Extracellular Microbial Polysaccharides

7 Xanthan Gum—Molecular Conformation and Interactions R. MOORHOUSE, M. D. WALKINSHAW, and S. ARNOTT

Downloaded by NORTH CAROLINA STATE UNIV on May 7, 2013 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0045.ch007

Department of Biological Sciences, Purdue University, West Lafayette, IN 47907

Xanthan Gum, the extracellular p o l y s a c c h a r i d e produced by the microorganism Xanthomonas campestris has found widespread industrial use (1,2,3) because o f its unique r h e o l o g i c a l properties. The p o l y s a c c h a r i d e forms homogeneous aqueous d i s p e r s i o n s and s o l u t i o n s e x h i b i t i n g high viscosity, as w e l l as having characteristics of both p s e u d o p l a s t i c and plastic polymer systems ( 4 , 5 ) . Of particular s i g n i f i c a n c e is the a t y p i c a l insensitivity of s o l u t i o n viscosity to s a l t e f f e c t s and to heat, e s p e c i a l l y at h i g h ionic s t r e n g t h . Molecular weight measurements (6) i n d i c a t e p o l y d i s p e r s e systems of h i g h molecular weight (>2x10 ). The primary s t r u c t u r e of xanthan has r e c e n t l y been r e i n v e s t i g a t e d (7,8) and found to c o n s i s t of pentasaccharide r e p e a t i n g u n i t s (I). 6

Pyruvate is attached on average to about o n e - h a l f of the t e r m i n a l mannose r e s i d u e s ; 0 - a c e t y l groups correspond to one residue f o r each pentassaccharide r e p e a t i n g u n i t . When p r e v i o u s l y detected in bacterial p o l y s a c c h a r i d e s , pyruvate has u s u a l l y been observed on every r e p e a t i n g u n i t ( 9 , 1 0 ) . However, the c l o s e l y r e l a t e d 90

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


7.

MOORHOUSE ET

AL.

Xanthan

Gum

Conformation

and

Interactions

91

polysaccharides from other Xanthomonas species (11,12) also show differing pyruvate contents (13). We have prepared f i b e r s of both xanthan and the r e l a t e d p o l y s a c c h a r i d e from Xanthomonas p h a s e o l i (14). Using e s t a b l i s h e d techniques f o r f i b e r d i f f r a c t i o n and computer aided model b u i l d i n g (15,16,17,18,19) we have been able to examine the p o s s i b l e molecular conformations of xanthan. The almost i n d e n t i c a l X-ray d i f f r a c t i o n patterns,from a l a r g e number of p o l y s a c c h a r i d e samples from both X. campestris and X. p h a s e o l i , i n d i c a t e s an o v e r a l l s i m i l a r i t y of molecular conformation and primary sequence. R e s u l t s and D i s c u s s i o n I t i s u s u a l l y p o s s i b l e to prepare specimens of long h e l i c a l polymers i n which the molecules are a l i g n e d with t h e i r long axes parallel. Often f u r t h e r o r g a n i z a t i o n occurs, but r a r e l y to the degree of a t h r e e - d i m e n s i o n a l l y ordered s i n g l e c r y s t a l . The xanthan X-ray d i f f r a c t i o n p a t t e r n (Figure 1) showing both c o n t i n uous i n t e n s i t y d i s t r i b u t i o n and Bragg maxima, i s c h a r a c t e r i s t i c of an ordered a r r a y of h e l i c e s which have t h e i r axes p a r a l l e l but are not f u r t h e r ordered (20). The presence of continuous d i f f r a c t i o n along the l a y e r l i n e s i n d i c a t e s that the i n d i v i d u a l molecules have random t r a n s l a t i o n s along and r o t a t i o n s about t h e i r axes and are not packed i n t o a w e l l developed c r y s t a l l a t t i c e . However, d e s t r u c t i v e i n t e r f e r e n c e has occurred near the center of the equator, l e a v i n g one broad Bragg r e f l e c t i o n of spacing 1.9 nm, the a r r a y of molecules t h e r e f o r e has some order when viewed down a molecular screw a x i s at s u f f i c i e n t l y low r e s o l u t i o n . The l a y e r l i n e spacing i s c o n s i s t e n t with a h e l i x of p i t c h 4.70 nm; the m e r i d i o n a l r e f l e c t i o n s (0,0,£) o c c u r r i n g only when Z=5n, suggests a 5-fold helix. T h i s gives a r i s e per backbone d i s a c c h a r i d e of 0.94 nm (Figure 2). The s t e r i c e f f e c t of the branching mannose r e s i d u e together with the consequent removal of the c e l l u l o s e 0(3)A—0(5) hydrogen bond across a l t e r n a t e 8-1,4 l i n k a g e s (u9ing the n o t a t i o n i n Figure 2) means that the backbone can no longer have the 2^ screw symmetry of c e l l u l o s e . Instead of the usual extended β-1,4 ribbon (21), a more sinuous h e l i x of the type shown i n F i g u r e 3 i s obtained. A p r i o r i we could have no preference f o r any of the four p o s s i b l e 5 - f o l d h e l i c a l models. The 5/1 and 5/4 conformations are r i g h t and left-handed r e s p e c t i v e l y and have a s i n g l e t u r n per h e l i x p i t c h while the two other (5/2 and 5/3) models a l s o d i f f e r by being r i g h t and left-handed and have two turns per h e l i x p i t c h . I n i t i a l l y molecular models f o r each of these f o u r s i n g l e h e l i c a l p o s s i b i l i t i e s , were examined assuming standard bondlengths, bond-angles and sugar r i n g conformation angles (15). The models were f u r t h e r c o n s t r a i n e d to e x h i b i t symmetry and p e r i o d i c i t y c o n s i s t e n t with the d i f f r a c t i o n p a t t e r n . On the b a s i s of a minimum s t e r i c compression comparison, the 5/1 (Figure 3) and 5/2 (Figure 4) right-handed h e l i c e s were most favored,



92

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MICROBIAL

POLYSACCHARIDES

Figure 1. Diffraction pattern typ ical for both Xanthomonas campestris and Xanthomonas phaseoli polysaccharides showing five-fold helical symmetry. The sharp Bragg reflection on the equator has a spacing of 1.9 nm.

Figure 2. The pentasaccharide repeat ing unit of xanthan showing atom label ing and aisaccharide backbone height. The unlettered residue and residue A are Ό-glucose, Β and Ε are O-mannose, and C is O-glucuronate.


MOORHOUSE

E TA L .

Xanthan

Gum Conformation

and

Interactions


7.

Figure 3.

The isolated 5/1 xanthan helix viewed perpendicular to the helix axis


93


Figure 4.

The isolated 5/2 xanthan helix viewed perpendicular to the helix axis



7.

MOORHOUSE E T A L .

Xanthan

Gum

Conformation

and

Interactions

95

having g l y c o s i d i c conformation angles w i t h i n the normal o l i g o saccharide ranges (Table 1) and no overshort non-bonded separat i o n s . With the left-handed h e l i c e s (5/3 and 5/4), minimization did not r e l i e v e a l l of the unacceptably short i n t e r a t o m i c cont r a c t s even a f t e r o p t i m i z a t i o n . In i s o l a t i o n there i s no d r i v i n g f o r c e to h o l d the s i d e chains c l o s e to the backbone and the i s o l a t e d chain models suggest a diameter of 3.8 nm as opposed to a value of 1.9 nm obtained from l a t e r a l p e r i o d i c i t i e s i n the d i f f r a c t i o n p a t t e r n . Studies on other branched polysaccharides favor the s i d e chains l y i n g roughly p a r a l l e l to the backbone (18), and we have t h e r e f o r e undertaken a second study i n which both packing and conformational v a r i a t i o n s were considered f o r each of the models. The most symmetrical and commonly observed c l o s e packing of polymeric molecules, having n e a r l y c i r c u l a r cross s e c t i o n , i s hexagonal packing i n which each molecule i s e q u i d i s t a n t from i t s 6 nearest neighbors but not n e c e s s a r i l y f u r t h e r r e l a t e d . We t h e r e f o r e placed one xanthan h e l i c a l chain i n a hexagonal u n i t c e l l of s i d e a. = 2.19nm, c_ = 4.70nm, that i s c o n s i s t a n t with the e q u a t o r i a l Bragg r e f l e c t i o n indexed as (100). Minimizing s t e r i c r e p u l s i o n i n t h i s environment causes the side chain to f o l d down against the backbone. Stereochemically both the 5/4 and 5/3 h e l i c e s are u n l i k e l y as an unacceptable number of i n t r a m o l e c u l a r overshort contacts p e r s i s t a f t e r refinement. This r e i n f o r c e s our previous c o n c l u s i o n of right-handedness f o r the i s o l a t e d chains. Although the 5/1 and 5/2 h e l i c e s are s t e r i c a l l y acceptable, the 5/1 e x h i b i t s the more f a v o r a b l e comparison with o l i g o s a c c h a r i d e conformation angles. I t i s of i n t e r e s t to note that the backbone conformation angles shown i n Table I have v a r i e d l i t t l e during the process of wrapping the s i d e chains around the backbone. Further, the 5/1 packed' h e l i x (Figure 5) shows a number of p o t e n t i a l intramolec u l a r hydrogen bonds (Table I I and Figure 6). Relaxing the a t t r a c t i v e i n t e r a c t i o n (hydrogen bond) terms i n the refinement did not a l t e r the molecular conformation. Only the a d d i t i o n a l i n f l u e n c e of small p e r t u r b a t i o n s to the conformation angles about the branching mannose l i n k a g e caused the s t a b i l i s i n g i n f l u e n c e of the hydrogen bonds to be l o s t . The 'packed 5/2 h e l i x presents a much t i g h t e r s t r u c t u r e than the 5/1 model while e x h i b i t i n g some overshort i n t r a m o l e c u l a r contacts and few p o t e n t i a l hydrogen bonds and was considered u n l i k e l y on the b a s i s of t h i s a n a l y s i s . Our reasoning so f a r has been based on the premise that the e q u a t o r i a l Bragg r e f l e c t i o n on the d i f f r a c t i o n p a t t e r n (Figure 1), a r i s e s from the packing of s i n g l e molecular e n t i t i e s , the p a t t e r n does not t e l l us what form these take. In our examination of i n t e r - c h a i n i n t e r a c t i o n s , we have thus considered those i n t e r a c t i o n s that can a r i s e from some side-by-side arrangement of the 5/1 h e l i c e s and a l s o the case of c o a x i a l m u l t i p l e h e l i c e s . 1

1


96

EXTRACELLULAR MICROBIAL POLYSACCHARIDES

TABLE I


Comparison o f backbone c o n f o r m a t i o n a n g l e s i n t h e i s o l a t e d and ' p a c k e d ' 5/1 a n d 5/2 h e l i c a l m o d e l s

Angle

-148

-119

-98

-63

-64

-76

-30

-10O>- -161

-111

-99

-98

-97

-92

-22

-81

-61

-78+

(d)

-78+

(a) - e [ c

( 1 ) A

"

e t 0

-

Using

9 [ 0

,o

-98

( 4 )

,c

( 4 )

,c

C

( 5 )

]

, C

(5)A, (l)A'°(4) (4)

(c) - 6 [ C (d)

helices 5/2

-121

(b) (c)

'Packed' 5/1

-136

-100+ -161

(a)

(b)

Isolated helices 5/1 5/2

Range

0

{ 1 )

> (4 C

) A

>

C

]

C

( 4 ) A

> (5) ] A

C

(5)' (1),°(4)A, (4)

] A

atom n o t a t i o n i n F i g u r e 2.


MOORHOUSE E T A L .

Xanthan

Gum Conformation

and

Interactions

97


7.

(a)

(b)

Figure 5. The 'packed' 5/1 helix viewed (a) perpendicular to and (b) down the helix axis




Figure 6. Possible hydrogen bonds ( ) that may stabilize the molecule. Some adjoining residues are omitted for clarity, the backbone having solid bonds. See also Table II.


Xanthan

MOORHOUSE ET AL.

Gum Conformation

and

Interactions

TABLE I I P o s s i b l e a t t r a c t i v e i n t e r a c t i o n i n the X and 5/2 h e l i c e s

aampestris


3

Model

Overshort

5/1

Potential Hydrogen bonds

contacts (nm)

none

5/1

°(3) 0

^ °(5)A

( 2 ) ~ -

°(8a)

D

°(6)

°(5)C

°(2)A

** °(7)B

°(3)B—* °(6) [

o

r

°(3)B

°(2)D 5/2

Η

°(5)Α··· (4)Α (0.195 nm)

°(3)~

Υ

" °(5)C

°(6b)C

> 0

(5)A

°(6)A

* °(5)

°(3)B

' °(5)C

°(3)C

°(5)D


]


100


P l a c i n g a s i n g l e 5/1 h e l i x i n o u r h e x a g o n a l c e l l r e v e a l s few i n t e r a c t i o n s w i t h i t s nearest neighbors. This suggests that t h e h e l i c e s a r e s l o t t i n g i n t o some g r o o v e t h a t i s w i d e enough t o a c c o m o d a t e them w i t h o u t s t e r i c c l a s h e s . A l t e r n a t i v e l y t h e molec u l e c o u l d be c o n s i d e r e d a s a r i g i d r o d o f p o l y s a c c h a r i d e s u r rounded by a c y l i n d e r o f w a t e r , i n which case v e r y few p o l y s a c c h a r i d e - p o l y s a c c h a r i d e i n t e r a c t i o n s would be a p p a r e n t . Furthermore, a s s u c h a s i t u a t i o n c l o s e l y m i m i c s t h e s o l u t i o n s t a t e , t h e unusual s o l u t i o n p r o p e r t i e s would p r o b a b l y a r i s e from i n t e r a c t i o n s b e t w e e n r e g i o n s o f O r d e r e d ' w a t e r some o f w h i c h may b e t i g h t l y bound t o t h e p o l y s a c c h a r i d e . Current X-ray f i b e r d i f f r a c t i o n t e c h n o l o g y c a n n o t e n a b l e u s t o l o c a t e t h i s amount o f w a t e r ( 1 8 ) , p o s s i b l y NMR s t u d i e s o n s o l u t i o n s may be a b l e t o l o c a t e ' o r d e r e d w a t e r b u t w i t h o u t t h e d e t a i l t h a t i s sometimes a v a i l a b l e f r o m d i f f r a c t i o n s t u d i e s ( 1 6 , 1 7 ) . On d r y i n g t h e s p e c i ment f o r p r o l o n g e d p e r i o d s we n o t e a r e d u c t i o n o f o v e r 5 0 % i n t h e c e l l volume c o n s i s t e n t w i t h a s h r i n k a g e i n t h e Bragg s p a c i n g on the e q u a t o r w h i l e t h e f i b e r a x i s d i m e n s i o n r e m a i n s u n a l t e r e d . Apparently the molecular conformation o f the xanthan molecule s u r v i v e s d r y i n g w i t h l i t t l e change a n d a s u b s t a n t i a l q u a n t i t y o f w a t e r w h i c h f i l l s o u t t h e s t r u c t u r e i s n o t f i r m l y bound. W h i l e i t i s p o s s i b l e t o c o n s t r u c t a double h e l i c a l model, u s i n g t h e 5/1 s i n g l e h e l i x a s p r e c u r s o r , i n w h i c h t h e s e c o n d c o a x i a l s t r a n d i s p a r a l l e l t o , a n d r e l a t e d t o , t h e f i r s t b y 180 r o t a t i o n some a p p a r e n t l y u n r e s o l v a b l e o v e r s h o r t i n t e r - s t r a n d contacts exist. I t i s p o s s i b l e t h a t r e l a x i n g t h e summetry s o t h a t the p a r a l l e l c o a x i a l s t r a n d s a r e n o t r e l a t e d b y a 180 f i b e r a x i s r o t a t i o n o r a r e a n t i p a r a l l e l t o one a n o t h e r , c o u l d r e s u l t i n a c c e p t a b l e i n t e r a c t i o n s between c h a i n s . Should t h i s be t h e case i t w i l l s t i l l be n e c e s s a r y t o o b t a i n s u p p o r t i n g e v i d e n c e from other sources t o demonstrate t h e e x i s t e n c e o f double h e l i c e s . N o r m a l l y t h i s would t a k e t h e form a comparison o f t h e model w i t h the X - r a y i n t e n s i t y d a t a f r o m a c r y s t a l l i n e d i f f r a c t i o n p a t t e r n (e.g. 16,17,18) p l u s e v i d e n c e f r o m s o l u t i o n s t u d i e s o f b i - m o l e c u l a r i t y ( e . g . 2 2 ) . We w o u l d s t r e s s however t h a t t h e r e i s no e v i d e n c e o f d o u b l e h e l i c e s e i t h e r i n s o l u t i o n (27) o r t h e s o l i d s t a t e . R e c e n t l y , we h a v e b e e n a b l e t o o b t a i n a d i f f r a c t i o n p a t t e r n t h a t e x h i b i t s i n c r e a s e d c r y s t a l l i n i t y and which h a s been t e n t a t i v e l y i n d e x e d o n a t e t r a g o n a l c e l l i n w h i c h f o u r 5/1 s i n g l e h e l i c e s w i l l p a c k w i t h t h e minimum o f s t e r i c c o m p r e s s i o n . A r e finement u s i n g b o t h s t e r e o c h e m i c a l and X-ray i n t e n s i t y d a t a has not y e t been completed. 1

Conclusions T h i s p r e l i m i n a r y s t u d y shows t h a t t h e o r d e r e d c o n f o r m a t i o n of xanthan i n t h e condensed s t a t e , and p r o b a b l y i n s o l u t i o n , i s r e l a t e d t o t h e 5/1 h e l i x o u t l i n e d h e r e .


7.

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Gum

Conformation

and Interactions

101

The Interactions t h a t o c c u r i n solution, giving rise t o viscosity effects showing t h e characteristic o f both flexible a n d stiff cross-linked r e g i o n s (4,5) must arise f r o m associations of t h e o r d e r e d 5/1 helical regions. The order/disorder transition s e e n with change o f t e m p e r a t u r e in solution ( 2 3 , 2 4 ) , w o u l d seem l i k e l y t o a r i s e f r o m c o n f o r m a t i o n a l changes p r i m a r i l y w i t h i n t h e s i d e c h a i n a s i t moves away f r o m i t s c l o s e a s s o c i a t i o n w i t h t h e o r d e r e d backbone e i t h e r accompanied b y , o r b e f o r e , c o n f o r m a t i o n a l changes i n t h e b a c k b o n e . T h i s s p r e a d i n g o f t h e 'arms o f t h e p o l y s a c c h a r i d e would cause a n i n c r e a s e d hydrodynamic volume and hence p r o v i d e t h e v i s c o s i t y s t a b i l i t y noted a t e l e v a t e d tempera tures (1,2,3,4). A s s o c i a t i o n , i n s o l u t i o n o f s i n g l e h e l i c e s does n o t r e q u i r e gel formation, a fact that points strongly i n favor o f single h e l i c a l x a n t h a n , w h i c h d o e s n o t show g e l a t i o n a t room t e m p e r a t u r e s . Weak g e l a t i o n o b s e r v e d a t t e m p e r a t u r e s c l o s e t o 0 C i s p r o b a b l y due t o a n a g g r e g a t i o n phenomenon. I t i s i n t e r e s t i n g t o n o t e t h a t t h e 5/1 h e l i x p r e s e n t s two d i s t i n c t f a c e s ; one h a v i n g t h e s i d e c h a i n s a n d c h a r g e d g r o u p s , the o t h e r e s s e n t i a l l y t h e c e l l u l o s e backbone. As xanthan i n t e r a c t s s y n e r g i s t i c a l l y w i t h t h e 3-1,4 l i n k e d g a l a c t o m a n n a n s l o c u s t b e a n a n d g u a r gums, i t i s p o s s i b l e t h a t t h i s t a k e s p l a c e a t t h e c e l l u l o s e ' g r o o v e ' i . e . b e t w e e n s i m i l a r 8-1,4 l i n k e d g l y c a n s . I t i s t h o u g h t t h a t 'smooth' u n s u b s t i t u t e d r e g i o n s o f t h e g a l a c t o mannan a r e i n v o l v e d i n t h e a s s o c i a t i o n ( 2 3 , 2 5 ) . More d e t a i l e d i n t e r p r e t a t i o n s o f t h i s c o n t i n u i n g w o r k w i l l be p u b l i s h e d e l s e w h e r e ( 2 6 ) .


1

Acknowledgement s We w i s h t o t h a n k D r s . A. J e a n e s a n d P.A. S a n f o r d , U.S.D.A,, P e o r i a a n d D r . I.W. C o t t r e l l , K e l c o , San D i e g o , f o r t h e i r g e n e r ous g i f t s o f s a m p l e s .

Literature Cited 1. Jeanes, A. (1973) In "proceedings of the ACS Conference on Water Soluble Polymers", ed. N.M. Bikales, Plenum Press, New York. pp. 227-242. 2. Jeanes, A. (1974) J . Polymer S c i . , Symp. No. 45, 209-227. 3. McNeely, W.H. and Kang, K.S. (1973) In "Industrial Gums" R.L. Whistler and J.N. BeMiller eds., pp. 473-497, Academic Press, New York. 4. Jeanes, Α., Pittsley, J.E. and Senti, F.R. (1961) J . Appl. Polymer S c i . , 5,519-526. 5. Jeanes, A. and Pittsley, J.E. (1973) J . Appl. Polymer S c i . , 17,1621-1624. 6. Dintzis, F.R., Babcock, G.E. and Tobin, R. (1970) Carbohyd. Res. 13,257-267.


102

7. 8. 9. 10.


11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

EXTRACELLULAR MICROBIAL

POLYSACCHARIDES

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Extracellular Microbial Polysaccharides

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