J. Biol. Macromol. 4(3), 83-90 (2004)

ArticleThe relationship between cellulase activity and oligosaccharides and cellulose
productions by Acetobacter xylinum ATCC23769
Fuyu Ito, Yoshihiko Amano*, Kouichi Nozaki, Inder M. Saxena1,
Malcolm R. Brown Jr.1, Takahisa Kanda
Department of Chemistry and Material Engineering, Faculty of Engineering, Shinshu
University (4-17-1, Wakasato, Nagano 380-8553, Japan)
1

School of Biological Science, the University of Texas at Austin(Austin, Texas 78712, USA)

Received August 13, 2004, accepted September 21, 2004
We have tried to investigate various carbohydrates production and cellulose degrading
enzyme activity in the culture broth of cellulose producing microorganism, Acetobacter
xylinum to clarify the role of cellulase for cellulose production. Carboxymethylcellulose(CMC) degrading activity and various oligosaccharides in addition to cellulose havealready been detected after one-day culture. These oligosaccharides increased graduallyduring the culture, were identified to be rhamnose, mannose, some kinds of β-linked
disaccharides such as β-1, 4-linked and β-1, 6-linked glucobiose (cellobiose andgentiobiose), cellotriose and cellotetraose by thin layer chromatogram analysis. It is
assumed that these oligosaccharides may be enzymatic degradation products from acetan
produced in the culture broth because they are the constitution sugar of acetan andcellulase produced by this microorganism could not degrade bacterial cellulose produced
by itself. Cellulase activity is correlated closely with cellulose production, and more closely
related to the changes in amount of oligosaccharides. In addition when A. xylinum wascultured in the presence of β-glucodisaccharides such as gentiobiose and cellobiose, we
observed the increases of cellulase activity and cellulose production.
Keywords: Acetobacter xylinum, cellulase, β-oligosaccharides*Corresponding author(Tel/Fax: +81-26-269-5394, E-mail: yoamano@gipwc.shinshu-u.ac.jp)

84

F. Ito, et al.

culture broth of A. xylinum in 1981. They

Introduction

thought this disaccharide was probably

Cellulose, the most abundant organic

gentiobiose, but the origin of these sugars haspolymer on the earth, occurs in plants and1)certain algae, fungi, and bacteria. It is an

not been clarified. In addition, it has been

extracellular polysaccharide, synthesized asreported that some polysaccharides such as

long β-1,4 glucan chains that associate to

CMC could be altered the structure ofform the microfibrils commonly observed incellulose produced.10, 11)cellulose-synthesizing organisms. It is wellIn this paper, the metabolic products suchknown that some strains of Acetobacter sp.

as sugars and cellulase in the culture medium

produce bacterial cellulose (BC).

2, 3)during culture of A. xylinum ATCC23769

The

gram-negative bacterium Acetobacter

have been investigated. In particular we

xylinum synthesizes an extracellular ribbon of

investigated the relationship between

cellulose microfibrils from

cellulase activity and oligosaccharides

membrane-localized cellulose synthesizing

produced in culture broth to clarify the role

protein complexes that are organized in aof cellulase for cellulose production.

linear row along the long axis of the bacterialcell. This BC is expected to be a new

Materials and Methods

industrial material because of its uniquecharacteristics such as ultra fine fibrils andOrganism and media. A. xylinum ATCC

the extremely high Young’s modulus of a

23769 was used in this study. For seed4)

sheet prepared from it. It is thought that

culture, Hestrin & Schramm (SH)12) medium

further investigations on cellulose producing

was used, in which SH was composed of 2%

mechanism are essential to dissolve the(w/v) glucose (Waco Chemicals, Tokyo),problem of cost performance for industrial

0.5% (w/v) yeast extract (Difco Laboratories,

use.

USA), 0.5% (w/v) peptone (Nihon seiyaku,
Tokyo), 0.27% (w/v) Na2HPO4 and 0.115%

Various reports have described the
production of cellulolytic enzymes by(w/v) citric acid (Waco). We have also5)prepared SH media containing 0.05 wt% of

The role of this enzyme, however, has not

β-glucodisaccharides such as cellobiose andbeen known clearly. Furthermore it isgentiobiose.

cellulose-producing Acetobacter strains.

reported that A. xylinum has also produced

Cultivation. This study was performed

various sugars and water-soluble

using 5-d-old agar “rough” colonies13) ofpolysaccharide called acetan in addition tostrain ATCC23769. To examine metabolic

6)

cellulose. The structure of acetan have been

products it was cultured in SH medium withinvestigated, which is similar to xanthanof β-glucodisaccharides such as cellobiose

produced by microorganism.

7, 8)

Valla et al.

9)and gentiobiose. After 40 ml of liquid

detected mannose, rhamnose, glucuronic acid,medium in 100 ml Erlenmeyer flask was

glucuronyl mannnose and disaccharide

sterilized at 121℃ for 15 min, single colony

consisting of two glucose monomers in

was inoculated to each flask. It was incubatedRelation of cellulase, oligosaccharides and BC by A. xylinum85

at 25℃ for 14 days, and 400 µl of culture

solutions were applied on charcoal column

broth was collected and then used for(3.0×75 cm), and washed with distilled

analysis. After 100 ml of liquid medium withwater and then eluted with 5, 10, 15, 20, 25,

β-glucodisaccharide in 300 ml Erlenmeyer

50% ethanol to detect a small amount offlask was sterilized at 121℃ for 15 min, andoligosaccharides. Thin-layer chromatography

a colony was inoculated to investigate(TLC) was performed by using thecellulase activity and cellulose production.

double-ascending method with a solvent

The culture condition was the same assystem of chloroform – methanol – water

mentioned above.

(90:65:15). TLC plates were sprayed
13)

Analytical methods.

The cell mass wasestimated by measuring the optical density at
660 nm after dissolving cellulose bycompletely with 30 wt% of

H2SO4 solution

and then heated at 130℃ for 5-10 min.
ABEE-conversion method. β-gluco-

enzymatic treatment with cellulase

disaccharides were converted to(Celluclast, Novo). Cellulose produced by A.

corresponding aminobenzoic acid ethyl ester

xylinum was treated with 2% NaOH at 100℃

as (ABEE)-converted β-glucodisaccharide byfor 30 min to dissolve cells and was washedreductive amination. An ABEE reagent

with distilled water, to measure the amount ofsolution (40 µl) was added to the culture

cellulose produced. The dry weight ofsolution (10 µl), and mixed with vortex andcellulose sheet was measured after drying

then cooled to room temperature. Equal

with filter papers at 50℃ for 24 h in vacuum

volumes of chloroform were added before

oven. The amounts of glucose, gluconic acidadding distilled water (0.2 ml). Afterand oligosaccharide in the culture broth werevigorous mixing, the mixtures were

determined using HPLC. They were detected

centrifuged for 3 min, and the upper aqueous

using a RI monitor (410; Waters, USA)layer was subjected to HPLC analysis.

equipped with a TSKgel G-Oligo-PW

ABEE-converted oligosaccharide analysis

column (7.8×300 mm, TOSOH, Tokyo).

was done using a UV monitor (SPD-6A,

Distilled water was used as a mobile phase at

Shimadzu, Kyoto) at the wavelength of 305

the flow rate of 0.5 ml/min (801, JASCO,

nm with an Honenpak C18 (4.6×75 mm,

Tokyo).

Seikagaku, Tokyo). The mobile phase was

Assay of CMCase activity. The activity

0.1 M ammonium acetate buffer (pH 4.0)

was assayed viscometrically in Ostwald-typecontaining 0.1 M acetonitril at the flow rate

viscometer at 30℃ with 1ml of the culture, 4of 1.0 ml/min.

ml of 10 mM sodium acetate buffer (pH 5.0)

Hydrolysis of oligosaccharides The

and 1 ml of 1 wt% CMC

reaction mixture consisted of 30 µl of the(carboxymethylcellulose). One unit offractions eluted with 5-15% ethanol, 30 µl of

cellulase activity was defined as amount of

5.0 Unit/mg β-glucosidase from almonds

enzyme causing a 0.1 decrease in relative(SIGMA, USA). After incubation at 37℃ forviscosity after 24 h incubation.

2 h, 30 µl aliquots of reaction mixtures wereThin-layer chromatography. The culture

spotted on analytical TLC plates.

86

F. Ito et al.

Results and Discussion
Cell growth and metabolic products in SH
mediumChanges in the amounts of glucose,
gluconic acid, cell mass and pH during theculture are shown in Fig. 1. The amounts of

40

2.5

30

2
1.5

20

1
10

0.5
0

glucose in the culture broth were measured

Cellulose (mg/flask)

Disaccharides (mg/ml)
Cellulase activity (unit)

3

0
0

2

4

6

8

10

12

14

Time (day)using HPLC analysis. In the initial stage,
glucose as the sole carbon source appeared
tube converted to gluconic acid. At 3rd day,Fig. 2. Changes in cellulase activity, cellulose

pH of the culture gradually began to decrease

production and the amounts offrom 6.0 to 4.0, where cell growth became

oligosaccharides during the culture of A.

active. After 5 days the pH of culture solution

xylinum ATCC23769.

stayed constant around 4.0 and cells began to

Symbols: ●, disaccharides; ■, cellulose; ▲,grow rapidly. Cell mass was reached at its

cellulase activity.

maximal at 1.4 mg/ml after 2 weeks
incubation, and the pH of the culture brothincreased gradually from the lowest value of

Changes in cellulase activity, cellulose

3.8 to 5.0.

production and the amounts of

25

7

20

6

in Fig. 2. 0.2 mg/ml of disaccharides were
produced after 1 day and increased gradually
thereafter. As compared with 5 and 6 days

5

15

4
10
5
0
0

2

4

6

8

10

12

pH

Glucose, Gluconic acidand Cell mass×10 -1(mg/ml)oligosaccharides during the culture are shown

culture, the accumulation of disaccharides

3

increased rapidly and the same phenomenon

2were observed between 8 and 10 days culture.

1

Cellulose production showed a linear

14

Culture (day)correlation with cell growth. At 14 days, theamount of cellulose was maximal at 40

Fig. 1. Changes in the amounts of glucose,

mg/flask. On the other hand, the level of the

gluconic acid, cell mass and pH during theextracellular cellulase activity increased

culture of A. xylinum ATCC23769.

rapidly after 1 day and was kept constant ofSymbols: ●, glucose; ■, gluconic acid; ▲, cell

the activity between 3 and 5 days culture.

mass; ✻, pH.

Then it increased rapidly again and thistendency was repeated. From these results it
was found that the increases of cellulase
activity, cellulose production and the amountof oligosaccharides are inclined to show theRelation of cellulase, oligosaccharides and BC by A. xylinum

87

similar changes during the culture. It has

xantan.3〜5) Acetan consists of glucose,been assumed that oligosaccharides produced

mannose, glucuronic acid, and rhamnose inin the culture broth are degradation products

the proportions of 4:1:1:1. However weof cellulose. However in the early phase ofconfirmed that the strain we used, A. xylinum

culture in this experiment, about 0.2 mg/ml

ATCC23769, did not produce acetan. Itof oligosaccharides were produced after 1

seems that this strain have no ability to

day incubation even though CMCase activity

polymerize the minimum unit of acetan.

was very low and little cellulose has

Therefore these various oligosaccharides may

produced. So we have carried out in detecting

be secreted as metabolic products in thea small amount of oligosaccharides in theculture without polymerization. In future weculture as describing in the proceeding

hope to describe what the role of these

section.

oligosaccharides during cellulose productionis.

Identification of β-glucodisaccharide in
SH medium
During the culture this bacterium
produced all sorts of sugars. Thin layer

G1chromatogram of oligosaccharides produced
is shown in Fig. 3. Rhamnose, mannose,

G2

disaccharide, cellotriose and cellotetraose
were detected in the culture medium.

G3

Furthermore, HPLC profiles by
ABEE-conversion of β-glucodisaccharide

G4

produced are shown in Fig. 4. Thesechromatograms showed the detection of
disaccharides such as gentiobiose, cellobiose
and a very little laminaribiose. Moreover

St

Rh

Mn

A

B

C

D

hydrolysis products from theseoligosaccharides by β-glucosidase were

Fig. 3 Thin layer chromatogram of

examined. These oligosaccharides were

oligosaccharides produced by A. xylinum

degraded to glucose (data not shown).

ATCC23769.

Consequently these oligosaccharides were

Symbols: S1: standard sugars, G1: glucose,

identified β-linked oligosaccharides.

G2:cellobiose, G3: cellotriose, G4:

It is not obvious what is real origin ofcellotetraose, Rh: rhamnose, Mn: mannose, A

those oligosaccharides. Various reports have

〜D: fractions eluted on charcoal column

described A. xylinum has also accumulated achromatography with various solvent as follows,

water-soluble polysaccharide called acetan,

A: 5% EtOH, B: 10% EtOH, C: 15% EtOH, D:

the structure of which is similar to that of

20% EtOH.

88

F. Ito et al.

(B)
Cellulase activity (Unit)

(A)

Gen
Lam
Sop
Cel
Glc

3
2
1
0
0

2

4

6

8

10

12

14

Culture (day)Standard

Culture Medium

Fig. 4. HPLC profiles of β-glucodisaccharides

Fig. 5. CMC-degrading activity in three

produced by A. xylinum ATCC23769.

kinds of culture broths of A. xylinum(A) profile of authentic sugars. (B) profile of

ATCC23769.

culture broth for 7 days incubation.

Symbols: ●, SH medium; ■, SH medium with

Symbols: Gen, gentiobiose; Lam, laminaribiose;cellobiose; ▲, SH medium with gentiobiose.

Sop, sophorose; Cel, cellobise; Glc, glucose.

Cell growth and cellulose production

containing in the medium with

cultured in the presence of

β-glucodisaccharides during the culture are

β-glucodisaccharides

shown in Fig. 6. In SH medium and SH

Moreover the metabolic products cultured

medium with cellobiose, a small amountin the presence of β-glucodisaccharides sucholigosaccharides are produced after 1day andas cellobiose and gentiobiose have been

began to increase gradually. Whereas theinvestigated. Changes in cellulase activity

production of these oligosaccharides in SH

during the culture are shown in Fig. 5.

medium with gentiobiose were not observed

Cellulase activity produced in SH medium

in the early stages. Nevertheless, thewith cellobiose or gentiobiose was higher

production of oligosaccharides among three

than that produced in SH medium withoutkinds of media exhibited very similar profiles

β-glucodisaccharide. The difference inwith each other.

cellulase activity was large in early stage ofculture.
The changes in amounts of glucose, pH

The changes in cellulose production
during the culture are shown in Fig. 7. The
cellulose production in the case ofand cell mass showed similar tendency whengentiobiose added medium was the smallest

A. xylinum ATCC23769 cultured in threeamong three kinds of medium in early stages.

different types of media (data not shown).

But at a later stage the production of

The changes in amount of oligosaccharides

cellulose in the medium with cellobiose andRelation of cellulase, oligosaccharides and BC by A. xylinum

100

1.5

Cellulose (mg)

Oligosaccharides (mg/ml).

2

1
0.5

80
60
40
20

0

0

0

2

4

6

8

10

12

14

0

Culture (day)Fig. 6.

89

Changes in the amounts of

2

4

6
8 10
Culture (day)12

14

Fig. 7. Changes in cellulose production indisaccharides in three kinds of culture

three kinds of culture broths of A. xylinum

broths of A. xylinum ATCC23769.

ATCC23769.

Symbols: ●, SH medium; ■, SH medium withBlack, white and stripe bars represent SH

cellobiose; ▲, SH medium with gentiobiose.

medium, SH medium with cellobiose andgentiobiose respectively.

gentiobiose increased rapidly and amounts ofReferences

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Communicated by Ohnishi Masatake