Structure and Enzymatic Degradation of Elsinan, a New - American

11

Structure and α-D-Glucan

E n z y m a t i c D e g r a d a t i o n of E l s i n a n , a

Produced

by

Elsinoe

New

leucospila

AKIRA MISAKI and YOICHI TSUMURAYA

Downloaded by EAST CAROLINA UNIV on December 15, 2017 | http://pubs.acs.org Publication Date: June 10, 1980 | doi: 10.1021/bk-1980-0126.ch011

Department of Food and Nutrition, Faculty of Science of Living, Osaka City University, Sumiyoshi, Osaka 558, Japan

Many microorganisms are known to accumulate s i g n i f i c a n t amounts o f e x t r a c e l l u l a r p o l y s a c c h a r i d e s during t h e i r growth under s u i t a b l e c u l t u r a l c o n d i t i o n s . C u r r e n t l y , however, commer c i a l production o f the m i c r o b i a l p o l y s a c c h a r i d e s are l i m i t e d to dextran, produced by Leuconostoc mescenteoides, s c l e r o g l u c a n (polytran) by Sclerotium species and the r e l a t e d f u n g i , xanthan by Xanthomonas campestrics (1), and p u l l u l a n by Aureobasidium pullulans (2). A few other p o l y s a c c h a r i d e s , such as 3-(l+3)D-glucan, l i k e curdlan produced by a mutant of Alcaligenes faecalis (S), appear to be under commercial e x p l o i t a t i o n . These p o l y s a c c h a r i d e s possess unique p r o p e r t i e s , u s e f u l i n food and pharmaceutical i n d u s t r i e s , as plasma expanders, food a d d i t i v e s , e d i b l e packing f i l m s and other a p p l i c a t i o n s . Apart from above i n d u s t r i a l u t i l i z a t i o n , the water i n s o l u b l e , and adherent a-(1+3)-linked D-glucans, elaborated by c a r i o g e n i c Streptococcus mutans, have r e c e n t l y a t t r a c t e d much a t e n t i o n , i n r e l a t i o n to the r o l e i n the formation o f dental c a r i e s (4). In the course of searches o f new m i c r o b i a l p o l y s a c c h a r i d e s having unique s t r u c t u r e s and u s e f u l p r o p e r t i e s , we became aware that a fungus i s o l a t e d from a spot o f the white scab o f the tea leaves, i d e n t i f i e d as Elsinoe leucospila (5), produces a mucosus l a y e r , when grown on sucrose-potato e x t r a c t agar. The chemical i n v e s t i g a t i o n s o f t h i s p o l y s a c c h a r i d e i n d i c a t e d i t to be a new type o f α-D-glucan c o n t a i n i n g both (1+4)- and (1+3)D - g l u c o s i d i c l i n k a g e s . The glucan was designated as El S i n a n , s i n c e other f u n g i belonging to Elsinoe species were found to produce s i m i l a r glucans. In t h i s paper, we d e s c r i b e the production, d e t a i l e d s t r u c t u r e , enzymatic degradation and i s o l a t i o n o f novel o l i g o saccharides, and a l s o d e r i v a t i z a t i o n , such as 3,6-anhydroelsinan. Some o f r h e o l o g i c a l p r o p e r t i e s and p o t e n t i a l u t i l i z a t i o n of t h i s glucan w i l l a l s o be d i s c u s s e d . 0-8412-0555-8/80/47-126-197$06.00/0 © 1980 A m e r i c a n C h e m i c a l Society

Sandford and Matsuda; Fungal Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

FUNGAL POLYSACCHARIDES

198


Production o f E l s i n a n In a p r e l i m i n a r y study, the c o n i d i a o f E. leucospila s t r a i n CS-1 were suspended i n s t e r i l i z e d water and i n o c u l a t e d on a cellophane sheet which covers the agar-plate c o n t a i n i n g 2% sucrose and p o t a t o - e x t r a c t . A f t e r growing at 24° f o r 4 - 5 days, the slimy c o l o n i e s on the cellophane sheet were c a r e f u l l y c o l l e c t e d , suspended i n water, c e n t r i f u g e d , and the polysacchar i d e i n the supernatant was p r e c i p i t a t e d by a d d i t i o n o f 3 volumes o f e t h a n o l . A f t e r p u r i f i c a t i o n by d i a l y s i s followed by repeated p r e c i p i t a t i o n s with ethanol, the p o l y s a c c h a r i d e was shown to comprise s o l e l y D-glucose. The glucan d i d not give any c o l o r when reacted with i o d i n e , although s t r u c t u r a l analyses showed the presence of a high p r o p o r t i o n o f a-(l->-4)D - g l u c o s i d i c l i n k a g e s . These f i n d i n g s prompted us to i n v e s t i g a t e the d e t a i l e d s t r u c t u r e and p r o p e r t i e s o f the p o l y s a c c h a r i d e . For the production o f e l s i n a n by submerged c u l t u r e , the fungus was grown i n a medium c o n t a i n i n g 5% sucrose, e i t h e r p o t a t o - e x t r a c t ( d i a l y z a t e o f hot water e x t r a c t o f f r e s h potato (300 g per 1 l i t e r medium)) o r 0.5% corn steep l i q u o r (CSL) p l u s 0.2% NaN0 , 0.1% K HP0 , 0.05% MgS0 , 0.05% KC1. The fermentation was c a r r i e d out i n shaking f l a s k s i n a small f e r mentor (10 l i t e r volume) with a e r a t i o n (equal volume to that of medium per min), at 24° f o r 4 - 6 days. The time course o f the production o f e l s i n a n by the submerged c u l t u r e i s shown i n Figure 1. The glucan formation reaches the maximum a f t e r 5 - 6 days with r a p i d consumption of sucrose. The reducing sugars r e l e a s e d during the fermentation contained both glucose and f r u c t o s e at the e a r l y stage, and are g r a d u a l l y u t i l i z e d . The viscous c u l t u r a l b r o t h was c l a r i f i e d by c e n t r i f u g a t i o n and the crude p o l y s a c c h a r i d e was prec i p i t a t e d from the supernatant by a d d i t i o n o f 3 volumes o f ethanol or acetone. I t was d i s s o l v e d i n water, d i a l y z e d and p r e c i p i t a t e d again with ethanol, and then l y o p h i l i z e d ; y i e l d 23 - 25 g per l i t e r o f broth. Figure 2 shows the e f f e c t o f v a r i o u s carbon-sources (each 5%) on the production o f e l s i n a n , when CSL and NaNU3 were used as n i t r o g e n source. Among the d i f f e r e n t carbohydrates or p o l y hydroxy a l c o h o l s t r i e d , sucrose and f r u c t o s e seem to be most e f f e c t i v e l y u t i l i z e d f o r production o f e l s i n a n (2.6 and 2.3 g, r e s p e c t i v e l y , from 100 ml medium). Glucose was the best carbon source f o r growth o f the fungus, but the y i e l d o f the glucan was l e s s than from sucrose. Galactose and g l u c i t o l were very poor carbon sources. 3

Chemical

2

4

4

P r o p e r t i e s o f E l s i n a n (6)

The p o l y s a c c h a r i d e p u r i f i e d from the c u l t u r a l f i l t r a t e i s e s s e n t i a l l y f r e e from n i t r o g e n compound (N < 0.1%), and i s composed s o l e l y o f D-glucose, as revealed by paper- and



MisAKi A N D TSUMURAYA

Ehinan:

Structure

b- Degradation

199

Culture time(day)

Figure 1. Time course of production of elsinan. Five liter medium containing sucrose was fermented with airation, 5—6.5 L/min and agitation, 300 rpm, at 24°C: (Φ) elsinan; (O) dry cell; ( — A - - ) sucrose; ( — X — ) pH; ( - ·) reducing sugar. m

Production of elsinan(g/100 ml) 0

1

2

none sucrose

ethylene glycol sodium acetate

Figure 2.

Effects of carbon sources on the production of elsinan


200


g a s - l i q u i d chromatography ( g . l . c . ) , a f t e r complete hydro l y s i s by heating with M - s u l f u r i c a c i d f o r 5 h. The homogeneity o f e l s i n a n was assessed by u l t r a c e n t r i f u g a l analysis ( S 5.92 X 1 0 ~ ) . The molecular 1 3

2 Q

weight of the n a t i v e glucan was i n a range o f 2 - 6 X 10^, as estimated by gel e x c l u s i o n chromatography on a Sepharose column. The high s p e c i f i c r o t a t i o n , [ a ] + 243° (c,0.8, water) and + 2 3 9 ° (c,0.8, M NaOH), and the c h a r a c t e r i s t i c absorbance at 840 cm i n i . r . spectrum i n d i c a t e that the D - g l u c o s i d i c linkages are o f aconfiguration. The glucan i s r e a d i l y s o l u b l e i n warm water to give a h i g h l y v i s c o u s s o l u t i o n , having an i n t r i n s i c v i s c o s i t y , [η] 1.86 at 25°. Although i t s aqueous s o l u t i o n s are s t a b l e at low c o n c e n t r a t i o n s , they tend to form g e l s at higher c o n c e n t r a t i o n . Some v i s c o s i t y p r o p e r t i e s w i l l be discussed l a t e r .


D

Structure of Elsinan

(6)

Mode o f D - g l u c o s i d i c linkages For the l i n k a g e a n a l y s i s , the glucan synthesized from sucrose was methylated by the method o f Hakomori (7), and the methylated glucan was hydrolyzed with a c i d . The p a r t i a l l y methylated sugars were analyzed by g.l.c-m.s. at 180° u s i n g a ECNSS-M column, a f t e r conversion i n t o t h e i r corre sponding a l d i t o l a c e t a t e s . The r e s u l t showed the presence o f 2,3,6-tri-(70.8%) and 2,4,6-tri-O-methyl-D-glucose (28%), together with small p r o p o r t i o n s o f 2,3,4,6-tetra-(0.7%) and 2,4-di-0-methylD-glucose (0.5). Although the r e t e n t i o n times o f 2,3,6- and 2,3,4-tri-O-methyl-D-glucose were very c l o s e to each other, under the c o n d i t i o n s employed, the 2,3,6-tri-0-methylglucose was i d e n t i f i e d as the methyl g l u c o s i d e by g . l . c . A l l the glucan preparations elaborated from d i f f e r e n t carbon sources were found by methylation a n a l y s i s to have e s s e n t i a l l y the same s t r u c t u r e s (Table I ) . The glucan produced from a medium c o n t a i n i n g glucose appears to have a s l i g h t l y higher con tent o f (1+4)-linkages than the p o l y s a c c h a r i d e s from other carbo hydrate sources. However, there may be no e s s e n t i a l s t r u c t u r a l d i f f e r e n c e between the glucans prepared from v a r i o u s sources. These g l u c o s i d i c linkages assigned were a l s o supported by the r e s u l t s o f p e r i o d a t e o x i d a t i o n and Smith degradation. E l s i n a n was o x i d i z e d with 0.03 M sodium periodate at 4°, and a f t e r complete o x i d a t i o n (5 days; p e r i o d a t e consumption, 0.80mole, and formic a c i d production, 0.07 mole per glucose r e s i d u e ) , the o x i d i z e d glucan was reduced with sodium borohydride. Erythritol, glucose and a t r a c e o f g l y c e r o l i n the h y d r o l y s i s product were q u a n t i t a t i v e l y analyzed by high performance l i q u i d chromatography ( h . p . l . c ) , and a l s o by g . l . c , a f t e r r e d u c t i o n with borohydride followed by a c e t y l a t i o n (Figure 3). T h e i r molar p r o p o r t i o n s were


Sandford and Matsuda; Fungal Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1980. 9

PRODUCED FROM VARIOUS

0.7 27.4

0-methyl-D-glucose(%) 0.4 2,3,4,6-tetra27.9 71.7 (trace)(trace)

2,4,6-tri-

2,3,6-tri-

2,4-di-

1.6 21.4 76.1 0.9

27.3 72.1 0.2

1.5

0.4

1.6

72.0

71.6

(trace) (trace) (trace)

72.0

27.6 28.0

27.6

0.4

0.9 0.4

1.0

0.4

1.4

the carbon source and 0.5% o f corn s t e e p - l i q u o r . Carbohyd. R e s . , 66 (1978) 53.

E l s i n a n was produced by shaking c u l t u r e i n a medium c o n t a i n i n g 5% of

71.9

2.3

sucrose f r u c t o s e mannose glucose maltose x y l o s e mannitol

Carbon source

2.6

ml of broth)

Yield of elsinan (g dry weight/100

CARBON SOURCES

YIELDS AND METHYLATION ANALYSES OF ELSINAN

TABLE I


202


(B)


(A)

4

8 12 Time(min)

20

*0

TV 60

Time(min)

Carbohydrate Research Figure 3. Identification of the products of complete Smith degradation of elsinan. (A) analyzed with a Yanaco liquid chromatograph Model L-1030 fitted with a refractive-index indicator, on a column of SCX-1001 (6 X 500 mm), with water as carrier, at 25°C; (B) analyzed by gas-liquid chromatography, on a column of 3% of ECNSS-M, programed from 100°-190°C (6°C/min): I, glucose; II, erythritol; III, glycerol (6).

Figure 4.

Smith degradation of elsinan


11.

MISAKI AND TSUMURAYA

Elsinan:

Structure

ir

Degradation

203


68.4%,

3 0 . 1 % and 1 . 5 % , r e s p e c t i v e l y , as estimated by g . l . c . The e r y t h r i t o l and glucose should have a r i s e n from ( 1 + 4 ) - and ( 1 + 3 ) l i n k e d D-glucose r e s i d u e s , and the t r a c e o f g l y c e r o l from the non-reducing terminal ends o f the s l i g h t l y branched glucan. Thus, both methylation and p e r i o d a t e o x i d a t i o n s t u d i e s c l e a r l y i n d i c a t e that e l s i n a n i s an e s s e n t i a l l y l i n e a r molecule c o n s i s t ing mainly o f a - ( 1 + 4 ) - and ( 1 + 3 ) - D - g l u c o s i d i c linkages i n a molar ratio of 2 . 3 - 2 . 5 : 1 . 0 . The presence o f very small proportions of 2 , 4 - and 2 , 3 , 4 , 6 - t e t r a - O - m e t h y l - D - g l u c o s e may be due to a l i m i t e d p r o p o r t i o n o f branching at 0 - 6 p o s i t i o n s o f the ( 1 + 3 ) l i n k e d D-glucose r e s i d u e s . There may be one branching p o i n t per ~ 1 4 0 sugar r e s i d u e s .

Sequence o f the D - g l u c o s i d i c linkages Since e l s i n a n contains both ( 1 + 3 ) - and ( 1 + 4 ) - l i n k a g e s , the glucan was subjected to m i l d Smith degradation. Paper and gas l i q u i d chromatography (as t r i m e t h y l s i l y l d e r i v a t i v e s ) showed the presence o f e r y t h r i t o l and 2 - 0 - a - D - g l u c o s y l - D - e r y t h r i t o l and t r a c e o f g l y c e r o l (Figures 3 and 4 ) . T h e i r molar r a t i o was 1 . 4 4 : 1 . 0 0 : 0 . 0 4 , as estimated by g . l . c I t i s evident that the e r y t h r i t o l a r i s e s from cons e c u t i v e a - ( 1 + 4 ) - l i n k e d D-glucose r e s i d u e s , such as + 4 ) - G l c - ( l + 4 ) G l c - ( 1 + , whereas 2 - 0 - a - D - g l u c o s y l - D - e r y t h r i t o l must a r i s e from a s i n g l e , a - ( 1 + 3 ) - l i n k e d D-glucose r e s i d u e flanked by ( 1 + 4 ) linkages. When the mild hydrolyzate from the glucan-polyalcohol was a p p l i e d to a column o f B i o - g e l P - 2 , no a p p r e c i a b l e peak corresponding to a p o l y s a c c h a r i d e o r o l i g o s a c c h a r i d e emerged. This r e s u l t suggests the e s s e n t i a l absence o f consecutive a - ( 1 + 3 ) l i n k e d D-glucose r e s i d u e s .

Fragmentation o f e l s i n a n by p a r t i a l a c i d h y d r o l y s i s For examination o f d e t a i l e d s t r u c t u r a l f e a t u r e s , e l s i n a n was subjected t o p a r t i a l , a c i d h y d r o l y s i s with 0 . 5 M s u l f u r i c a c i d f o r 4 h at 8 5 ° . The degradation products were f r a c t i o n a t e d by a Charcoal column, followed by p r e p a r a t i v e , paper chromatography. Table II shows the r e s u l t s o f the f r a c t i o n a t i o n . Disaccharide Two d i s a c c h a r i d e s were detected by paper chromatography. By methylation a n a l y s i s , they were i d e n t i f i e d as nigerose and maltose, r e s p e c t i v e l y . Trisaccharide Two t r i s a c c h a r i d e components were separated by paper chromatography. One component ( M 1 5 2 ° ) was i d e n t i f i e d as m a l t o t r i o s e . The other t r i s a c c h a r i d e components ( t r i s a c c h a r i d e A ) , which gave a s i n g l e spot on a paper chromatogram ( R 0 . 5 3 ; butanol-pyridine-water, 6 : 4 : 3 ) were f u r t h e r f r a c t ionated by p r e p a r a t i v e l i q u i d chromatography with borate b u f f e r , according t o T o r i i et_ al_. ( 8 ) . T h i s procedure gave two d i s t i n c t components, t r i s a c c h a r i d e A - l (MQI 0 . 2 8 ) and A - 2 ( M Q 0 . 6 0 ) . +

n

G

C

1 C



II 3

59 22

4 , 7 , and 9%(2 l i t e r s )

9(liters),

13(2 l i t e r s )

15%(2 l i t e r s )

20%

25%

warm 50%

2

3

4

5

6

7

8

90

28

55

208

higher o l i g o s a c c h a r i d e s

p e n t a - , h e x a - , and hepta-saccharides

t e t r a - and penta-saccharides

tetrasaccharides

t r i - and t e t r a - s a c c h a r i d e s

t r i saccharides

d i saccharides

glucose

Components

e l u t i o n by 4 - l i t e r p o r t i o n s o f aqueous e t h a n o l . Carbohydrate Research

O l i g o s a c c h a r i d e s were separated by a charcoal column ( 4 . 2 X 57 cm) w i t h stepwise

and 1 5 ï ( 2 l i t e r s )

1 1 , and 13%(2 l i t e r s )

1160

water

1 400

Weight (mgi

F r a c t i o n Eluent(% ethanol)

YIELDS OF OLIGOSACCHARIDE FRACTIONS FROM PARTIAL ACID HYDROLYSIS OF ELSINAN

TABLE


11.

MisAKi AND TSUMURAYA

Elsinan:

Structure

&

205

Degradation

From the r e s u l t s o f methylation a n a l y s i s and the a c t i o n o f yeast α-D-glucosidase, t r i s a c c h a r i d e A - l was i d e n t i f i e d as O-a-Dg l u c o s y l -(1+3) -O-a-D-glucosyl- (1+4) -D-glucose ( I ) . T r i s a c c h a r i d e A-2 was c h a r a c t e r i z e d i n a s i m i l a r manner, as O-a-D-glucosyl(1+4)-O-a-D-glucosyl-(1+3)-glucose (II) . Tetrasaccharide At l e a s t , two t e t r a s a c c h a r i d e s were de t e c t e d by paper chromatography, one having R 0.30 and the other Rg which corresponds to maltotetraose. They were separated from each other on a f i l t e r - p a p e r sheet. The f i r s t component (R 0.30), which c o n s i s t s o f (1+3)- and (l+4)-D-glucosidic linkages i n the r a t i o o f 1 : 2, was found by paper e l e c t r o p h o r e s i s to be a mixture o f two t e t r a s a c c h a r i d e s . One ( M 0.22) was i d e n t i c a l t o O-a-Dglucosyl-(1+4)-O-a-D-glucosyl-(1+3)-O-a-D-glucosyl-(1+4)-Dglucose ( I I I ) , which has been i s o l a t e d from a d i g e s t o f e l s i n a n with human s a l i v a r y α-amylase. The other component (Mçi 0.51) may be e i t h e r O-a-D-glucosyl-(1+4)-O-a-D-glucosyl-(1+4)-O-a-Dg l u c o s y l - (1+3)-D-glucose o r O-a-D-glucosyl-(1+3)-O-a-D-glucosyl(1+4)-O-a-D-glucosyl-(1+4)-D-glucose. The other t e t r a s a c c h a r i d e component (Rg 0.26) had the same Rg value as that o f maltotetraose, which was i d e n t i f i e d by methyl a t i o n and the a c t i o n o f beta amylase. g


g

G l c

c

Acetolysis A c e t o l y s i s o f e l s i n a n was performed according to the method o f Matsuda et_ a l . (9). The glucan (3 g) was added to a mixture o f a c e t i c anhydride (14.4 ml), a c e t i c a c i d (9.6 ml), and s u l f u r i c a c i d (1.8 ml) at 25°. A f t e r s t i r r i n g f o r 7 days at 25°, the a c e t o l y s i s product was obtained by pouring i n t o i c e d water, and then e x t r a c t i o n with chloroform ( y i e l d , 4.5 g). The syrup was deacetylated with sodium methoxide i n methanol ( y i e l d 2.1 g ) . The mixture o f o l i g o s a c c h a r i d e s was f r a c t i o n a t e d on a charcoal column followed by paper chromâtοtraphy. Among v a r i o u s o l i g o s a c c h a r i d e s , nigerose and maltose were i d e n t i f i e d as d i s a c c h a r i d e s . The t r i s a c c h a r i d e f r a c t i o n con t a i n e d at l e a s t two components, m a l t o t r i o s e , O-a-D-glucosyl-(1+3)O-a-D-glucosyl-(1+4)-D-glucose, and O-a-D-glucosyl-(1+4)-O-aD-glucosyl- (1+3)-D-glucose. The t e t r a s a c c h a r i d e f r a c t i o n appeared to be a mixture o f maltotetraose and a t e t r a s a c c h a r i d e composed o f one a-(1+3)- and two a-(1+4)-linked D-glucose r e s i dues. Thus, the methylation and fragmentation data e s t a b l i s h that e l s i n a n c o n s i s t s o f three consecutive a-(1+4)-linked Dglucose r e s i d u e s , joined by a-(1+3)-linkages. However, the r a t i o of (1+4)- to (1+3)-linkages i s 2.3 - 2.5 :1, suggesting the presence o f a monor p r o p o r t i o n o f maltotetraose residues i n the glucan molecule. T h i s s u p p o s i t i o n was confirmed by the i s o l a t i o n of maltotetraose i n the p a r t i a l , a c i d h y d r o l y z a t e . A c e t o l y s i s o f e l s i n a n a l s o gave o l i g o s a c c h a r i d e products s i m i l a r to those o b t a i n ed from the p a r t i a l , a c i d h y d r o l y s i s . The proposed s t r u c t u r e o f e l s i n a n , and the formation o f o l i g o s a c c h a r i d e s by p a r t i a l , a c i d h y d r o l y s i s i s i l l u s t r a t e d i n Figure 5.




V ',' 1 ' '! Ί ι——il ι — — ι ll

ι VI

I

V

I III

VII Carbohydrate Research Figure 5. Repeating unit of elsinan: I, maltose; II, nigerose; III, maltotriose; IV, 3-O-a-O-glucosylmaltose; V, 4-O-a-O-glycosylnigerose; VI, maltotetraose (6).



11.


Elsinan:

Structure

b-

Degradation

207

E l s i n a n has some p o i n t s o f s t r u c t u r a l resemblance with p u l l u l a n , an e x t r a c e l l u l a r α-D-glucan o f a black yeast, Audiobasidium pullulans (2), and a l s o with nigeran, the α-glucan o f Aspergillus niger (10). P u l l u l a n i s a water-soluble, l i n e a r polymer composed of m a l t o t r i o s e j o i n e d by a-(1+6)-linkages, instead o f the (1+3)linkages found i n e l s i n a n . Furthermore, the s t r u c t u r e o f p u l l u l a n , which contains about 6% o f maltotetraose (11), i s again r e miniscent o f the presence o f maltotetraose i n e l s i n a n , although f u r t h e r s t r u c t u r a l a n a l y s i s would be necessary t o determine the exact r a t i o o f maltotetraose to m a l t o t r i o s e residues i n e l s i n a n . Nigeran, obtained by hot-water e x t r a c t i o n from the mycelia o f Aspergillus niger, contains both a-(1+3)- and (1+4)-D-glucosidic linkages, i n an a l t e r n a t i n g sequence. I n t e r e s t i n g l y , the s o l u b i l i t y o f e l s i n a n d i f f e r s from that o f nigeran. I t may be noted that lichenan, a water-soluble 3-D-glucan extracted from Iceland moss (Cetraria islandica), i s a l i n e a r polysaccharide having almost the same s t r u c t u r a l sequences as e l s i n a n , except f o r the opposite anomeric c o n f i g u r a t i o n s , namely c e l l o t r i o s e and c e l l o t e t r a o s e residues j o i n e d by (1+3)-3-D-glucosidic linkages. In r e l a t i o n to chemical taxonomy, other plant pathogenic fun g i belonging to the Elsinoe species, such as Ε. fawetti, responsi b l e f o r c i t r u s scab, have a l s o been found to produce e x t r a c e l l u l a r α-D-glucans s i m i l a r t o the e l s i n a n o f E. leucospila.

Enzymatic Degadation o f E l s i n a n (12) Since e l s i n a n has a unique s t r u c t u r a l feature, i t s d i g e s t a b i l i t y and also methanism o f the enzymatic degradation have a t t r a cted our a t t e n t i o n . In the searches o f enzymes capable o f degrad ing t h i s glucan, we became aware o f s u s c e p t i b i l i t y o f e l s i n a n to p a r t i c u l a r type o f α-amylase, such as s a l i v a r y and pancreas amylase. T h i s prompted us to examine various a m y l o l y t i c enzymes, and i s o l a t i o n o f the degradation products. Enzymes S a l i v a r y a-amylase was p u r i f i e d from the human s a l i v a o f adult males by ammonium s u l f a t e p r e c i p i t a t i o n (25 - 80% s a t u r a t i o n ) , followed by g e l - f i l t r a t i o n on Sephadex G-200 column, and r e p r e c i p i t a t i o n with ammonium s u l f a t e (0-45% s a t u r a t i o n ) . The enzyme was d i s s o l v e d i n 0.05 M phosphate b u f f e r , pH 6.8, c o n t a i n ing 0.05 M sodium c h l o r i d e . Other enzymes used were hog pancreas α-amylase (type I-A; Sigma Co.), Bacillus subtilis α-amylase ( l i q u e f y i n g type), Aspergillus oryzae α-amylase (Take amylase, p u r i f i e d ) , Termamyl amylase (Novo Ind. Co.), isoamylase o f Pseudomonas amylodermosa, Pullulanase o f Aerobacter aerogenes, Bacillus subtilis α-amylase ( s a c c h a r i f y i n g type, type II-A, Sigma Co.), glucoamylase o f Rhizopus niveous and sweet potato 3-amylase.



III

y

m e

0 8

dramylase ( B a c i l l u s s u b t i l i s , Siqma)

tf-amylase 0 0 0 0

glucoamylase (Rhizopus n i v e u s , Seikaqaku Κ ο α ν ο )

f - a m y l a s e (Sweet p o t a t o , Sigma)

isoamylase (Pseudomonas amyloderamosa, Hayashibara)

p u l l u l a n a s e (Aerobacter aeroaenes,

Hayashibara)

0

d-amylase (Termamyl, Novo)

( A s p e r g i l l u s o r y z a e , Sankvo)

0

35

(t-amylase ( B a c i l l u s s u b t i l i s . l i a u e f v i n a . Seikagaku Kogyo)

43

33

de-amylase (porcine pancreas, Sigma)

Ct-amylase ( B a c i l l u s s u b t i l i s . s a c c h a r i f v i n a . Seikagaku Kogyo)

4

4

30

98

17

34

39

37

67

44

0

0

0

0

0

4

0

0

66

5.5

4

Hvdro1vsis(%) Velocity elsinan soluble (soluble starch starch =100) 29

(ji amylase ( s a l i v a )

E n z

ACTION OF SEVERAL ENZYME ON ELSINAN

TABLE


11.


Elsinan:

Structure

b-

Degradation

209


A c t i v i t y o f s a l i v a r y α-amylase was measured by incubation o f the enzyme with 0.5% s o l u b l e s t a r c h i n 0.05 M phosphate b u f f e r (0.2 ml), pH 6.8, containing 0.005 M sodium c h l o r i d e at 37° f o r 20 min, and the reducing sugars were measured by the method o f Nelson-Somogyi. One u n i t was defined as the amount o f enzyme which produces one ymole o f reducing group (as glucose) per min. A c t i v i t i e s of other enzymes were assayed by s i m i l a r methods. A c t i o n of Starch-degrading Enzymes S u s c e p t i b i l i t i e s of e l s i n a n toward several a m y l o l y t i c enzymes were examined, and the degrees o f h y d r o l y s i s at the f i n a l stages and the i n i t i a l v e l o c i t i e s o f h y d r o l y s i s o f e l s i n a n and of s o l u b l e s t a r c h are compared (Table I I I ) . It i s apparent that e l s i n a n i s degraded by some aamylase, e_.g^*, s a l i v a r y , pancreas, B. subtilis (saccharifying), and Taka amylase, but not with other enzymes t e s t e d here. Among these enzymes, b a c t e r i a l s a c c h a r i f y i n g amylase appears to be most e f f e c t i v e i n terms o f both i n i t i a l h y d r o l y s i s v e l o c i t y and the degree o f h y d r o l y s i s (35% estimated as glucose). Nevertheless, the apparent h y d r o l y s i s r a t e o f e l s i n a n by t h i s enzyme i s one h a l f toward the s o l u b l e s t a r c h . The d i s t r i b u t i o n p a t t e r n o f the enzymatic d i g e s t i o n products a f t e r 24 h incubation with several α-amylases are compared i n Table IV. These o l i g o s a c c h a r i d e s were analyzed by h.p.I.e. It must be noted that the main products by the a c t i o n s of s a l i v a r y , hog pan creas and b a c t e r i a l s a c c h a r i f y i n g α-amylase appear to be i d e n t i cal to each other. It was c h a r a c t e r i z e d as 4-0-a-nigerosyl-Dglucose. The mode o f a c t i o n with Taka amylase seems to d i f f e r from those with s a l i v a r y and pancreas amylases. Degradation o f E l s i n a with Human S a l i v a r y Amylase When the p a r t i a l l y p u r i f i e d s a l i v a r y amylase was acted on e l s i n a n , i t was g r a d u a l l y hydrolyzed as shown i n Figure 6. The h y d r o l y s i s of s o l u b l e starch reachs the maximum a f t e r 5 h, with apparent hydro l y s i s o f 44%(as glucose), while e l s i n a n was more slowly hydroly zed (apparent h y d r o l y s i s , 29%, a f t e r 25 h ) . Km value and V of s a l i v a r y amylase with e l s i n a n were 6.9 Χ 10~ M (glucose u n i t ) and 0.12 umole/min. u n i t enzyme, r e s p e c t i v e l y . Under the same c o n d i t i o n , Km 6.8 Χ Ι Ο " M and V 0.96 ymole were obtained f o r soluble starch. The enzyme d i g e s t from e l s i n a n was shown by paper chromato graphy to contain glucose, d i s a c c h a r i d e , t r i s a c c h a r i d e and tetrasaccharide. Figure 7 shows the p r o f i l e of h . p . l . c . of these oligosaccharides. For c h a r a c t e r i z a t i o n o f major oligosaccharide's, the enzyme d i g e s t was f r a c t i o n a t e d using a charcoal column, and each sugar f r a c t i o n eluted with aqueous ethanol (0 to 25%) was p u r i f i e d by p r e p a r a t i v e paper chromatography (Whatman 3 MM, butanol-pyridinewater, 6:4:3). 2

1 3

m a x


210


TABLE IV


REACTION PRODUCT OF ELSINAN WITH SEVERAL QC-AMYLASE Enzyme Saliva

H y d r o l y s i s Reaction product(%) G G G (*) 4 5 29 12.0 0.6 75.3 12.1 0

Porcine pancreas

33

Bacillus subtilis (saccharifying)

35

1

A s p e r g i l l u s oryzae

8

2

3

12.4 0.4 85.2 7.5 0 0

0

92.5 0

G

G

6 0

7 0

G

G

2.0 0

0

0

0

0

0

0

56.6 0

0

43.4


11.


Elsinan:

Structure

&

The d i s a c c h a r i d e f r a c t i o n ([a]rj + 130°) was i d e n t i f i e d as maltose. The t r i s a c c h a r i d e ( [ a ] + 164°, R 0.9 and M 0.25) was shown by methylation a n a l y s i s t o c o n t a i n equal p a r t s o f (1+3)and (1+4)- D - g l u c o s i d i c l i n k a g e s . Since the methylation a n a l y s i s of the borohydride-reduced t r i s a c c h a r i d e i n d i c a t e d that the redu c i n g terminal r e s i d u e i s j o i n e d by (1+4)-linkage, t h i s t r i s a c c h a r i d e was c h a r a c t e r i z e d as O-a-D-glucosyl-(1+3)-Ο-α-D-glucosyl(1+4)-D-glucose [4-0-a-nigerosyl-D-glucose] (see, formula I ) . The t e t r a s a c c h a r i d e showed [ a ] + 184°, R 0.28 and M 0.23. Although chromatographic m i g r a t i o n was c l o s e to that o f maltote traose, i t c o n s i s t s o f one mole o f (1+3)- and two moles o f (1"M)D - g l u c o s i d i c linkages per non-reducing terminal r e s i d u e . Reduc t i o n followed by methylation i n d i c a t e d that the reducing terminal i s j o i n e d by (1+4)-bond. By the a c t i o n o f α-D-glucosidase, g l u cose and 4-0-a-nitrosyl-D-glucose were produced. Thus, the t e t r a s a c c h a r i d e was c h a r a c t e r i z e d as O-a-D-glucosyl-(1+4)-O-a-Dg l u c o s y l -(1+3)-O-a-D-glucosyl-(1+4)-D-glucose (3 - m a l t o s y l maltose) (see, formula I I I ) . From the i s o l a t i o n o f 4-0-a-nigerosyl-D-glucose (74.3%) and 3-maltosyl-maltose (12.1%), s a l i v a r y amylase may attack, p r e f e r e n t i a l l y , the g l u c o s i d i c bonds between the a-(1+4)-linked sugar r e s i d u e s , as i n d i c a t e d i n Figure 8. The formation o f 3-maltosylmaltose can be explained by the clevage o f a-(1+4)-D-glucosidic linkages i n the maltotetraose u n i t s . The glucose and maltose, i n a d d i t i o n t o the above o l i g o s a c c h a r i d e s might be a t t r i b u t e d t o f u r t h e r h y d r o l y s i s o f the higher saccharides. The a c t i o n p a t t e r n o f s a l i v a r y , hog pancreas and B. Subtilis ( s a c c h a r i f y i n g ) enzyme seem t o be very s i m i l a r i n respect t o t h e i r i n i t i a l v e l o c i t i e s , the extents o f h y d r o l y s i s and the o l i g o s a c c h a r i d e s formed by the enzymatic a c t i o n s . D

g

G l c

D


211

Degradation

g

G l c

A c t i o n o f Taka Amylase Taka amylase has a lower a c t i v i t y on e l s i n a n , compared with other mamalian and b a c t e r i a l aamylases, and forms t e t r a - and heptasaccharide, suggesting that the enzyme possesses d i f f e r e n t s p e c i f i c i t y from other amylases toward e l s i n a n , probably, i t r e q u i r e s c e r t a i n length o f the con s e c u t i v e a- (1+4)-linked D-glucose r e s i d u e s . The Taka amylase d i gest o f e l s i n a n (2 g) was c e n t r i f u g e d , and the higher saccharides i n the §upernatant was p r e c i p i t a t e d with ethanol (800 mg), l e a v i n g a mixture o f o l i g o s a c c h a r i d e s ( 9 7 6 mg) i n the solution.The o l i g o saccharide mixture was f r a c t i o n a t e d by paper chromatography, which gave t e t r a s a c c h a r i d e and heptasaccharide f r a c t i o n s . Tetrasaccharide; [ a ] + 185° (water), R 0.34 and M G I 0.25 The t e t r a s a c c h a r i d e was shown by methylation a n a l y s i s t o con s i s t o f (1+4)- and (1+3)-D-glucosidic linkages i n the r a t i o o f 2:1, and a f t e r borohydride-reduction the r a t i o was changed t o 1:1, i n d i c a t i n g that the reducing terminal i s j o i n e d by (1+4)l i n k a g e s . T h i s t e t r a s a c c h a r i d e y i e l d e d on p a r t i a l , a c i d hydroly s i s , m a l t o t r i o s e and 4-0-a-nigerosyl-D-glucose. In a d d i t i o n , the D

g


c



212

4

Figure 7. High performance liquid chromatography of reaction product of elsinan with human salivary a-amylase.

1

10

Τ 3Gv-*Gif^Gi Journal of Applied Biochemistry

OH

Tetrasacchari de

8

Journal of Applied Biochemistry

Figure 8. Action pattern of human salivary α-amylase on elsinan: (^) repre sents the sites cleaved by the enzyme; G, α-Ό-glucopyranosyl residue

OH

6 Time (min)

,L

-

OH

OH

1 Heptasacchari de

Figure 9. Possible action pattern of Taka amylase, and release of tetra- and heptasaccharide: site cleaved by Taka amylase; (O), α-Ό-glucopyranosyl unit; ( ), (1 -» 4)-O-glucosidic linkage; (f), (1 -» 3)-O-glucosidic linkage.


11.


Elsinan:

Structure

6·

Degradation

213

t e t r a s a c c h a r i d e was found to be r e s i s t a n t to a-D-glucosidase. Therefore, i t was c h a r a c t e r i z e d as O-a-D-glucosyl-(1+3)-O-a-Dg l u c o s y l - (1+4)-O-a-D-glucosyl-(1+4)-D-glucose (see, formula I V ) . Heptasaccharide; Rg 0.11 and M G I 0.23. A c i d h y d r o l y s i s of the methylated heptasaccharide gave 2,3,4,6-tetra-, 2 , 4 , 6 - t r i - and 2,3,6-tri-0-methyl-D-glucose, i n the molar r a t i o o f 1 : 2 : 4. The a c t i o n o f s a l i v a r y α-amylase r e s u l t e d i n the formation of 4-0-a-nigerosyl-D-glucose and glucose (approximate r a t i o , 2 : 1 ) . From these f i n d i n g s the hepta saccharide i s most probably made up o f the sequences shown i n Figure 9. The f a c t that the s t r u c t u r e o f the t e r a s a c c h a r i d e r e leased by Taka amylase d i f f e r s from that produced by the a c t i o n of s a l i v a r y amylase, and the heptasaccharide may have the sequen ce shown i n Figure 9, s t r o n g l y suggest that Taka amylase a t t a c k s , s e l e c t i v e l y , a-(1+4)-D-glucosidic l i n k a g e s , adjacent to the terminal end i n the maltotetraose u n i t s which are probably l o c a t e d as short blocks (see, Figure 9 ) . Thus, the v a r i a b i l i t y o f the a c t i o n s o f s e v e r a l α-amylases from d i f f e r e n t o r i g i n s on e l s i n a n i s c o n s i s t e n t with the con v e n t i o n a l c l a s s i f i c a t i o n o f amylases. The enzymes having high a c t i v i t i e s on the r e l a t i v e l y lower maltosaccharides, such as maltotetraose and maltopentaose, are l i k e l y to hydrolyze e l s i n a n . On the other hand, the i n c a p a b i l i t y o f c e r t a i n α-amylases, , the l i q u e f y i n g type amylases, may be due to t h e i r a f f i n i t i e s to higher maltosaccharides. T h i s can be supported by the f a c t t h a t Taka amylase shows a lower a c t i v i t y to y i e l d p a r t i c u l a r t e t r a saccharide and heptasaccharide. As regards the enzymatic degradation o f e l s i n a n , i t may be i n t e r e s t i n g to examine the s u s c e p t i b i l i t y o f t h i s glucan to mycodextranase, which has been known to be s p e c i f i c to nigeran. The i n v e s t i g a t i o n s on the a c t i o n o f d i f f e r e n t enzymes on e l s i n a n should provide more knowledges on the substrate s p e c i f i c i t i e s of these enzymes.


c

P h y s i c a l P r o p e r t i e s of E l s i n a n As described already, e l s i n a n i s a high molecular, l i n e a r polysaccharide c o n s i s t i n g o f the r e g u l a r arrangements o f a-(1+4)and (1+3)-D-glucosidic l i n k a g e s . T h i s glucan was found to have a high v i s c o s i t y , much greater than that o f p u l l u l a n , and to form a strong and a c i d - r e s i s t a n t f i l m . These p r o p e r t i e s may be u s e f u l f o r a p p l i c a t i o n s o f t h i s p o l y s a c c h a r i d e to the f i e l d s o f food and pharmaceutical i n d u s t r i e s . Viscosity E l s i n a n i s r e a d i l y s o l u b l e i n hot water and gives a high v i s c o u s s o l u t i o n , at a low c o n c e n t r a t i o n , approxi mately 10 times than that o f p u l l u l a n . For instance, 3% aqueous




214

e l s i n a n s o l u t i o n gives 100 CP. Figure 10 shows the r e l a t i o n s h i p between concentration and v i s c o s i t y o f aqueous s o l u t i o n , measured at 30°, a f t e r d i s s o l v e d at 80°. Unlike p u l l u l a n , e l s i n a n tends to form a g e l , at 5% or higher concentration. Elsinan solutions are h i g h l y pseudoplastic, and v i s c o s i t y decreases r a p i d l y with i n c r e a s i n g the shear r a t e (Figure 11). With regard to the e f f e c t o f temperature on v i s c o s i t y , when a 2% aqueous s o l u t i o n of e l s i n a n i s dispersed at 30°, and the s o l u t i o n i s g r a d u a l l y heated, 5° per min, a s l i g h t decrease i n the v i s c o s i t y i s seen at the temperature up to 45°, a f t e r which the v i s c o s i t y increased to reach a maximum at 60°, then decreased r a p i d l y with i n c r e a s i n g temperature (Figure 12). However, when the e l s i n a n d i s p e r s i o n i s preheated at 90° f o r 30 min, the v i s c o s i t y i s lower than that without preheating; there i s no v i s c o s i t y peak. These v i s c o s i t y c h a r a c t e r i s t i c s might be due, at l e a s t , to i r r e v e r s i b l e changes i n the inter-molecular a s s o c i a t i o n o f the e l s i n a n chains. The e f f e c t s of pH and e l e c t r o l y t e s on the v i s c o s i t y o f e l sinan are shown i n Figure 13 and 14. The v i s c o s i t y (2% s o l u t i o n ) of e l s i n a n appears not to be a l t e r e d i n a wide range o f pH (3 - 11) . The c o m p a t i b i l i t y with s a l t s i s e x h i b i t e d over a wide range o f s a l t concentration. The non-ionic, n e u t r a l nature of the e l s i n a n molecule may be r e s p o n s i b l e f o r i t s s t a b i l i t y to s a l t s and pH. F i l m Formation E l s i n i n forms strong and r e s i l i e n t f i l m s on evaporation of i t s aqueous s o l u t i o n . Some o f the p r o p e r t i e s o f the f i l m are l i s t e d i n Table V. It was found that, l i k e p u l l ulan f i l m , e l s i n a n f i l m i s impervious to oxygen, s u i t a b l e as coating or packing f i l m o f food. For instance, when o l e i c a c i d was packed i n the e l s i n a n f i l m , there was no c o l o r i n g observed, even a f t e r three months. In another experiment, when f r e s h sardines were coated with e l s i n a n f i l m s and then a i r - d r i e d , no c o l o r a t i o n due to auto-oxidation was observed over four months. Another c h a r a c t e r i s t i c property o f the e l s i n a n f i l m may be r e l a t i v e l y s t a b l e i n a d i l u t e a c i d i c s o l u t i o n (pH 1 - 4 ) , probably due to i t s l i n e a r s t r u c t u r e c o n s i s t i n g of a-(1+4)- and (1+3)-D-gluco-

Chemical M o d i f i c a t i o n o f E l s i n a n

(13)

Recently various chemical m o d i f i c a t i o n s and d e r i v a t i z a t i o n s of polysaccharides have been developed to meet new needs. However, i n t r o d u c t i o n o f 3,6-anhydro-linkages to the polysaccharides appear to be l i m i t e d to 3,6-anhydro-amylose (14). Our i n t e r e s t has been drawn to the i n t r o d u c t i o n o f 3,6-anhydro-linkages i n t o a-(1+4)-linked D-glucose u n i t s of e l s i n a n , whereby some a l t e r a t i o n o f the p h y s i c a l p r o p e r t i e s would be expected.


Ehinan:



4

Structure

i?

Degradation

215

Figure 10. Effect of concentration on viscosity of elsinan dispersed at 100° C, measured with a rotational, shear-type viscometer at 2.5 rpm: cp represents centipoise.

6 Elsinan (%)

1000

Heat

0

5

10

treatment

15

Figure

20

Rotaling rate (rpm)

11.

Pseudoplasticity of elsinan solution

60

20

40

60

Temperature ( ° C )

80

Figure 12. Effect of temperature on vis cosity of elsinan (2% w/w) dispersed at (Φ) 30°C or at (O) 100°C, measured at 20 rpm, programmed from 10° to 80°C (5°/min).




216

Figure 13. Effect of pH on the viscosity of elsinan (2% w/w) dispersed at 100°C, measured at20°C

5

7

PH

40

£20

„

L

Figure 14. Effect of ionic strength (NaCl) on the viscoity of elsinan (2% w/w) dispersed at 100°C, measured at 20° C

10

20

30

NaCl conc.(%)

TABLE V PROPERTIES OF ELSINAN FILM P e r m e a b i l i t y of oxygen

I. 0 ml/m , 24 h, atm

Bending strength

973 times

Tensile strength

950 kg/cm

Hygroscopicity

I I . 1% ( R e l a t i v e humidity 33%)

2

15.2% ( R e l a t i v e humidity 65%) 19.5% ( R e l a t i v e humidity 90%) Transparency

Excellent

H e a t - s e a l i n g property

Excellent

Digestibility

P a r t i a l l degraded by s a l i v a r y and pancreas o(-amylases


40


11.


Elsinan:

Structure

&

Degradation

217

The synthesis o f 3,6-anhydro-elsinan was achieved by a simple method, which i n v o l v e s m i l d s u l f a t i o n p o s s i b l y at 0-6 p o s i t i o n s by treatment with dimethylsulfoxide-S03, and then a l k a l i treatment of the p a r t i a l l y s u l f a t e d glucan with 2 Ν sodium hydro xide at 80°. The d e s u l f a t i o n and simultaneous anhydro-ring forma t i o n were checked by measurement o f the change i n the o p t i c a l r o t a t i o n . Scheme I shows a s e r i e s o f the r e a c t i o n s . Table VI summarizes the p r o p e r t i e s o f the n a t i v e , p a r t i a l l y s u l f a t e d , and 3,6-anhydro- d e r i v a t i v e o f e l s i n a n . The 3,6anhydro-elsinan contained 3,6-anhydro-glucose r e s i d u e s , approxima t e l y a h a l f o f the o r i g i n a l (1+4)-linked glucose r e s i d u e s . The anhydro-elsinan showed a low o p t i c a l r o t a t i o n . In i . r . spectrum the formation o f a new absorption band at 895 cm" was recognized. These r e s u l t s suggest the changes i n the conformation of a-(1+4)l i n k e d glucose r e s i d u e s from C - l to 1-C forms. The i n t r o d u c t i o n of 3,6-anhydroring t o e l s i n a n gives very low v i s c o s i t y , and a l s o the r e s i s t a n c e to the a c t i o n o f s a l i v a r y amylase. The formation o f 3,6-anhydro-glucose r e s i d u e s was confirmed by a n a l y s i s o f the a c i d h y d r o l y s i s products. Paper chromatography as w e l l as g . l . c . revealed the presence o f 3,6-anhydro g l u c o f u r a nose and glucose. I t was found that the g l y c o s i d i c linkages o f 3,6-anhydro glucose r e s i d u e s are s e n s i t i v e to a c i d . For i n s t a n c e , when 3,6-anhydro e l s i n a n was heated with 0.1 Ν s u l f u r i c a c i d at 80° f o r 30 min, c o n s i d e r b l e h y d r o l y s i s occured, with formation of anhydro glucose, 4-0-glucosyl-3,6-anhydro-D-glucose, 4-0maltosyl-3,6-anhydro-D-glucose, O-a-D-glucosyl-(1+3)-O-a-D-gluco s y l - (1+4)-3,6-anhydro-D-glucose e t c . Although our primary purpose to o b t a i n somewhat a g a r - l i k e p h y s i c a l property f a i l e d , b e t t e r c o n t r o l o f the r e a c t i o n con d i t i o n s and d i s t r i b u t i o n o f 3,6-anhydro-linkages, may yet provide a unique d e r i v a t i v e o f e l s i n a n or other p o l y s a c c h a r i d e s . 1

Abstract E l s i n a n i s an e x t r a c e l l u l a r α-D-glucan produced by a s t r a i n of Elsinoe leucospila from sucrose or other carbohydrates i n an aerobic c o n d i t i o n . Methylation s t u d i e s , Smith degradation, p a r t i a l a c i d h y d r o l y s i s and a c e t o l y s i s e s t a b l i s h e d that e l s i n a n i s composed mainly o f m a l t o t r i o s e r e s i d u e s and maltotetraose r e sidues (minor) j o i n e d by α-(1->3)-linkages. E l s i n a n i s partially hydrolyzed by c e r t a i n α-amylases, e.g., s a l i v a r y , pancreas and b a c t e r i a l ( s a c c h a r i f y i n g ) α-amylase to r e l e a s e mainly 4-O-α-nigerosyl-D-glucose. The a c t i o n o f Taka amylase, which hydrolyzes e l s i n a n more slowly, r e s u l t s i n the formation o f a t e t r a s a c c h a r i d e , O-α-D-glucosyl-(1->3)-O-α-D -glucosyl-(1->4)-O-α-D-glucosyl-(1->4)-D-glucose, and a heptasaccha r i d e composed o f (1->4)- and (1->3)-linked D-glucose r e s i d u e s .



CH 0H

CH 0H


2

2

OH Elsinan DMSO-SO

3

3-5 moles/glucose residue CH 0S0 Na 2

CH 0S0 Na 2

3

3

o OH

CH 0S0 Na 2

3

J — 0

Λ- _|(θΗ

)L J_

0

0

OH OH P a r t i a l l y sulfated elsinan

η

OH ' 3,6,-anhydro-elsinan

η

CH 0H 2

OH

Scheme 1.

OH

Synthesis of 3,6-anhydro-ehinan



3 Χ 10

M o l e c u l a r weight 5

2 Χ 10

2 Χ 10

—

0.029

0.052

5

(0.53/(U4)-linkage)

(S, 8.39%)

5

0.38

0.61 ^ a

+71.7°

3,6-anhydroelsinan

+126.3°

P a r t i a l l y sulfated elsinan

Presumably s u b s t i t u t e d a t 0-6 p o s i t i o n .

A c t i o n o f s a l i v a r y QC-amylase

1.86

+243°

I n t r i n s i c v i s c o s i t y (25°C)

Degree of s u b s i t u t i o n

Optical rotation

Elsinan

COMPARISON OF PROPERTIES OF NATIVE AND MODIFIED ELSINAN

TABLE VI



220


Alpha-(1->4)-linked D-glucose residues i n the e l s i n a n mole c u l e can be converted partially i n t o 3,6-anhydro-D-glucose r e s i d u e s through p r e f e r e n t i a l s u l f a t i o n at the O-6-position, and subsequent alkali treatment. the i n t r o d u c t i o n o f 3,6-anhydro groups was confirmed by methylation a n a l y s i s and a l s o by i s o l a t i o n o f o l i g o s a c c h a r i d e s from mild, a c i d hydrolyzate. S o l u t i o n s o f e l s i n a n gives high v i s c o s i t y and pseudoplasticity. The v i s c o s i t y e x h i b i t s e x c e l l e n t pH stability, and c o m p a t i b i l i t y with the presence o f s a l t s . E l s i n a n forms strong and r e s i l i e n t f i l m s that a r e imprevious t o oxygen. These p r o p e r t i e s and p a r t i a l digestibility by α-amylases may propose wide utilization o f e l s i n a n i n food, pharmaceutical and other industries.

Literature Cited (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14)

W. H. McNeely, and K. S. Kang, " I n d u s t r i a l Gums", ed. by R. L. W h i s t l e r , P.486, Academic Press Inc., 1973. H. Bender, J. Lehmann and K. W a l l e n f e l s , Biochim. Biophy. Acta, 36 (1959) 309. T. Harada, A. Misaki and H. S a i t o , Arch. Biochem. Biophys., 124 (1968) 292. S. Ebisu, A. M i s a k i , K. Kato and S. Kotani, Carbohyd. Res. 38 (1974) 374. S. Takaya, T. Fukuda and Y. Oihe, Chagyo-Gijutsu-kenkyu (Study o f Tea), 49 (1975) 79. Y. Tsumuraya, A. M i s a k i , S. Takaya and M. Torii, Carbohyd. Res., 66 (1978) 53. S. Hakomori, J . Biochem. (Tokyo), 55 (1964) 205. M. Torii and K. Sakakibara, J . Chromatοgr., 96 (1974) 255. K. Matsuda, H. Watanabe, Κ. Fujimoto and K. Aso, Nature, 191 (1961) 1951. S. Barker, E. J. Bourne and M. Stacey, J. Chem. Soc., (1953) 3084. B. J. Catley and W. J . Whelan, Arch. Biochem. Biophys., 143 (1971) 138. Y. Tsumuraya and A. M i s a k i , J . Appl. Biochem., ( i n press) Y. Ohe,and A. M i s a k i , Ann. Meeting o f A g r i c u l t u r a l Bio chemical Soc. Japan, A p r i l , 1978, Nagoya. R. L. W h i s t l e r and S. H i r a s a , J . Org. Chem., 26 (1961) 4600.

RECEIVED January

29,

1980.


Structure and Enzymatic Degradation of Elsinan, a New - American

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