Extracellular Microbial Polysaccharides

20 Production, Properties, and Application of Curdlan

Downloaded by UCSF LIB CKM RSCS MGMT on November 19, 2014 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch020

TOKUYA HARADA Institute of Scientific and Industrial Research, Osaka University, Yamadakami, Suita-shi, Osaka, Japan (565)

Curdlan was produced in high yield by cultures of a newly isolated and improved mutant strain of Alcaligenes faecalis var. myxogenes. Curdlan forms a gel with specific properties and it should be a useful, new polymer not only as a food additive, but also for industrial purposes. I. Findings The history of the discovery of curdlan is interesting. In 1962, Harada and his colleagues made great efforts to obtain microorganisms which could utilize petrochemical materials. They isolated an organism from soil, capable of growing on medium containing 10% ethylene-glycol as the sole carbon source (1) and named it Alcaligenes faecalis var. myxogenes 10C3 (2, 3). They found that this organism produced a new β-glucan which contained about 10% succinic acid and named it succinoglucan (4, 5) . The structure of the polysaccharide moiety of succinoglucan (6, 7) is shown below: -->Glcl-->4Glcl-->3Glcl-->3Glcl-->6Glcl-->4Glcl-->3Glcl-->3Glcl-->4Glcl-->3Gall-->4Glcl--> During investigations on the production of succinoglucan, one day they found that the culture medium did not become viscous and no succinoglucan was formed, but almost all the added glucose was consumed. They thought that some special compound(s), must have been produced instead of succinoglucan in the culture. So they examined the product and found that it was a neutral polysaccharide (8, 9). They named it curdlan in 1966 (10). Curdlan is composed of β-l,3-glucosidic linkages. A mutant strain 10C3K was isolated from the stock culture 10C3, which produced only curdlan. Strain 10C3K is a spontaneous mutant and it has stable ability to produce the exocellular polysaccharide whereas the ability of strain 10C3 is unstable(11). Thus, by chance, they succeeded in obtaining a suitable organism for pro duction of curdlan. Later Takeda Chemical Industries Ltd.isolated 265

In Extracellular Microbial Polysaccharides; Sandford, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

266

a u r a c i l - l e s s mutant o f s t r a i n 10C3K named s t r a i n * 13140 as a b e t t e r gel-forming B-l,3-glucan producer (12). The polymer from the s t r a i n , designated as p o l y s a c c h a r i d e 13140, i s a k i n d o f curdlan i n a broad sense or a curdlan type p o l y s a c c h a r i d e .


II.

Mutation

The d e t e c t i o n o f c u r d l a n using A n i l i n e Blue was t e s t e d using s t r a i n 10C3 and i t s mutant s t r a i n s 10C3k and 22 as shown i n Figure 1.(13). The c u l t u r e medium used i n t h i s p l a t e , c o n s i s t e d o f 1% glucose, 0.5% yeast e x t r a c t , 0.005% A n i l i n e Blue and 2% agar. The middle colony i s t h a t o f 10C3. The s u r r o u d i n g - c l e a r zone i s due to the formation of succinoglucan which i s a s o l u b l e , viscous polymer. Succinoglucan does not s t a i n with A n i l i n e Blue. Curdlan can form a complex with t h i s dye which i s b l u e . The r a t e o f i n t e r a c t i o n o f the polymers with A n i l i n e Blue was shown by Nakanishi and h i s colleagues to be p r o p o r t i o n a l to t h e i r concent r a t i o n s and degrees o f p o l y m e r i z a t i o n (14). The l e f t colony i s that o f a spontaneous mutant o f the parent s t r a i n which produces only curdlan. The complex o f the polymer with the dye can e a s i l y be s t r i p p e d o f f . The remaining c e l l s do not s t a i n with the dye. The r i g h t colony i s that o f mutant s t r a i n 22, d e r i v e d from 10C3 by treatment with N-methyl-N -nitro-N-nitrosoguanidine . This s t r a i n produces only succinoglucan. Mutation o f s t r a i n 10C3 to s t r a i n s s t a i n i n g with A n i l i n e Blue was a l s o induced by treatment with mutagens such as NTG, and ethylmethane-sulfonate and u l t r a v i o l e t l i g h t , but not by t r e a t ment with Mitomycin C, ethidium bromide or A c r i d i n e Orange which are reagents causing e l i m i n a t i o n of plasmids (Table 1) (11). Experiments on t r a n s f e r of genes concerned with production o f succinoglucan and(or) curdlan between d i f f e r e n t mutant s t r a i n s have not been s u c c e s s f u l . Thus, a plasmid may not be d i r e c t l y i n v o l v e d i n the production o f the p o l y s a c c h a r i d e s . f

III.

Structure

Curdlan i s composed o f 3-1,3-glucosidic linkages ([ot] +18° IN NaOH) (10, 15). S a i t o and h i s colleagues (15) i n d i c a t e d the presence of two i n t e r n a l 6 - l , 6 - g l u c o s i d i c linkages i n o r i g i n a l curdlan (DPn 455) while Ebata (16) detected one p a r t of g e n t i b i o s e to 360 p a r t s of glucose i n the hydrolyzate o f the glucan by the a c t i o n o f exo-B-l,3-glucanase, although Nakanishi and h i s colleagues could not detect any other g l u c o s i d i c linkages besides 6-1,3-glucosidic linkages i n p o l y s a c c h a r i d e 13140 (12). C e l l u l o s e f i b e r , which i s composed o f 6 - l , 4 - g l u c o s i d i c l i n k a g e s , does not s w e l l i n the presence o f water whereas curdlan swells i n water and can form a r e s i l i e n t g e l on heating. T h i s i s an important and i n t e r e s t i n g f a c t . C a l l o s e and pachyman are g-1,3-glucans which are l a r g e l y composed o f g - l , 3 - g l u c o s i d i c l i n k a g e s . C a l l o s e cont a i n s a l i t t l e g l u c u r o n i c a c i d (17). Pachyman has other glucoD


20.

Curdlan

HARADA

267

Table 1 E f f e c t s o f Mutagens on Mutation o f S t r a i n 10C3 with A n i l i n e Blue (11)


~~ Mutagen

ConcenGrowth tration inhibition (per ml) (%)

None

R a t

io w

h

to S t r a i n s S t a i n i n g

o f blue c o l o n i e s i

t

e

c

o

l

o

n

i

e

s

( % )

1.0

X

10"

1.4

X

10"

7

f

N-Methyl-N nitro-N-nitrosoguanidine

- 3

30 jig

Ethylmethanesulfonate 5-Bromouracil

25

3 5

1.4

X

10"

3

&

2

Q

1.0

X

10"

7

&

Ultraviolet light irradiation

2 0 X

10"

3

s i d i c linkages and does not form a r e s i l i e n t gel on h e a t i n g (15). Cal l o s e has been found i n a v a r i e t y o f l o c a t i o n s i n the tissues" o f h i g h e r p l a n t s , such as i n s i e v e tubes, young t r a c h e i d e s , p o l l e n , root h a i r s , stem h a i r s and root endodermis. No other p o l y saccharides besides curdlan composed e n t i r e l y o f g - l , 3 - g l u c o s i d i c linkages have yet been found. IV.

Production

Now i t has become p o s s i b l e to o b t a i n h e a t - g e l a b l e g-l,3-glucan e a s i l y from glucose and many carbon compounds. The y i e l d o f the polymer from added glucose i s about 50%. About 5 g o f curdlan can be produced from 10 g o f glucose i n 100 ml of simple defined medium, i f the pH i s maintained at n e u t r a l i t y (_9 , 12_, 18) · Curdlan can a l s o be produced using a c e l l suspension i n medium c o n t a i n i n g only glucose and calcium carbonate (19). A p i l o t p l a n t f o r p r o d u c t i o n o f p o l y s a c c h a r i d e 13140 has been accomplished i n Takeda Chemical I n d u s t r i e s L t d . Nakanishi and h i s colleagues examined the occurrence of curdlan type polysaccharides i n microorganisms, u s i n g the A n i l i n e Blue method (Table 2) (13). Four s t r a i n s o f Agrobacterium r a d i o b a c t e r , one strain~o"f Agrobacterium rhizogenes and a s t r a i n o f Agrobacterium sp. were found to produce curdlan type p o l y saccharides with water s o l u b l e β-glucans (11, 13). Spontaneous mutant s t r a i n s which produce p r i n c i p a l l y curdIan-type p o l y saccharides i n high y i e l d were a l s o induced from the r e s p e c t i v e parent s t r a i n s .


268

EXTRACELLULAR

MICROBIAL

POLYSACCHARIDES

Table 2 Curdlan Type Polysaccharides (Curdlan i n a Broad Sense) Alcaligenes faecalis var. myxogenes 10C3K


Alcaligenes faecalis var. myxogenes IFO 13140

r

uirttian

Polysaccharide 10C3K

Polysaccharide 13140

Agrobacterium radiobacter IFO IFO IFO IFO

12607 12665 13127 13256

Polysaccharide Polysaccharide Polysaccharide Po lys acch ari de

12607 12665 13127 13256

Agrobacterium rhizogenes IFO 13259 Agrobacterium sp. IFO 13660

Polysaccharide 13259 Polysaccharide 13660

The structure of the polysaccharide moiety of a water soluble polymer from strain A. radiobacter IFO 12665 seems to be like that of succinoglucan because a specific 3-glucanase, succinoglucan depolymerase from Flavobacterium sp. M64 (20), can attack the polymer to release oligosaccharide with similar Rf value to that of the product released from succinoglucan by the enzyme (21). Succinic acid may be not contained i n the polymer. V.

Rheology

Excretion of curdlan as microfibrils from the cells of strain 10C3K, i s seen by electron microscopy (Figure 2). When a 2% suspension of curdlan i s heated, i t becomes clear at about 54°C and gel forms at higher temperature (22). Agar gel i s formed when the sol of agar obtained by heating i t s suspension i s cooled. This i s a difference between curdlan and agar. Figure 3 i s a photograph of the gel of curdlan obtained by heating a 2% suspension at 90°C. The gel of curdlan i s very elastic and r e s i l i e n t and does not break, whereas agar gel breaks when i t i s pressed between the fingers. The gel as seen i n Figure 4 i s easy to make using curdlan but i t i s not easy to make such gels using agar. It has also been found that curdlan forms a gel when an alkaline solution i s dialyzed i n a cellophan bag(S.0kamoto unpublished), when an aqueous solution of 0.2 - 0.63 M dimethylsulfoxide i s cooled (23)or when calcium ions are added to a weakly alkaline solution (H. Kimura, unpublished). Maeda and his colleagues investigated the effect of temperature on gel formation using a curdmeter. They heated 3% sus-



HARADA

Curdlan

Journal of General and Applied Microbiology

Figure 1. Photograph of colonies of strains (left to right) 10C3K, 10C3, and 22 grown on glucose-yeast extract medium containing water-soluble aniline blue (0.005% ) (13)

Figure 2.

Curdlan excreted from the cells of 10C3k as microfibrils, negatively stained with uranyl acetate




Figure 3.

Photograph of curdlan gel. Aqueous suspension (2%) of this polymer was heated at90°C for 10 min.

Figure 4. Photograph of curdlan gel. Aqueous suspension (2%) of this polymer was heated in special vessel at90°C for a few min.



20.

HARADA

Curdlan

271

pensions of curdlan f o r 10 min and then measured the strength o f the r e s u l t i n g gels at 30°C (Figure 5). T h i s curve i s c u r i o u s : the g e l strength i s about the same between 60°C and 80°C and then i t increases from 80°C. The g e l strength depends on the temperature but i s independent o f the i n c u b a t i o n time at 70°C (22). Urea breaks hydrogen bonds and i t s e f f e c t on g e l formation was i n v e s t i g a t e d using a Shimadzu Microviscograph (Figure 6). The s t a r t i n g temperature f o r g e l formation decreased with increase i n the concentration o f urea added. I t i s i n t e r e s t i n g that f o r mation o f g e l i n the second stage was not observed with above 5 M urea. Thus, g e l formation i n the f i r s t stage seems t o r e q u i r e the breakage o f hydrogen bonds whereas that i n the second stage does not. I t i s a l s o i n t e r e s t i n g that the v i s c o s i t y increased markedly from 39°C to 20°C with 2 t o 8 M urea when the temperature was decreased. The formation o f g e l at low temperature may be due to formation of hydrogen bonds. E t h y l e n e - g l y c o l a c c e l e r a t e s formation o f hydrogen bonds and i t s e f f e c t on g e l formation was examined i n the same way. The r e s u l t s i n Figure 7 show that i t a l s o decreased the s t a r t i n g temperature f o r g e l formation. However, i n the presence o f a high concentration (5 M to 7 M) o f e t h y l e n e - g l y c o l , no g e l was formed. These r e s u l t s suggest that at the s t a r t i n g temperature f o r g e l formation some o r a l l the hydrogen bonds must be broken. The formation o f g e l o f the polymer was i n v e s t i g a t e d using a Rotovisca Viscometer (Haake) by the members o f Takeda Chemical I n d u s t r i e s Ltd.(24). The s p e c i f i c v i s c o s i t i e s were determined continuously as tEe temperature was r a i s e d t o 60°C and then decreased (Figure 8). From 54°C to 60°C s w e l l i n g occurred due to breakage o f hydrogen bonds. On c o o l i n g the g e l t o about 40°C, the v i s c o s i t y r a p i d l y increased and low-set gel was obtained (25). The e f f e c t o f temperature on transmittance was examined under tïïe same conditions (Figure 9). The transmittance increased on h e a t i n g to 60°C and decreased on c o o l i n g from 60°C (25). Figure 10 shows that the s p e c i f i c v i s c o s i t y a l s o i n c r e a s e d to some extent on c o o l i n g from 85°C with formation o f h i g h - s e t g e l (22, 24). As shown i n Figure 11, the transmittance decreased g r a d u a l l y with increase i n temperature from 60°C to 100°C (25). T h i s was probably due to formation o f hydrophobic bonds during formation o f cross l i n k s . Acetone powders were prepared from gels formed by heating at 60°C, 70°C and 90°C. The two formers formed s i m i l a r gels t o that d e r i v e d from the o r i g i n a l polymer, but a powder from g e l heated at 90°C d i d not. T h i s i n d i c a t e s that gels obtained a f t e r the second stage o f g e l formation have a d i f f e r e n t molecular arrangement from that o f the o r i g i n a l polymer. VI.

Conformation

Figure 12 shows some r e s u l t s o f Ogawa and h i s colleagues. They s t u d i e d the conformational behavior o f polysaccharide 13140 i n a l k a l i n e s o l u t i o n by measuring the o p t i c a l r o t a t o r y d i s p e r s i o n ,



EXTRACELLULAR MICROBIAL

50

POLYSACCHARIDES

60 70 80 9 0 100 Heating temperature V

Agricultural and Biological Chemistry

Figure 5. Effect of heating temperature on gel strength of curdhn(22)

Temperature

Figure 6. Effects of urea (0-8 M) on gel formation of curdlan (1% ). Shimazu microviscograph type SN1 was used.


HARADA


20.

Curdlan

273

Temperature

Figure 7. Effects of ethylene-glycol (0-7 M) on gel formation of curdlan (1%). Shimazu microviscograph type SN1 was used.

Figure 8. Effect of heating temperature on specific viscosity of polysaccharide 13140(1%)

Temperature

o| 0

, 30

, , 40 50 Temperature (°C)

1 60

Figure 9. Effect of heating temperature transmittance of polysaccharide 13140 (1%)

o

n


274


>.10


Journal of Food Science

Figure 10. Ε feet of heating temperature on specific viscosity of polysaccharide 13140(1%) (24)

20

40 60 Temperature (°C)

80

Ν

\

/

\ \

/

Extracellular Microbial Polysaccharides

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