Xanthan GumAcetolysis as a Tool for the Elucidation of Structure

14 X a n t h a n Gum—Acetolysis as a T o o l for the Elucidation of Structure

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C. J. LAWSON and K. C. SYMES Tate and Lyle Ltd., Group Research and Development, P.O. Box 68, Reading, U.K.

The development of microbial gums is now moving at an ever increasing pace and it appears likely that in the forseeable future a whole range of products will be available with properties not only reflecting and improving upon those found in many plant gums, but also of a novel nature to be exploited in as yet undeveloped applications. The most successful microbial gum to date is undoubtedly xanthan gum produced by Xanthomonas campestris, and this polymer is now commanding a market of several thousand tons per annum. The market position for xanthan gum has been developed through the unique physical properties which it shows, which are exploited for example in oil recovery and food applications. These properties are briefly, high viscosity, extreme pseudoplasticity stability to extremes of pH, salt tolerance and synergistic gelation in the presence of locust bean gum. The above properties are of course dictated by the primary, secondary and tertiary structures of the gum and it is necessary to determine these if any real understanding of the relationship between function and structure is to be obtained. Early reports on the structure of xanthan gum, presented the repeating unit as being made up of glucose, glucuronic acid, mannose and the substituents pyruvate and acetate, in a 14 or 16 residue repeating unit. (1) (2) A repeating unit as large as this is unusual as most microbial gums have tri, tetra or pentasaccharide repeats. Also some of the chemical evidence was somewhat ambiguous, for example the assignment of the pyruvate as being linked to a glucose residue when it could equally have been associated with mannose. More recently two papers have been published, revising the structure and proposing a new pentasaccharide repeating unit containing the same sugar residues as before. We now provide further supporting evidence for the revised structure and suggest an approach to a rapid and convenient qualitative analysis of aspects of covalent structure of this and similar polysaccharides. The interest of Tate and Lyle in microbial gums was originally connected only with microbial alginate, (3) but as a natural consequence of involvement with gums generally, it was decided to examine 183

Sandford and Laskin; Extracellular Microbial Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

184

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

the possibilities of d e v e l o p i n g other m i c r o b i a l p o l y s a c c h a r i d e s for i n c o r p o r a t i o n into a possible range of p r o d u c t s .

O n e c a n d i d a t e for e x a m i n a t i o n was

Xanthomonas campestrls as the properties shown by xanthan gum were c o n s i d e r e d to be c o m p l e m e n t a r y to m i c r o b i a l a l g i n a t e . T r a d i t i o n a l l y , three b a s i c lines of a p p r o a c h c a n be a d o p t e d in the e l u c i d a t i o n of p o l y s a c c h a r i d e structure.

These are m e t h y l a t i o n a n a l y s i s ,


p e r i o d a t e o x i d a t i o n a n d i s o l a t i o n of fragments w h i c h c a n be c h a r a c t e r i s e d ; the last a p p r o a c h o n l y b e i n g of use when the p o l y s a c c h a r i d e s have a r e p e a t ing u n i t .

The first two a p p r o a c h e s had b e e n reported in the previous papers

a n d therefore the third was the l o g i c a l c h o i c e .

A c e t o l y s i s was used because

aqueous a c i d hydrolysis often g i v e s a c i d i c oligomers from u r o n i c a c i d c o n t a i n i n g p o l y s a c c h a r i d e s a n d these are more d i f f i c u l t to c h a r a c t e r i s e than neutral fragments.

A l s o a t the time it was c o n s i d e r e d that a c e t o l y s i s might

g i v e a c o m p l e m e n t a r y result to p a r t i a l a c i d hydrolysis w h i c h was a l s o under consideration.

A c e t o l y s i s i s , p r a c t i c a l l y , a r e l a t i v e l y straightforward

process performed a t room temperature in a p p r o x i m a t e l y two days using commonly a v a i l a b l e reagents.

A sample of xanthan fermentation broth

o b t a i n e d in b a t c h c u l t u r e of NRRL B1459 was t a k e n .

P u r i f i e d x a n t h a n gum

was r e c o v e r e d after b a c t e r i a l c e l l s were r e m o v e d using high speed c e n t r i f u g a t i o n a n d trypsin d i g e s t i o n , by a l c o h o l p r e c i p i t a t i o n . The c a r e f u l l y d r i e d gum was shaken w i t h the a c e t o l y s i s mixture o f reagents used b y M o r g a n a n d O ' N e i l l , (4) in studies on desulphated carrageenan.

λ-

The a c e t o l y s a t e was then poured into w a t e r , the a c e t y l a t e d

products e x t r a c t e d into c h l o r o f o r m , a n d d e a c e t y l a t e d using m e t h a n o l i c sodium m e t h o x i d e in the usual w a y .

T h e p a l e y e l l o w syrup o b t a i n e d in

h i g h y i e l d r e v e a l e d , on c h r o m a t o g r a p h i c e x a m i n a t i o n , a number o f spots in a d d i t i o n to the e x p e c t e d m o n o s a c c h a r i d e s .

The p r o d u c t was then

r e s o l v e d into a c i d i c a n d neutral fractions b y separation on ion e x c h a n g e resin in the a c e t a t e f o r m .

(5)

A s h o p e d , the major proportion o f o l i g o -

m e r i c m a t e r i a l was in the neutral f r a c t i o n .

The oligomers Β to Ε were then

o b t a i n e d in a p u r i f i e d state from the neutral f r a c t i o n by a c o m b i n a t i o n of c e l l u l o s e c o l u m n a n d t h i c k paper c h r o m a t o g r a p h y .

(Figure 1) (Figure 2)

A t this p o i n t in the work it was l e a r n e d from Professor Rees o f results o b t a i n e d b y his group (6) a n d o f Professor Lindbergs group (7) proposing the r e v i s e d structure o f xanthan gum w h i c h has b e e n m e n t i o n e d e a r l i e r . The r e v i s e d r e p e a t i n g unit is based upon a c e l l u l o s i c b a c k b o n e w i t h t r i s a c c h a r i d e side c h a i n s o c c u r r i n g on a l t e r n a t e g l u c o s e residues.

Analysis

o f the a c e t o l y s i s oligomers was therefore c o n t i n u e d in order to a s c e r t a i n , whether they were consistent w i t h the a b o v e structure. T h e structure o f o l i g o s a c c h a r i d e C w i l l be used as a n e x a m p l e o f the a p p r o a c h a d o p t e d , a n d the other o l i g o s a c c h a r i d e s w i l l o n l y be m e n t i o n e d for the purpose o f m e n t i o n i n g s p e c i f i c points of d i f f e r e n c e in their a n a l y s i s . This o l i g o s a c c h a r i d e was found to consist of glucose a n d mannose in a 2:1 r a t i o after hydrolysis a n d g l c o f the d e r i v e d a l d i t o l a c e t a t e s .

This was


14.

LAWSON A N D SYMES

Structure

Elucidation

of Xanthan

Gum

185

XANTHAN G U M Ac 0/AcOH/H S0 2

2

4

t ACETYLATED

PRODUCTS


NoOMe/MeOH

PRODUCTS Acetate resin

NEUTRAL

FRACTION

Cellulose column/PC OLIGOSACCHARIDES B,C,D&E

ACIDIC FRACTION HV Paper electrophoresis ALDOBIOURONIC ACID A

Figure

1.

Figure

The

acetolysis of gum

xanthan

2. Xanthan acetolysate neutral oligosaccharides


EXTRACELLULAR MICROBIAL

186

POLYSACCHARIDES

c o n f i r m e d by α c o l o r î m e t r î c assay o f the r a t i o o f carbohydrates to g l u c o s e w h i c h a l s o showed that 5 0 % g l u c o s e was lost on r e d u c t i o n with b o r o h y d r i d e . T h e o l i g o s a c c h a r i d e was therefore shown to be t r i s a c c h a r i d e h a v i n g g l u c o s e as the r e d u c i n g m o i e t y .

T h e mass spectrum of the T . M . S . ether o f r e d u c e d

C h a d ions a t 451 as e x p e c t e d for fission of the substituted terminal hexose a n d 525 from the a l d i t o l m o i e t y , thus p r o v i d i n g e v i d e n c e that the t r i s a c c -


h a r i d e is not b r a n c h e d .

Further more the series o f fragments o b t a i n e d a t

m / e ratios 1 0 3 , 205 a n d 3 0 7 were those p r e d i c t e d from a 4 - l i n k e d r e d u c e d g l u c o s e residue a n d this was c o n f i r m e d w i t h a deuterium l a b e l l i n g e x p e r i ment.

(Figure 3)

The r e d u c e d o l i g o s a c c h a r i d e was then c o n v e r t e d into

the p a r t i a l l y m e t h y l a t e d a l d i t o l a c e t a t e s o f its c o m p o n e n t sugars w h i c h w e r e a n a l y s e d b y gas c h r o m a t o g r a p h y .

The retention times o f the three

resulting peaks were c o m p a r e d w i t h l i t e r a t u r e values a n d in this w a y the substitution pattern of the c e n t r a l hexose in the t r i s a c c h a r i d e was e s t a b lished as e i t h e r 2-substituted mannose or 3-substituted g l u c o s e .

(Figure 4)

The s e q u e n c e o f sugar residues in the t r i s a c c h a r i d e a n d their a n o m e r i c c o n f i g u r a t i o n was then c l e a r l y shown by the use o f the e n z y m e sidase.

a-manno-

This e n z y m e c l e a v e d the sugar into mannose a n d e e l I o b î o s e d e m o n -

strating that it is i n d e e d

a-mannosyl eel I o b i ose o f the structure s h o w n .

(Figure 5) U s i n g a s i m i l a r a p p r o a c h the structures o f the other mannose c o n t a i n i n g o l i g o s a c c h a r i d e s were e l u c i d a t e d .

In the case o f the mannose c o n t a i n i n g

d î s a c c h a r î d e (E) the position of the mannosyl substituent was d e t e r m i n e d using the l e a d t e t r a a c e t a t e o x i d a t i o n method d e s c r i b e d b y Perl i n . (8) O n hydrolysis o f the o x i d i s e d d î s a c c h a r î d e , arabinose was d e t e c t e d , s h o w i n g that mannose was l i n k e d to O 3 . (Figure 6) The b r a n c h e d t r i s a c c h a r i d e (B) was u n e x p e c t e d l y resistant to the a c t i o n of both

a-mannosidase

and

β - g l u c o s î d a s e presumably through s t e r i c h i n d e r a n c e o f a d j a c e n t hexoses on O 3 a n d O 4 a n d therefore p a r t i a l a c i d hydrolysis was used for this f a c e t o f the structural i n v e s t i g a t i o n .

The a c i d i c d î s a c c h a r î d e (A) was shown to

c o n t a i n g l u c u r o n i c a c i d a n d mannose in r o u g h l y e q u a l proportions a n d was assumed to be the a l d o b i u r o n i c a c i d p r e v i o u s l y i s o l a t e d from the g u m . O l i g o s a c c h a r i d e D was shown to be c e 11 o b i ose by c o - c h r o m a tography w i t h a n a u t h e n t i c sample on paper a n d gas c h r o m a t o g r a p h y .

(Figure 7)

A l l o f the sugars in the n e w l y proposed r e p e a t i n g unit o f the p o l y s a c c h a r i d e w i t h the s i n g l e e x c e p t i o n o f the terminal mannose residue a r e represented in a t least one o f the o l i g o m e r s , a n d our results are e n t i r e l y consistent w i t h the r e v i s e d s t r u c t u r e .

(Figure 8)

It is possible that the c o v a l e n t structures o f gums p r o d u c e d under d i f f e r e n t c o n d i t i o n s may vary i n some w a y , for e x a m p l e , a f t e r c h e m i c a l treatment.

(10)

T h e r e is e v i d e n c e a l s o that structural v a r i a t i o n may o c c u r

in gums from d i f f e r e n t species o f xanthomonas (9).

V a r i a t i o n in structure

is l i k e l y to be a s s o c i a t e d w i t h v a r i a t i o n in p h y s i c a l properties a n d it is possible that a range o f xanthan gum types c o u l d be d e v e l o p e d to g i v e a


14.

Structure

LAWSON AND SYMES

Elucidation

of Xanthan

187

Gum

CH OTMS 2

TMSOHEX .

HEX -

307(308) -OTMS

_205


ITMSO-

103 CH OTMS 2

451 (452)

451

Figure 3. Mass spectroscopy of the perO-trimethyhilyl ether of the derived glycitol from oligosaccharide C (Figures in parentheses are after NaBD reduction) h

[Man]1or >3[Glc]1->

[GlclH CH OAc

ÇH OAc

2

2

-Ο Me

+

AcO-OMe

OR

-OAc CH OMe 2

CH OMe 3

Ο Me MeO-OAc -OMe CH OMe

CH OMe

2

2

•2[Man]1->

•UGlucitol)

Figure 4. Partially methylated alditol acetates possible from gas chromato graphic evidence

oc-Mannosidase Ψ MANNOSE +

CELL0BI0SE

Figure 5. Action of a-mannosidase on oligosaccharide C


188

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

-OHpH

OH


|Pb(OAc)

4

O H C ^ Î Î )

OA^CHO

H CT 3

HOH C 2

HO? OH Η Figure 6.

OH

Lead tetraacetate oxidation of disaccharide Ε

Arabinose

A.

p-P-GlcAp-(l->2)-P-Mang

B.

B-D-Glcp-(B4)-D-Glcp

i< - D - M a n p

C.

B - D - G l c p - ( l-*4> - D - G l c p

3

Î l I a. - D - M a n p

D. Figure 7. Oligosaccharides from acetolysis of xanthan gum

Ε.

p-D-Glcp-(l->4)-D-Glcp

* -D-Manp-(l->3) - D - G I cp


14.

LAWSON

AND

SYMES

Structure

Elucidation

of Xanthan

Ε

189

Gum

C

- ——ι

• GA«

11

D

1


— M-

»GA-

•Μ ι

M

JL

1

I

!

•M

-,

G-4i

— .

> GA-

A Figure 8.

GA

Xanthan GumAcetolysis as a Tool for the Elucidation of Structure

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