme solutions on sucrose and on glucose-l-phosphate and with the chemical properties of the products of their action on sucrose.

EXPERIMENTAL

Preparation of Enzyme Solutions

Strain 19-34 of Neisseria pe$ava, known to produce large amounts of amylopectin-like material from sucrose, was cultivated in broth comprising 0.1 per cent Bacto-peptone, 0.15 per cent sodium citrate, 0.02 per cent yeast extract, 0.06 per cent KH2P0+ 0.15 per cent Na2HP04, and 0.05 per cent glucose. Flasks containing 800 ml. of the medium were inoculated with 10 ml. of a 1 day culture grown in the same medium, and incubated

* Aided by a grant from the Sugar Research Foundation, Inc.

267

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268 POLYSACCHARIDE SYNTHESIS BY AMYLOSUCRASE

at 37” for 5 days. In order to precipitate the enzymes, each flask of culture was treated with 300 gm. of ammonium sulfate and the mixture centrifuged in the cold for 1 hour at 1500 R.P.M. The sediments, which contained the bacterial cells as well as the enzymes, were separated from the super- natant fluids, drained, and further freed from phosphates and other soluble constituents of the culture medium by washing in half saturated ammonium sulfate solution that had been adjustled to pH 6.4 with ammonia. The washed precipitates were extracted with 0.025 nf maleate buffer (pH 6.4) by using lfj ml. of buffer for each 800 ml. of original culture, and the ex- tracts were clarified in a 14 inch angle-head centrifuge at 2500 R.P.M. for 2 hours. The final solutions were entirely free of bacterial cells and cell frag- ments; and, from the many control tests made on the incubated enzyme- substrate mixtures, we are certain that all the reactions observed in the present paper occurred in the complete absence of bacteria.

Action of Neisseria Enzymes on Sucrose and on Glucose-l-phosphate

Material and Methods-Mixtures of enzyme plus sucrose or glucose-l- phosphate buffered at pH 6.4 were used throughout. The sucrose was a selected sample of beet sugar essentially free of the traces of dextran, amylopectin, and other alcohol-precipitable material present in many lots of reagent and commercial sucrose (3, 4). The glucose-l-phosphate was the dipotassium salt prepared from an enzymatic digest of potato starch by the method of McCready and Hassid (5); though recrystallized, it contained a trace of accompanying starch.

The enzyme-substrate mixtures were examined for opalescence, turbidity on addition of 2.0 volumes of alcohol, kind and intensity of color with iodine, and contents of free reducing sugar, polysaccharide, inorganic phosphorus, and total acid-soluble phosphorus. For determining the intensity of color with iodine, 0.1. ml. of 1 per cent iodine and 2 per cent potassium iodide was added to 5.0 ml. of a 1: 10 dilution of the enzyme- substrate mixture and measurements were made with a Klett-Summerson photoelectric calorimeter with green Filter 54 (spectral range 500 to 570 mp); the zero point was the iodine-iodide reagent blank. Free reducing sugar contents of the mixtures were determined by the Hagedorn and Jen- sen (6) method and are expressed as mu of fructose’ per liter. For the polysaccharide determinations, 2.0 ml. of the enzyme-substrate mixture were placed in a large test-tube and treated with 8.0 ml. of 0.6 M acetate buffer (pH 5.0) and 20.0 ml. of alcohol. (Adjustment to pH 5.0 was essen- tial in order to prevent precipitation of glucose-l-phosphate from systems containing that sugar.) After storage overnight at 4”, the mixture was centrifuged; 2.0 ml. of 1.0 N KC1 were added to the precipitate, and the

1 Evidence that the free reducing sugar is’fructose and that the alcohol-precipitable material is a polyglucoside will be presented later in the paper.

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E. J. HEHRE 269

tube fitted with an air condenser and immersed in boiling water for 2.5 hours. Reducing sugars were determined on the neutralized hydrolysate by the Hagedorn and Jensen (6) method and calculated as mM of glucose’ per liter. The contents of inorganic phosphorus and of total acid-soluble phosphorus after mineralization with sulfuric and nitric acids were de- termined by the Fiske and Subbarow (7) procedure. All figures given for the production of polysaccharide, free reducing sugar, or inorganic phos- phorus represent the difference between the contents determined before and after incubation of the enzyme-substrate mixtures.

Over-All Action upon Sucrose-A representative experiment showing the action of the Neisseria enzymes upon sucrose is shown in Table I. Equal

TABLE I

Changes Produced by Action of Neisseria Enzymes upon Sucrose

Time of incuba- tion at 10’

hrs.

0 3 6

12 24 48 96

Opalescence

f +

+X!Z ++* +++

+++zt ++++

f +

+* ++zt +++

+++zt ++++

Intensity of color with

iodine

6 27

‘58 100 173 250 370

- Polysaccharide

produced*

?m.f m&l 0.01 o.ot 1.1 1.0 2.0 2.2 4.2 4.2 7.8 7.8

13.8 14.0 24.1 25.6

* Since the initial concentration of the sucrose substrate was 100 rn~, the values correspond to the per cent conversion of sucrose to polysaccharide and fructose.

t The unincubated mixture contained 0.3 mM of reducing sugar and 0.6 mM of polysaccharide, calculated as hexose; the nature of the reducing substance and polysaccharide is unknown.

volumes (250 ml.) of enzyme solution and of 0.2 M sucrose in 0.025 M

maleate buffer, pH 6.4, were mixed and maintained at 10”. At regular intervals, small samples were removed and examined for opalescence, precipitation with alcohol, color with iodine, and contents of polysac- charide and free reducing sugar.

Beginning with the first observations at 3 hours, there was an orderly increase in opalescence, in amount of alcohol-precipitable material, and in amounts of polysaccharide and of free reducing sugar. At each period there was excellent agreement between the quantity of polysaccharide and of free reducing sugar that had been produced, indicating that the over-all equation for the action of the Neisseria enzymes upon sucrose, as pre- viously suggested (1, 2), is:

nC12H2201t-(C6H1006)n + dXMh Sucrose Polyglucoside Fructose

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270 POLYSACCHARIDE SYNTHESIS BY AMYLOSUCRASE

The enzyme-sucrose mixture also showed an increasingly intense maroon coloration on treatment with iodine. However, it was noted that after the 1st day the increase in intensity of the color with iodine did not keep pace with the increases in polysaccharide and reducing sugar contents. This phencmenon, which probably is the result of alteration of the newly formed polysaccharide by some enzyme present in the Neisseria extracts, is being further investigated.2

InjEuence of High Concentrations of Phosphate-Although entirely non- reactive with glucose, glucose plus fructose, or other common sugars, the enzyme preparations, as previously found for the living bacteria, syn- thesized polysaccharide from glucose-l-phosphate. In the case of whole bacterial cells, the synthesis from glucose-l-phosphate was suppressed by addition of high concentrations of inorganic phosphate which did not inhibit the synthesis from sucrose (I, 2). A similar experiment was made with the cell-free enzyme system.

Four enzyme-substrate mixtures were prepared: 5.0 ml. of enzyme solu- tion were added to 5.0 ml. of (1) 40 mM sucrose in 320 mM maleate buffer, (2) 40 mM glucose-l-phosphate in 320 mM maleate buffer, (3) 40 mM sucrose in 320 InM phosphate buffer, and (4) 40 mM glucose-l-phosphate in 320 rntir phosphate buffer. The mixtures were held at 10” for 24 hours and then analyzed. The results are given in Table II.

In the systems buffered with maleate, polysaecharide was formed from glucose-l-phosphate as well as from sucrose, but in the case of the synthesis from sucrose the low content of total P and the absence of any change in the inorganic phosphate make it unlikely that glucose-l-phosphate served as an intermediate substance in that reaction. More conclusive evidence is presented by the data from the systems buffered with phosphate. In these systems, which contained 8 moles of phosphate to 1 of substrate, the synthesis from glucose-l-phosphate was entirely suppressed, whereas the synthesis from sucrose was not inhibited at all. That the inhibition ob- served in the glucose-l-phosphate system was not due to any loss of the substrate or to any inactivation of the enzyme was proved by supple-

2 It is unlikely that the alteration was due to hydrolysis by an LY- or p-amylase, since the reducing sugar liberated in the enzyme-sucrose mixture did not exceed the amount expected from a sucrose condensation reaction; nor did it contain any appreciable amount of aldose, e.g. maltose. It also is unlikely that phosphorolysis was involved, since the mixture did not contain any appreciable amount of free inorganic phosphate. The change in character of the polysaccharide product may have been due to some enzyme like the cross-linking enzymes in liver (8) and in potatoes (9) reported to bring about the synthesis of glycogen and of amylopectin, or, perhaps, to adsorption of some fatty acid. The former explanation seems more likely, since Neisseria extracts convert crystalline corn amylose into a glycogen-like polysaccharide without the release of reducing sugars, but fail to do so if heated.

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E. J. HEHRE 271

mentary tests: at the end of the experiment, when solid sucrose was added to one portion, polysaccharide synthesis occurred at a rate equal to that in t,he maleate-buffered system, and chemical analyses on another portion showed that glucose-l-phosphate was present in its original concentration.

InJEuence of Heat and Gas Treatment--The data from the following exper- iment show that the enzyme solutions prepared from Neisseria contain two systems: an amylosucrase, active preferentially if not solely upon sucrose, and a phosphorylase, active upon glucose-l-phosphate but not upon sucrose.

A sample of enzyme solution was divided into three parts. The first was untreated, the second was heated at 45” for 10 minutes (pH 6.4), while the third, after adjustment to pH 5.9 with 1.0 N HCl, was exposed in a thin film to pure COZ gas for 5 minutes at 15” and atmospheric pressure, and

TABLE II Action of Neisseria Enzymes on Sucrose and on Glucose-l-phosphate in Absence

and in Presence of High Concentrations of Inorganic Phosphate

llZM n&M ?nM

Sucrose. Maleate ++-I-+ 7.5 3.3 o.oo* Glucose-l-phosphate. “ + 3.1 0.0 3.5 Sucrose. . Phosphate +++ 7.7 11.5 Glucose-l-phosphate. “ 0 0.0 0.0 t

* This mixture contained 0.10 mM of inorganic phosphorus and 0.20 mM of total acid-soluble phosphorus both before and after incubation.

t The glucose-l-phosphate content of this mixture, measured both before and after incubation by the method of Hassid and McCready (5), showed no change.

then readjusted to pH 6.4 with 1.0 N NaOH. The three enzyme solutions were tested for activity in mixtures containing final concentrations of 20 mM of sucrose and of glucose-l-phosphate in maleate buffer.

It is apparent (Table III) that heat treatment sufficient to cause almost complete loss of the capacity of the original enzyme solution to convert sucrose to polysaccharide and fructose resulted in only slight impairment of the capacity to convert glucose-l-phosphate to polysaccharide and free phosphate, whereas gas treatments sufficient to cause over 90 per cent loss of activity for glucose-l-phosphate brought about loss of only about half of the activity toward sucrose.

3 The inactivation produced by CO2 in this experiment has been duplicated in other experiments with air, 02, or Hz gas. The pH is apparently an important factor, since the inactivation which occurs in enzyme solutions adjusted to pH 5.9 did not occur in solutions at pH 6.4.

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272 POLYSACCHARiDE SYPU’THESIS BY AMYLOSUCRASE

Analysis of Products Formed from Sucrose by Neisseria Enzymes-The starting material consisted of 440 ml. of the enzyme-sucrose mixture which had been incubated at 10” for 4 days in the experiment in Table I. The mixture was first partially deproteinized by shaking with chloroform and removing the chloroform emulsion layer. The fluid then was treated with 22 gm. of crystalline sodium acetate and 1.5 volumes of alcohol and was centrifuged after storage overnight at 4”. The precipitate was used for experiments on the nature of the polysaccharide, while the alcoholic superna- tant fluid was used for experiments on the nature of the free reducing sugar.

Identijkation of Free Reducing Sugar-The alcoholic supernatant fluid was evaporated in vacua at a temperature below 40”, and the dry residue dissolved in water. Analyses for reducing capacity (7) before and after oxidation with iodine (10) showed that all of the reducing sugar was ketose

TABLE III Effect of Heat and of Gas Treatment upon Activity of Enzyme Solutions for Sucrose

and for Glucose-l-phosphate

Treatment of enzyme solution

Untreated ..................... Heated at 45”, 10 min .......... Gas-treated ....................

I SUCIWZ I

Glucose-l-phosphate

Polysaccharide Fructose

WZM nzM

5.5 5.8 0.1 0.1 2.3 2.6

Polysaccharide Inorganic phosphorus

mnr ?mM

6.1 7.0 5.1 6.1 0.6 0.6

sugar; aldose was not detected. Proof that the ketose sugar was fructose was obtained by treatment of another portion of the solution with cy- methylphenylhydrazine sulfate, according to the method of Neuberg and Mandl (11) (the cu-methylphenylhydrazine sulfate was kindly supplied by Dr. Neuberg). Well formed, needle-shaped crystals of fructose methyl- phenylosazone, identical in appearance with those prepared from a sample of fructose, were isolated. The yield of osazone (113 mg. after recrystal- lization from ethyl acetate), obtained by treatment of a solution containing 92 mg. of reducing sugar, was approximately that expected from a similar amount of pure fructose (cf. (11)).

Isolation and Analysis of Polysaccharide-The alcohol-precipitated ma- terial from Dhe enzyme-substrate mixture was extracted with 5 per cent sodium acetate solution, insoluble material was removed by centrifuga- tion, and the solutions treated with 1.5 volumes of alcohol to precipitate the polysaccharide. Solution of the polysaccharide and precipitation with alcohol were repeated twice; the last precipitation was from a solution in distilled water at room temperature in order to insure a minimal ash

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E. J. HEHRE 273

content. The final precipitate was ground under absolute alcohol and dried in vacua at 25” over CaC&. The yield was 1.58 gm. (moisture-free basis), which represented 92 per cent of the polysaccharide present in the starting material.

The isolated product was a white amorphous powder, with an ash content (as NazS04) of 1.2 per cent, micro-Kjeldahl nitrogen (12) 0.45 per cent, phosphorus (7) 0.03 per cent, and a reducing power (13) of less than 0.2 per cent that of glucose. Calculated on an ash- and moisture-free basis, the optical rotation was [a] ,% = i-175” (c = 0.3 in 0.5 N NaOH); after boiling for 2.5 hours in 1.0 N HCl, with correction for the entry of water, the hydrolysate had [a] & = +49.3” (c = 0.5) and 91 per cent reducing sugar, as glucose. Abundant glucose phenylosazone (m.p. 205’, uncor- rected) was obtained from the hydrolysate; fructose, if present at all, amounted to less than 1 per cent of the sugar, according to determinations made by the Roe procedure (14); Bial’s test for pentoses was negative.

The presence of maltosidic linkages in the enzymatically synthesized polysaccharide was established by the isolation of maltose. A 382 mg. sample of ash- and moisture-free polysaccharide in 7 ml. of water was treated with 10 mg. of P-amylase prepared from wheat flour (15) and suf- ficient dilute HzS04 to bring the pH to 4.8; after 16 hours at 30”, the reduc- ing value indicated that 31 per cent conversion had occurred. The digest, when treated with methanol according to the procedure described by Haworth et al. (16), yielded 98 mg. of crystalline p-maltose monohydrate, bl 5& = + 111.9” -+ f126.9” (c = 1.5 in HzO). The optical measure- ments were made with a Schmidt and Haensch polarimeter kindly made available by Dr. du Vigneaud.

The formation of the maltose through the action of P-amylase on poly- saccharide material which had been synthesized by the action of Neisseria enzymes on sucrose represents the first demonstration of an enzymatic conversion of sucrose to maltose in the absence of cells. This conversion seems of fundamental interest, even though the enzymes that accomplished it were of different biological origins.

Comparison with Other Polyglucosides-The enzymatically synthesized polysaccharide was compared with a number of reference polyglucosides: a sample of amylopectin-like polysaccharide produced from sucrose by living cultures of the strain of Neisseria used in the preparation of the enzyme (1); commercial liver and oyster glycogens; purified amylopect,in C-107-B and recrystallized amylose C-107/111-A prepared from a speci- men of corn-starch by the pentasol method (kindly supplied by Dr. T. J. Schoch) ; and dextran prepared from the sucrose broth cultures of Leuco- nostoc mesenteroides, strain B (17). All of the polysaccharidex were sub- jected simultaneously to the following procedures.

The limits of conversion to maltose by cr- and /3-amylases were determined

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274 POLYSACCHARIDE SYNTHESIS BY AMYLOSUCRASE

by methods described in a succeeding paper (18) ; all the digestions were at 30” for 24 hours. Reactions with iodine were examined in several ways: (1) the color was observed when 5.0 ml. of a 0.04 per cent solution of polysaccharide at p1-I 5.8 were treated with 0.2 ml. of 1 per cent Iz and 2 per cent KI; (2) the “blue value” was determined by a slight modification (18) of the method of McCready and Hassid (19) ; (3) the percentage of iodine bound in complex formation (mg. of IZ per 100 mg. of polysaccharide) was measured by Schoch’s modification (18) of the potentiometric method of Bates, French, and Rundle (20) ; and (4) the capacity of iodine to precipi- tate each polysaccharide from dilute (0.05 per cent) solution was tested (18). The polysaccharides were tested also for their capacities to dissolve in cold water, to “retrograde” (i.e., to precipitate as particles that do not redissolve on heating) from 0.5 per cent solutions stored overnight at 8”, and to be precipitated from solution by pentasol and butanol (18). They were examined also for their opalescence in 1 :lOOO solution at pH 6.2, for their capacity to give serological precipitation in dilutions from 1: 1000 to 1: 10 million with a standardized, dextran-reactive, type 2 pneumococcus antiserum (17), and for their speed of hydrolysis by hydrochloric acid. For the latter determination, solutions of polysaccharide in 0.5 N HCl in sealed tubes were heated for 15, 20, and 45 minutes in boiling water, then cooled, neutralized, and tested for content of reducing sugar, as glucose (6). The rate of hydrolysis was expressed as the first order velocity constant, Kl(21).

From the data summarized in Table IV, the polysaccharide synthesized by the cell-free enzyme system from Neisseria per$ava appears to be a glycogen-like substance. Although differing in that it gave much more opalescent solutions, it was similar to the glycogen from oysters and liver in respect to the extent of hydrolysis by the several CY- and /J-amylases tested, the kind of color given with iodine, including the absence of any measurable “blue value,” the slight amount of iodine bound in complex formation, the failure to be precipitated by iodine, pentasol, or butanol, and the likeness in the rate of hydrolysis by hydrochloric acid. In com- parison to corn amylopectin it showed considerably less complete hy- drolysis by the ac- and P-amylases, had less intense and less blue color with iodine, and a lower capacity to form an iodine complex. The product of the enzyme differed from the amylopectin-like polysaccharide from the living culture of the Neisseria in the same manner as from the corn amylo- pectin.4

4 A further difference between the products of enzymatic and cultural source was evident from tests made by Dr. S. Hestrin of their capacity to undergo phosphoroly- sis with purified crystalline muscle phosphorylase. He found (personal communica- tion) that the cultural product was split to the extent of about 40 per cent, while the productrformed by the cell-free enzyme underwent a reaction that was considerably smaller and slower.

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Comp

ariso

n of

Po

lysac

char

ide,

Synth

esize

d fro

m Su

crose

by

Ce

ll-Fre

e Ne

isser

ia En

zyme

s, wi

th

Polys

acch

aride

El

abor

ated

by

Liv

ing

Cultu

res

of Ba

cteria

fro

m

Whic

h En

zyme

s W

ere

Deriv

ed,

and

with

Po

lygluc

oside

s fro

m Se

vera

l Ot

her

Biolo

gical

Sour

ces*

TABL

E IV

Prop

erty

N. p

erfla

va

polys

acch

aride

s So

urce

and

kind

of

refer

ence

po

lygluc

oside

s

Enzy

me-su

crose

mi

xture

oy

ster

glyco

gen

Mamm

alian

liv

er

glyco

gen

Conv

ersio

n to

malto

se

by

amyla

ses,

per

cent

a-Am

ylase

(sa

liva)

,%

Amyla

se

(ung

ermi

nated

ba

rley)

@-A

mylas

e (w

heat)

Reac

tions

wi

th

iodine

Color

wi

th

iodine

“B

lue

value

” I2

boun

d in

comp

lex

forma

tion,

y0

Pptn

. by

iod

ine

Red-

brow

n Ma

roon

Lig

ht

red-

brow

n Lig

ht

red-

brow

n Ma

roon

0

11

0 0

32

0.1

Cu.

0.2

Ca.

0.1

Cu.

0.1

Cu.

0.6

0 0

0 0

0

Inten

se

blue

None

31

5 0

18.9

0.0

+++

0

Solub

ility

in co

ld wa

ter

“Retr

ogra

datio

n”

tende

ncy

Pptn

. by

pe

ntaso

l or

buta

nol

Opale

scen

ce

of 1:

1000

so

lution

Se

rolog

ical

prec

ipitat

ionf

Acid

hydro

lysis

(Kr),

mi

n.-r

Othe

r pr

oper

ties

+++

+++

+++

+++

’ ++

+ ot

ot 0

0 0

0 0

0 0

+++

+++

0 zk

0 0

0 0

0.04

1 0.0

5 j

0.04

0.05

i +++

+++

0 0 0.06

+++

0 0 + +++

0.01

* Al

l the

da

ta ar

e ca

lculat

ed

to the

mo

istur

e-fre

e ba

sis.

t Bo

th

the

N.

perJl

ava

polys

acch

aride

s do

ten

d to

prec

ipitat

e ou

t of

solut

ions

store

d for

lon

g pe

riods

in

the

cold,

bu

t the

y red

is-

solve

re

adily

wi

thou

t he

ating

. $0

, no

pr

ecipi

tation

in

tests

of 1:l

OOO

or hig

her

dilut

ions

of the

po

lysac

char

ides

with

typ

e 2

pneu

moco

ccus

an

tiser

um;

+++,

% pr

ecipi

tation

in

tests

of 1:l

O mi

llion

and

lower

dil

ution

s of

the

polys

acch

aride

. cn

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276 POLYSACCHARIDE SYNTHI!%IS BY AMYLOSUCRASE

The Neisseria polysaccharides, both of enzymatic and of living culture origins, differed from corn amylose in nearly all of the properties listed in Table IV; furthermore, the magnitude of the differences, especially in the iodine reactions, was sufficient to show that little or no amylose was present as an accompanying substance in either of the bact.erial products.

The sharp distinction between the Neisselia polysaccharides and the dextran of Leuconostoc mesenteroides is of special interest because they are derived from the same substrate (sucrose). The dextran failed to show any hydrolysis by the amylases, failed to give any of the reactions with iodine, and was hydrolyzed by acid at a rate one-fourth to one-fifth that of the Neisseria products. That dextran was not contained, even in trace amounts, in either Neisseria polysaccharide was shown by the failure of the Neisseria products to give serological precipitation in dilutions as low as 1: 1000 with an antiserum capable of revealing the presence of dextran in dilutions as high as 1: 10 million.

DISCUSSION

Glycogen-like polysaccharides were synthesized from sucrose and from glucose-I-phosphate (and apparently also from amylose2) through the action of soluble enzymes obtained from cultures of Neisseria per$ava. We believe that this is the first instance in which the conversion of sucrose to polysaccharide ma,terial of the starch-glycogen class has been demon- strated in a system free of living cells.

The data on the nature and quantities of the products formed from sucrose established that the over-all reaction consists of the conversion of r~ molecules of sucrose to a glycogen-like polymer of n (glucose - HzO) residues plus n molecules of free fructose, apparently by direct polycon- densation. The fact that glucose-l-phosphate also served as a substrate raised the possibility that that phosphorylated sugar might be required as an intermediate substance in the synthesis of the polysaccharide from sucrose. However, only very small amounts of phosphorus were present in active enzyme-sucrose systems, and no detectable change in the phos- phorus partition occurred during polysaccharide synthesis from sucrose; moreover, the presence of inorganic phosphate in concentrations sufficiently high (8 moles of phosphate to 1 of substra.te) to prevent all synthesis from glucose-I-phosphate had no inhibitory effect whatsoever upon the syn- thesis from sucrose. Furthermore, the activity for sucrose and the activity for glucose-l-phosphate were at least partially separated by mild heating treatment and by gas treatment. Thus, the evidence as a whole indicates strongly that the Neisseria extracts contain two systems: an amylosucrase, active preferentially if not solely upon sucrose, and a phosphorylase, active upon glucose-l-phosphate but not upon sucrose; and that, when the

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E. 5. HEHRE 277

amylosucrase acts upon sucrose, the synthesis of polysaccharide of the starch-glycogen &ss proceeds by a pathway that does not involve glucose- l-phosphate. This mechanism is contrary to the general belief (expressed, for example, by Cori (22) and by Hassid, Doudoroff, and Barker (23)) that all members of the starch or glycogen class are always formed from glucose- 1 -phosphate through the action of phosphorylases.

The syntheses of dextrans and levans from sucrose by enzymes from Leuconosfoc mesenteroides (24-26) and Aerobacter levanicum (27, 28) also apparently proceed without mediation of glucose-l-phosphate or of any other phosphorylated sugar, according to the equations:

12 sucrosewdextran + n fructose n sucrose---+levan + n glucose

We have suggested the name amylosucrase for the enzyme system of Neisseria that converts sucrose to glycogen- or amylopectin-like material in order to indicate its close relationship to dextra.nsucrase and levansucrase, as well as to emphasize its distinction from the phosphorylase plus cross- linking enzyme systems which bring about the synthesis of amylopectins and glycogens from glucose-l-phosphate (1, 29). However, in spite of the apparent differences, the enzymatic syntheses of polysaccharides from sucrose and from glucose-l-phosphate are fundamentally much alike in that the substrate in each instance contains the basic unit of the final polymer product in the form of a glycoside radical that is exceedingly easily split off by acids. This point of similarity was first observed (25) in a comparison of dextran and levan formation by bacterial enzymes with starch formation by plant and animal phosphorylases, in which instances the polysaccharide products were different. The similarity is even more prominent in the present example in which the product formed from sucrose by Neisseria amylosucrase is essentially like the glycogen formed from glucose-l-phosphate by tissue phosphorylases. The activity of glucose-l-phosphate as a polysaccharide precursor thus would appear to depend not upon its bejng a phosphate “ester,” as is commonly believed, but upon a structural feature which is possessed also by the sucrose molecule (cf. (29)). Sucrose not only enters enzymatic reactions .in which its glucoside and fructoside units are transferred to polysaccharides of several types (dextrans, levans, glycogen-like polysaccharides), but also enters reactions in which its glucoside group is transferred to glucose-l-phosphate and to several disaccharides (30,31). This versatility in capacity to donate glycoside groups, though so far observed only with bacterial enzymes, deserves recognition as one of the most important biochemical properties of sucrose.

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278 POLYSACCHARIDE SYNTHESIS BY AMYLOSUCRASE

SUMMARY

Cell-free extracts derived from Neisseria per$ava, a variety of bacteria that occurs commonly in human throats, synthesized a glycogen-like polysaccharide from sucrose. The extracts also acted upon glucose-l- phosphate, but the actions on the two substrates could be distinguished on the basis of differences in the influence of high concentrations of inorganic phosphate and in the susceptibilities to heat and to gas treatment. The Neisseria extracts apparently contained a phosphorylase system, active upon glucose-l-phosphate but not upon sucrose, in addition to a new, heat-labile, enzyme system (amylosucrase) which has the capacity of forming polysaccharide material of the starch-glycogen class from sucrose by a pathway (direct polycondensation) that does not involve glucose-l- phosphate.

The polysaccharide formed by the Neisseria enzymes was more like glycogen than like the amylopectin formed by the living Neisseria perJava cultures or the amylopectin component of plant starch; it was much dif- ferent, from the dextran which Leuconostoc mesenteroides forms from sucrose.

The in vitro conversion of sucrose to maltose was accomplished by the successive action of Neisseria enzymes and of @-amylase.

BIBLIOGRAPHY

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E. J. HEHRE 279

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Doris M. Hamilton and Arthur S. CarlsonEdward J. Hehre and With the assistance of

(AMYLOSUCRASE)BACTERIAL ENZYME SYSTEM

FROM SUCROSE BY A CELL-FREE,OF THE STARCH-GLYCOGEN CLASS SYNTHESIS OF A POLYSACCHARIDE

1949, 177:267-279.J. Biol. Chem. 

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