THE BIOLOGICAL OXIDATION OF SORBITOL*

BY JOSEPHT. CUMMINS, TSOO E. KING, AND

VERNON H. CHELDELIN
(From the Department of Chemistry and the Science Research Institute,
Oregon State College, Corvallis, Oregon)
(Received for publication, June 18, 1956)

Sorbitol is fermented in good yields to sorbose by some of the members

of the Acetobacter species (1, 2). Resting cells of Acetobacter suboxydans
may, in the absence of a source of nitrogen, oxidize sorbitol with the con-
sumption of over 4 atoms of oxygen per molecule of substrate (3, 4).
From a study of dinitrophenol action on this organism, the first step in
sorbitol oxidation was shown to be non-phosphorylative (4, 5).
Recently, a cyclic mechanism, the Horecker pentose cycle, has been
demonstrated in A. suboxydans which can oxidize sugars to COz and water
and can also lead to the formation of cellular materials (6).
The present paper will demonstrate dual pathways for the oxidation of
sorbitol by soluble extracts of A. suboxydans. In the presence of triphos-
phopyridine nucleotide (TPN), sorbose is formed; in the presence of di-
phosphopyridine nucleotide (DPN), fructose is produced.’ The fructose
can then be phosphorylated and oxidized via the pentose cycle. Details
of the properties of these TPN- and DPN-linked dehydrogenases will be
given in a later paper.

EXPERIMENTAL

Organism, A. suboxydans, ATCC No. 621, was used for all experiments.
It was maintained on yeast-glycerol agar slants (7), and the culture was
transferred every 2 weeks.
Preparation of Cells-All cells used were grown in 10 liter batches at
300, with extremely rapid aeration. The medium was prepared as follows:
To 10 liters of water were added 500 gm. of sorbitol, 100 gm. of yeast extract,
* This work was supported by grants from the Nutrition Foundation, Inc., Swift
and Company, and the Division of Research Grants and Fellowships, National Insti-
tutes of Health, United States Public Health Service. Published with the approval
of the Monographs Publications Committee, Research paper No. 303, School of
Science, Department of Chemistry. Taken from the dissertation for the degree of
Doctor of Philosophy of Joseph T. Cummins, Oregon State College, 1957.
1 The following abbreviations will be used throughout this paper: CFE, cell-free
extract; ATP, adenosine triphosphate; DPN, diphosphopyridine nucleotide; TPN,
triphosphopyridine nucleotide; TPP, thiamine pyrophosphate; TTZ, triphenyltetra-
zolium chloride; TTZH, reduced TTZ (formazan); Tris, tris(hydroxymethyl)amino-
methane; TCA, trichloroacetic acid.
323

This is an Open Access article under the CC BY license.

324 OXIDATION OF SORBITOL

50 gm. of KH2POd, and 3 ml. of Dow antifoam. The pH was then adjusted
to 6, and the mixture was autoclaved at 15 pounds pressure for 40 minutes.
The medium was inoculated with 100 ml. of an actively growing A. subozy-
dons culture, and after 40 hours the cells were harvested in a Sharples
supercentrifuge. The cells were starved for 1 hour by shaking in 1 liter of
0.05 M phosphate buffer at pH 6 and were then washed twice with water.
Finally the cells were frozen-dried in 2rucuo and stored at - 10”. Yields
of 1.1 to 1.3 gm. of dry weight were obtained per liter of medium. The
cultures were found to be free of contamination. The cells were grown on
sorbitol instead of glycerol as in the previous work (8), since this treatment
increased the concentration of the enzymes studied.
Preparation of Cell-Free E&act--The cell-free extract (CFE) was ob-
tained by grinding together 4 gm. of dry cells, 8 gm. of alumina (Alcoa
A-301 (8)), and 4 ml. of water. Ordinarily the preparation was centrifuged
at 25,000 X g and dialyzed against distilled water for 8 hours, although in
some experiments the dialysis was carried out against Dowex 50 to remove
thiamine pyrophosphate (TPP). This procedure (9) involved dialysis of
crude CFE in a cellophane bag against a suspension of the resin in the hy-
drogen form, which had been washed until the pH of the suspending water
remained unchanged. The protein precipitated within the bag. After
18 hours at l-3” the precipitated protein was centrifuged at 2000 X g
and redissolved in 50 ml. of 0.01 M Tris buffer at pH 8.2. The supernatant
solution was used after centrifuging again to remove traces of undissolved
protein.
Reagents-The following substances were obtained commercially and
used without further purification: adenosine triphosphate (ATP) and di-
and triphosphopyridine nucleotides from the Sigma Chemical Company;
sorbitol from Matheson, Coleman, and Bell, Inc.; TPP from Hoffmann-
La Roche and Company, Ltd.; sorbose from the Pfanstiehl Laboratories,
Inc.; and orcinol from the Eastman Kodak Company. Sorbose-l-phos-
phate was kindly supplied by Dr. H. A. Lardy.
Analytical Methods--For calorimetric determinations, a Bausch and
Lomb monochromatic calorimeter was employed. Experiments with
oxygen as the final electron acceptor were performed by means of Warburg
manometry, whereas those in which triphenyltetrazolium chloride (TTZ)
was used were carried out as described previously (6). The latter method
was adopted for larger volumes in some experiments in order to obtain
greater quantities of products.
Ribose was determined calorimetrically by the orcinol test at 660 rnp,
according to the method of LePage (10). Hexose was measured by the
cyst&e-HzS04 method (ll), at 420 mp. The Fiske-Subbarow method
(12) was employed for organic and inorganic phosphorus determinations.
Fructose and sorbose were measured by a method of Roe (13).
 J. T. CUMMINS, T. E. KING, AND V. H. CHELDELIN 325

Paper Cknnatogru~hy-Whatman filter paper No. 1 was used. Phos-

phate esters were prepared as previously described (6) and identified on
paper chromatograms by the method of Hanes and Isherwood (14). For
the identification of fructose and sorbose, digestion mixtures containing the
sugars were concentrated to 0.1 ml. by lyophiliaation before the mixture
was placed on paper. The sugars were located by the aniline phthalate
method (15).

Results
Sorbose OxkiXen-Resting cells in the presence of ATP were able to
oxidize sorbose with the consumption of 3 atoms of oxygen (see Fig. 1).

Fructose

I TIME (minuted

FIG. 1. Oxidation of glucose, fructose, sorbose, and sorbitol by resting A. sub-

oxydans cells. Each Warburg vessel contained 20 pmoles of MgCl,, 6 pmoles of ATP,
0.2 pmole of TPN, 6 pmoles of substrate, 70 pmoles of phosphate buffer at pH 6.0,
20 mg. of suspended cells. Total volume, 3.0 ml.; atmosphere, air; temperature, 29”.

However, without ATP only 1 atom of oxygen was consumed, and there
was a 2 hour lag period before oxidation began. This is in line with earlier
observations (4) on the non-phosphorylative character of the fnst sorbitol
oxidation step, as contrasted to the subsequent steps which are phosphate-
dependent. With cell-free extracts, with either O2 or TTZ as the final
electron acceptor, no oxidation was obtained, even when ATP was supplied.
Sorbose-l-phosphate was also not significantly oxidized. With paper
chromatography, it was not possible to show the formation of any new
phosphate esters after the incubation of sorbose with ATP and CFE;
neither was any inorganic P formed. Warburg manometry in bicarbonate
buffer with sorbose, ATP, and CFE did not show any increase in acid that
would have reflected a phosphorylation process.
Sorbitol Oxidation-Resting cells oxidized sorbitol with the consumption
of up to 5.5 atoms of oxygen per molecule, either with or without ATP.
326 OXIDATION OF SORBITOL

The cell-free extract without ATP formed 1 molecule of TTZH per molecule
of sorbitol, with either DPN or TPN. However, in the presence of ATP,
DPN materially improved the oxidation. When compared to the behavior
of these systems upon glucose and glycerol (Table I) (cf. (6)), it was ap-
parent that this preferential enhancement of oxidation by DPN was pecul-
iar to sorbitol.
IdentiJication of Products-Since the extent of the oxidation of sorbitol
depended upon the pyridine nucleotide employed, it was thought that the
products of DPN and TPN oxidation might be different. This proved to
be correct, as shown in Table II, where fructose and sorbose were identi-
fied BS the respective products. A small amount of fructose was formed

TABLE I
Phosphorylative and Non-Phosphorylative Oxidations with DPN
and TPN by Cell-Free Extract of A. suboxydans
TPN DPN
Substrate
-ATP +ATP -ATP +ATP

Sorbitol. . . . . . . . . . .. 0.98 0.98 0.95 1.60

Sorbose.. . . . . . . . 0 0 0 0
Glycerol.. . . . . .. 0 0.40 0.15 0.40
Glucose. . . . . . . 1.30 2.40 1.10 1.95

The tubes were incubated in vacua for 4 hours with 2 pmoles of substrate, 2 pmoles
of ATP, 0.1 pmole of TPN or DPN, 50 pmoles of MgC12, 100 rmoles of Tris buffer at
pH 8.5, 10 rmoles of TTZ, 0.2 ml. of CFE. The total volume was 1 ml.; tempera-
ture, 30’. The figures are in micromoles.

along with sorbosein the TPN system; this may have resulted from traces
of DPN remaining in the enzyme preparations. Positive identification of
the sugars was made by optical rotation, by melting points, and by the
crystalline form of the osazones. In order to obtain satisfactory melting
points, the osazoneswere fist purified by means of a talcum powder column
according to the procedure of J@rgensen(18).
Fructose Oxidation; Pentose Cycle Activity-In contrast to sorbose,
which was not oxidized in cell-free extracts, fructose was dissimilated to the
extent of about 3 atoms of oxygen per molecule in soluble extracts and up to
8.8 atoms by whole cells, as shown in Fig. 1. The intermediate oxygen
consumption by resting cells in the sorbitol oxidation (5.5 atoms) is pre-
sumably due to the summation of the dual pathways through which a part
of the substrate goes to sorbosewith only 1 atom of oxygen consumed per
mole of sorbitol.
Upon oxidation of sorbitol with DPN, the fructose formed could be dis-
similated via the pentose cycle. Table III demonstrates the quantitative
 J. T. CUMMINS, T. E. KING, AND V. H. CHELDELIN 327

formation of pentose and hexose for both the DPN and TPN oxidations.
Pentose accumulated in the digestion mixture because TPP had been re-
moved from the enzyme and transketolase could therefore not function.

TABLE II
Zdentification of Products of Sorbitol Oxidation by A. suboxydans
-
Pyridiie nucleotide added
- n-Fructose
standard
lcS.OdXlSe
standard
DPN TPN
_- --
Position constant in phe-
nol-H,O (4: l)* . . . . . . . . 0.93 0.69 1.00 0.73
[a]: (16), degrees. . . . . . . . . -102’ -48 -92 -42
Crystal form of osazone
(17). . . . . . . . . . . . . . . . . . . . Rosettes Amorphous Rosettes Amorphous
Melting point of osazone
(16), “C.. . . . . . . . . . . . . . 206 161.5-163 206 163
I -
The reaction mixtures contained the following: 180 pmoles of sorbitol, 10 pmoles
of TPN or DPN, 3 ml. of CFE, 100 amoles of TTZ, 500 &moles of MgClz, 1 mmole of
Tris buffer, pH 8.5. The total volume was 10 ml.; temperature, 30°; time, 4 hours.
1 ml. of 50 per cent TCA was added, and the mixture was centrifuged and extracted
with ether. The [a]? was calculated on the basis of sorbose and fructose deter-
mined both by the cysteine-HeSOd and resorcinol methods.
* The position constant is the distance traveled by the compound divided by the
distance traveled by fructose.
TABLE III
Formation of Hexose and Pentose from Sorbitol in Presence of ATP
DPN observed DPN calculated* TPN observed l-PN calculated’

pmeles pwwles ptnoke pmeles

TTZ reduced. . . . . . . . . . . . 31 25 7 7
Hexose formed. . . . . . . . . . 1 7
Pentose formed.. . . . . . . . 8 0

The tubes were incubated in vucuo with 20 pmoles of sorbitol, 20 rmoles of ATP,
1 rmole of TPN or DPN, 0.5 mmole of MgCL, 1 mmole of Tris buffer at pH 8.5, 100
pmoles of TTZ, 3 ml. of CFE. The total volume was 10 ml.; temperature, 30’; time,
2 hours. The reaction was stopped with 1 ml. of 50 per cent TCA. Pentose and
hexose were assayed as described in the text, with ribose and fructose (or sorbose)
as standards.
* Based on quantities of hexose and pentose formed (3 amoles of TTZ should
be reduced per amole of pentose obtained).

Fig. 2 indicates the rate of formation of hexose and pentose by the cell-
free extract during sorbitol oxidation, with TTZ as electron acceptor.
TPPdeficient enzyme was used, and the TPP added again after 2 hours.
In the oxidation of sorbitol with DPN and ATP, pentose formed to the
323 0xIDmIoN 037 SORBITOL

extent of 0.4 mole per mole of sorbitol, then dropped to zero after the ad-
dition of TPP; hexose accumulat,ed in the mixture until TPP was added,
then dropped slightly to a constant value. In the incubation of sorbitol
with TPN and ATP, 0.1 mole of pentose was formed per mole of sorbitol.
This also decreased to zero upon the addition of TPP, whereas hexose
reached a peak of 0.3 mole per mole of sorbitol and was unaffected by the
addition of TPP. The small amount of pentose formed during the TPN
oxidation may have been due to some DPN in the system, or it may have

TIME (Hours)
FIQ. 2. Conversion of sorbitol to hexose and pentose with a TPP-deficient enzyme
in the presence of either DPN or TPN. CFE was dialyzed against Dowex 50 for 20
hours. The reaction mixture contained 3 ml. of dialyzed CFE, 20 pmoles of sorbitol,
20 rmoles of ATP, 100 pmoles of TTZ, 2 Icmoles of DPN or TPN as indicated, and
200 pmoles of Tris buffer, pH 8.5. Total volume, 20 ml.; temperature, 30”. After
2 hours incubation, 5 pmoles of TPP were added to each tube. 3 ml. aliquots were
removed at the indicated time intervals, and the reaction was stopped with 0.5 ml.
of 50 per cent trichloroacetic acid (TCA). Pentose and hexose were assayed as
described in the text with ribose and fructose (or sorbose) as standards. A blank of
the digestion mixtures containing no sorbitol was subtracted to give the plotted
values.

been in part due to as yet unidentified intermediates (related to sorbose?)

which also characterize the partial dissimilation of sorbose in resting cells.

DISCUSSXON

Cell-free preparations of A. subozz~&z~spossessthree pathways for the

oxidation of sorbitol. In addition to the particulate dehydrogenase (19)
which catalyzes a one-step oxidation of sorbitol (presumably to hexose),
the soluble portions of the cells contain the two alternative enzyme systems
described herein. Since sorboseis formed in the presence of TPN and fruc-
tose with DPN, pyridine nucleotide appears to determine which pathway
will be employed, i.e. which end of the sorbitol molecule will undergo
 J. T. CUMMINS, T. E. KING, AND V. H. CHELDELIN 329

oxidation. This unexpected influence of pyridine nucleotide in guiding

metabolic “traffic” has also been noted in certain animal systems under
investigation in this laboratory (R. L. Jolley, R. W. Newburgh, and V. H.
Cheldelin, unpublished results; also Wenner and Weinhouse (20)).
The present observations also demonstrate clearly that in the first step
oxidation of sorbitol ATP is produced. The latter, in turn, phosphorylates
the fructose formed to channel the pathway into the pentose cycle. On
the other hand, sorbose cannot be further phosphorylated or oxidized with
CFE. Only in whole cells in the presence of an energy source can this
sugar be extensively dissimilated. This process, including the intermediate
products arising during its operation, is still unknown.

SUMMARY

The oxidation of sorbitol by soluble extracts of Acetobacter mboxydans

proceeds by two pathways, depending upon which pyridine nucleotide is
present. In the presence of triphosphopyridine nucleotide, sorbose is
formed. In the presence of diphosphopyridine nucleotide fructose is pro-
duced. The fructose can t.hen- be phosphorylated or further oxidized by
cell-free extracts. These two newly defined pathways occur in addition to
the previously demonstrated sorbitol dehydrogenase which is found in the
particulate fraction of the cells.

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