Structural Studies of Apple Pectins with Pectolytic Enzymes - American

4 Structural Studies of Apple Pectins with Pectolytic Enzymes 1

Downloaded by UNIV OF ARIZONA on December 16, 2012 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch004

J. A . de Vries , A. G. J . Voragen, F. M . Rombouts, and W. Pilnik Department of Food Science, Agricultural University, De Dreijen 12, 6703 BC Wageningen, The Netherlands

Apple pectic substances consist of chains of partially esterified galacturonic acid residues with side chains composed of arabinose, galactose and xylose. Degradation with purified pectolytic enzymes shows that the side chains are present in blocks ("hairy regions"). Enzymic and chemical degradation of the hairy regions reveals that they consist of arabinogalactan side chains and short xylose side chains. It can be concluded that apple pectic substances are constructed of homogalacturonan, xylogalacturonan and rhamnogalacturonan regions with side chains of arabinogalactan. About 95% of the uronic acid residues is present in the homogalacturonan regions. The arabinogalactans are highly branched. HPLC analysis of enzymic degradation products suggests that the methoxyl groups of the uronic acid residues are randomly distributed. There i s consensus about t h e b a s i c f i n e s t r u c t u r e o f p e c t i n m o l e c u l e s . Everyone agrees w i t h t h e i d e a o f an a - 1 , 4 - D - g a l a c t u r o n a n w i t h few a-1,2 bound L-rhamnose r e s i d u e s i n t h e main c h a i n , t h e g a l a c t u r o n a n b e i n g p a r t l y e s t e r i f i e d w i t h methanol and p a r t l y a c e t y l a t e d . The main c h a i n f u r t h e r c a r r i e s c o v a l e n t l y a t t a c h e d n e u t r a l sugars as s i d e groups and/or as s i d e c h a i n s which may be s u b s t i t u t e d w i t h a c e t y l and p h e n o l s , many o f t h e s e s i d e c h a i n s b e i n g bound t o L-rhamnose. There i s a l s o agreement t h a t a p e c t i n p r e p a r a t i o n c o n s i s t s o f a m i x t u r e o f v a r i o u s m o l e c u l e s . Many o f them a r e a r t i f a c t s i n t h e case o f e x t r a c t i o n with c e l l wall modifying reagents. S t r u c t u r a l features also depend on o r i g i n and degree o f r i p e n e s s o f t h e p l a n t s o u r c e . These views have been o b t a i n e d and s u b s t a n t i a t e d over 30 y e a r s . Much work was done on d e g r a d a t i o n , work up o f o l i g o m e r i c fragments f o l l o w e d by d e t e r m i n a t i o n o f s t r u c t u r e w i t h c l a s s i c a l methods and r e c e n t l y by GC and GC/MS a f t e r c h e m i c a l m o d i f i c a t i o n ( r e d u c t i o n ) and d e r i v a t i o n . 1Current address: The Copenhagen Pectin Factory, Ltd., DK-4623 Lille Skensved, Denmark. 0097-6156/ 86/0310-0038506.00/0 © 1986 American Chemical Society

In Chemistry and Function of Pectins; Fishman, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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DE VRIES ET AL.

Structural Studies of Apple Pectins

39

A t p r e s e n t , a number of q u e s t i o n s r e m a i n open. There i s the s p e c i f i c q u e s t i o n on the s t r u c t u r e o f the o f t e n branched s i d e c h a i n s and the more g e n e r a l q u e s t i o n on whether s t r u c t u r a l f e a t u r e s a r e i n t r a - o r i n t e r m o l e c u l a r phenomena. Do t h e m o l e c u l e s o f a p e c t i n p r e p a r a t i o n have smooth and h a i r y r e g i o n s o r i s t h e r e a m i x t u r e o f pure g a l a c t u r o n a n m o l e c u l e s and g a l a c t u r o n a n m o l e c u l e s w i t h many s i d e c h a i n s ? How i s the i n t e r - and i n t r a m o l e c u l a r d i s t r i b u t i o n o f m e t h o x y l a t e d and f r e e c a r b o x y l groups? The a v a i l a b i l i t y o f pure p e c t o l y t i c enzymes w i t h known s p e c i f i c i t y p l u s chromatography t e c h n i q u e s p r o v i d e s the p o s s i b i l i t y f o r r e l a t i v e l y s i m p l e experiments to answer some o f these q u e s t i o n s . I n r e c e n t y e a r s we have used t h e s e p o s s i b i l i t i e s i n our s t u d i e s on the s t r u c t u r a l f e a t u r e s o f a p p l e p e c t i c s u b s t a n c e s ( 1 - 6 ) . T h i s work i s r e v i e w e d h e r e and complemented w i t h r e s u l t s o f o t h e r i n v e s t i g a t o r s . F o r d e t a i l e d d e s c r i p t i o n s o f e x p e r i m e n t a l work the r e a d e r i s r e f e r r e d to ( 1 - 4 ) . The a p p l e p e c t i n s s t u d i e d were e x t r a c t e d from Golden D e l i c i o u s a p p l e s as s c h e m a t i c a l l y shown i n F i g . 1. I n t o t a l l e s s than 50% o f the p e c t i n p r e s e n t i n the A l c o h o l I n s o l u b l e S o l i d s (AIS) p r e p a r e d from p e e l e d and c o r e d a p p l e s c o u l d be e x t r a c t e d under the m i l d c o n d i t i o n s u s e d . The amounts o f p e c t i n i n the v a r i o u s ex t r a c t s a r e i n d i c a t e d i n F i g . 1. Neutral

Sugar D i s t r i b u t i o n

To e s t a b l i s h the d i s t r i b u t i o n of the n e u t r a l s u g a r s among the p e c t i n m o l e c u l e s we (J_) f r a c t i o n a t e d the f o u r p e c t i n e x t r a c t s on DEAEc e l l u l o s e . F i g . 2 shows a t y p i c a l e l u t i o n p r o f i l e o b t a i n e d w i t h a l i n e a r g r a d i e n t o f 5-500 mM Na-phosphate b u f f e r s o f pH 5.1 f o r the c o l d - b u f f e r e x t r a c t o f r i p e a p p l e AIS. The p e c t i c m a t e r i a l was found t o e l u t e as one t a i l i n g peak. About 10% o f the p e c t i n d i d n o t b i n d t o the column and c o u l d o n l y be f r a c t i o n a t e d a f t e r p a r t i a l c o l d a l k a l i s a p o n i f i c a t i o n . A s m a l l p e r c e n t a g e was r e t a i n e d on the column and c o u l d o n l y be e l u t e d w i t h 0.01 M NaOH. The p e c t i n c o n t a i n i n g f r a c t i o n s from each e x t r a c t were c o l l e c t e d i n 10 p o o l s , each p o o l c o n t a i n i n g 10% o f the a n h y d r o g a l a c t u r o n i c a c i d (AUA) a p p l i e d t o t h e column ( F i g . 2 ) . In t o t a l 48 p o o l s were o b t a i n e d f o r the f o u r ex t r a c t s , t h i s i n c l u d e s the p e c t i n f r a c t i o n s which d i d n o t b i n d t o the column and the ones t h a t had t o be e l u t e d w i t h a l k a l i . The n e u t r a l sugar c o n t e n t i n each p o o l was e s t i m a t e d and c a l c u l a t e d as moles n e u t r a l sugar r e s i d u e s per mole of g a l a c t u r o n o s y l r e s i d u e s . I t was found t o v a r y between 0.04 t o 1.7 moles/mole AUA, p o o l s w i t h r a t i o ' s o f 0.15, 0.24 and 0.53 were p r e v a i l i n g . By a r r a n g i n g the p o o l s i n a s c e n d i n g o r d e r o f moles n e u t r a l sugars p e r mole AUA and p l o t t i n g the AUA c o n t e n t o f p o o l s w i t h a c e r t a i n r a t i o i n % of AUA p r e s e n t i n a l l p o o l s c u m u l a t i v e l y a g a i n s t the r a t i o , a c u m u l a t i v e n e u t r a l sugar d i s t r i b u t i o n c u r v e was c o n s t r u c t e d . N u m e r i c a l d i f f e r e n t i a t i o n o f t h i s c u r v e r e s u l t s i n the n e u t r a l sugar d i s t r i b u t i o n c u r v e as shown i n F i g . 3 f o r e x t r a c t s of r i p e and u n r i p e a p p l e s . These c u r v e s suggest that i n p e c t i n a discontinuous r a t h e r than a continuous d i s t r i b u t i o n of the n e u t r a l sugars i s p r e s e n t . F i v e t y p e s o f p e c t i n s (A t o E) a r e i n d i c a t e d by the n e u t r a l sugar d i s t r i b u t i o n c u r v e s . The n e u t r a l sugar c o n t e n t s o f the p e c t i n s of t y p e s B, C, D and Ε have r a t i o ' s of 1:1.7:3.7:10. C o l d b u f f e r e x t r a c t s were found t o c o n t a i n m a i n l y p e c t i n s of type A, Β and C, i n r i p e a p p l e s a l s o type Ε was present.


40

CHEMISTRY AND FUNCTION OF PECTINS

CAIS v

)

'

1 1

NaAc buffer pH 5.2, ambient temp, 30 mi η

centrifuged

-^29 m g / g \

NaAc buffer pH 5.2 70°C, 30 min


centrifuged (^PELLETJ

rΓ

centrifuged centrifuged

Γ

^PELLET ^ centrifuged-

-^28 mg/g^•filtered and precipitated with ethanol

0.05M EDTA + 0.05M NH- oxalate in NaAc buffer pH 5.2 4

7 Q

° ' C

3

0

m

i

n

(22 mg/g)-

•enzymic degradation •chemical degradation

•gelfiltration chromatography •ion-exchange chromatography

HC1 solution, pH 2.5 70°C, 30 min -ζ]9

mq/qj

i ^PELLET ^

Figure 1. E x t r a c t i o n ( p e e l e d and c o r e d ) .

of pectin

fractions

o f AIS o f a p p l e s

AUA

Figure 2. Fractionation of the pectin from the cold buffer extract of the ripe apple AIS on DEAE-cellulose. AUA, anhydrouronic a c i d , mg/ml; Ve, e l u t i o n volume, ml. The numbers i n the figure indicate the neutral sugar residue content of the f r a c t i o n s , expressed as moles of neutral sugar residues/mole of galacturonate residues. (Reproduced with permission from reference 1. Copyright 1981 Carbohydrate Polymer).



4.

D E VR1ES E T A L .


41

Hot b u f f e r e x t r a c t s c o n t a i n e d m a i n l y types B, C and D, o x a l a t e ex t r a c t s types A, B, C and D and i n a c i d e x t r a c t s t y p e s C, D and Ε dominated. Ion-exchange chromatography has been s u c c e s s f u l l y a p p l i e d by v a r i o u s i n v e s t i g a t o r s to f r a c t i o n a t e p e c t i n s (7, 8, 9 ) . The f r a c t i o n a t i o n i s m a i n l y based on the degree of e s t e r i f i c a t i o n of p e c t i n s ( 1 0 ) . However, i t i s l i k e l y t h a t c o v a l e n t l y l i n k e d n e u t r a l s u g a r s and the m o l e c u l a r weight of the p e c t i n a l s o a f f e c t the e l u t i o n p r o f i l e ( J J ) . To v e r i f y , i f the p o s s i b l e i n t e r a c t i o n s between degree of e s t e r i f i c a t i o n , m o l e c u l a r weight and n e u t r a l sugar c o n t e n t i n t e r f e r e d i n the D E A E - c e l l u l o s e f r a c t i o n a t i o n , p o o l s were f u r t h e r f r a c t i o n a t e d by g e l f i l t r a t i o n and rechromatographed on DEAE-cellulose. The e x i s t e n c e of the d i f f e r e n t t y p e s o f p e c t i n m o l e c u l e s was c o n firmed. Important a d d i t i o n a l i n f o r m a t i o n on the f i n e s t r u c t u r e was ob t a i n e d by s u b j e c t i n g i s o l a t e d p e c t i n s t o enzymic d e g r a d a t i o n by pure p e c t i n l y a s e o r p e c t a t e l y a s e . The d i g e s t s were t h e n f r a c t i o n a t e d by g e l f i l t r a t i o n chromatography on S e p h a c r y l S-300. A t y p i c a l p r o f i l e f o r a p e c t i n f r a c t i o n i s o l a t e d from c o l d b u f f e r e x t r a c t of AIS from r i p e a p p l e s w i t h 4% of the g l y c o s i d i c l i n k a g e s s p l i t by p e c t a t e l y a s e i s shown i n F i g . 4. F r a c t i o n s were c o l l e c t e d i n 4 p o o l s as i n d i c a t e d , the degree o f e s t e r i f i c a t i o n and n e u t r a l sugar c o m p o s i t i o n were e s t a b l i s h e d and are g i v e n i n the f i g u r e . P o o l α was found t o c o n t a i n o n l y 5% of the g a l a c t u r o n i c a c i d r e s i d u e s but c o n t a i n e d 90% o f the n e u t r a l sugar r e s i d u e s . The p e c t i n i n t h i s p o o l was h i g h l y e s t e r i f i e d . F r a c t i o n s (3, γ and 6 had a h i g h AUA c o n t e n t and a low l e v e l o f n e u t r a l s u g a r s . The degree of e s t e r i f i c a t i o n of the p e c t i n fragments i n t h e s e p o o l s v a r i e d between 50 and 81%. S i m i l a r o b s e r v a t i o n s were made f o r o t h e r i s o l a t e d p e c t i n s degraded w i t h p e c t i n l y a s e o r p e c t a t e l y a s e . From t h e s e r e s u l t s i t wae c o n c l u d e d t h a t a l most a l l n e u t r a l sugar r e s i d u e s a r e l i n k e d t o r e l a t i v e l y s h o r t s e g ments of the r h a m n o g a l a c t u r o n a n backbone ('Tiairy" r e g i o n s ) l e a v i n g l a r g e p a r t s of the r h a m n o g a l a c t u r o n a n backbone u n s u b s t i t u t e d ("smooth" r e g i o n s ) . E v i d e n c e f o r the c o n c e p t i o n of p e c t i n as c o n s i s t i n g of "smooth" and " h a i r y " r e g i o n s has a l s o been produced by o t h e r i n v e s t i g a t o r s (7, 12, 13) and t h i s c o n c e p t i o n i s a l s o i n a g r e e ment w i t h the e x i s t e n c e of f i v e t y p e s of p e c t i n s i n d i c a t e d by the n e u t r a l sugar d i s t r i b u t i o n c u r v e . T h i s can be v i s u a l i z e d by the s i m p l e model o f p e c t i n s of t y p e A t o Ε shown i n F i g . 5. Type Β was found t o be the dominant p e c t i n type i n the e x t r a c t s , t y p e A and Ε can be c o n s i d e r e d to be degraded p e c t i n s . T a b l e I shows the n e u t r a l sugar c o m p o s i t i o n as e s t i m a t e d f o r p e c t i n types A to Ε from r i p e a p p l e s e x p r e s s e d as moles sugar per mole a r a b i n o s e . F o r the p e c t i n s from u n r i p e a p p l e s s i m i l a r r e s u l t s were o b t a i n e d . I t can be seen t h a t the n e u t r a l sugar c o m p o s i t i o n f o r a l l p e c t i n t y p e s i s f a i r l y c o n s t a n t w i t h the e x c e p t i o n o f g a l a c t o s e . The same i s t r u e f o r p o o l s α t o δ o f F i g . 4; h e r e g a l a c t o s e i s p r a c t i c a l l y o n l y p r e s e n t i n p o o l a . From t h e s e r e s u l t s the c o n c l u s i o n was d e r i v e d t h a t w i t h i n the n e u t r a l sugar s i d e c h a i n b l o c k s a r e peating pattern i s present. I s o l a t e d " h a i r y " r e g i o n f r a c t i o n s were found to be r a t h e r r e s i s t a n t a g a i n s t d e g r a d a t i o n by p e c t i c enzymes. E n d o - 3 - 1 , 4 - g a l a c t a n a s e could release arabinogalactans and o l i g o m e r i c g a l a c t o s e from h a i r y r e g i o n f r a c t i o n s . They c o u l d a l s o be p a r t i a l l y degraded when




A B C

D

Ε

moles neutral sugar/mole A U A

Figure 3 . Neutral sugar d i s t r i b u t i o n curves of pectins extracted from ripe and unripe apple AIS. The t o t a l area represents 100$ of the AUA present i n the four extracts. ( — ) Unripe apples; ( ) ripe apples. (Reproduced with permission from reference 1. Copyright 1981 Carbohydrate Polymer).

AUA(mg/ml) 0 6-,

α Degree o f e s t e r i f i c a t i o n

(%)

% o f AUA Neutral

sugar

β

Ύ

δ

95

81

75

50

7

31

54

1 .33

.03

.02

.01

.08

.06

.10

.05

.09

.09

.10

.07

.87

.71

3.0

6.1

.10

.06

.10

.09

8

content

(moles/mole o f g a l a c t u r o n i c acid

residues)

moles rhamnose/mole moles xylose/mole

moles g a l a c t o s e / m o l e moles

arabinose

arabinose arabinose

glucose/molearabinose

Figure 4. G e l f i l t r a t i o n o f a p e c t a t e l y a s e degraded pectin. AUA = a n h y d r o - u r o n i c a c i d c o n t e n t , Ve = e l u t i o n volume, t h e e l u e n t was water.


4.

DE VRIES ET AL.

type A

type Ε

i Downloaded by UNIV OF ARIZONA on December 16, 2012 | http://pubs.acs.org Publication Date: June 5, 1986 | doi: 10.1021/bk-1986-0310.ch004

43


—I-

type Β

I-

H

type C

type D

Figure 5. Model of the pectins of types A-E. Horizontal l i n e s : rhamnogalacturonan backbone of the pectin molecule. Black areas: blocks of neutral sugar side chains. (Reproduced with permission from reference 2. Copyright 1982 Carbohydrate Polymer).

T a b l e I . N e u t r a l sugar c o m p o s i t i o n o f p e c t i n s e x t r a c t e d from r i p e and u n r i p e a p p l e s ( A I S ) . The c a p i t a l s A-E r e f e r t o F i g . 2. The n e u t r a l sugar c o m p o s i t i o n i s e x p r e s s e d as moles s u g a r p e r mole a r a b i n o s e . The v a l u e s a r e averages o f t h o s e f o r p e c t i n s w i t h about t h e same n e u t r a l s u g a r c o n t e n t i n t h e f o u r e x t r a c t s . Mannose was absent i n a l l c a s e s ( 1 ) . ripe

A

Β

C

D

Ε

rhamnose xylose galactose glucose

0.15 0.09 1.42 0.17

0.07 0.10 0.80 0.13

0.08 0.09 0.69 0.12

0.07 0.08 0.38 0.12

0.09 0.08 0.29 0.03

sugar content (moles/mole g a l . A . )

0.08

0.15

0.24

0.54

1.42

on

average

0.09 0.09 0.10

Source: Reproduced with permission from reference 1. Copyright 1981 Carbohydrate Polymer.


44


s u b j e c t e d to 3 - e l i m i n a t i o n a t pH 6, 100°C f o r 2 hours i n 0.05 M s o dium phosphate b u f f e r . F i g . 6 shows the g e l f i l t r a t i o n p r o f i l e s o f h a i r y r e g i o n f r a c t i o n s b e f o r e and a f t e r 3 - e l i m i n a t i o n and the sugar c o m p o s i t i o n o f degraded p e c t i n f r a c t i o n s . A h i g h p r o p o r t i o n o f x y l o s e r e s i d u e s was p r e s e n t i n the lower m o l e c u l a r weight f r a c t i o n s and t h i s p r o v i d e s e v i d e n c e f o r the p r e s e n c e of x y l o g a l a c t u r o n a n r e g i o n s a l s o r e p o r t e d by o t h e r i n v e s t i g a t o r s (12, 14).


D i s t r i b u t i o n o f M e t h o x y l a t e d and F r e e C a r b o x y l

Groups

We have a l r e a d y seen t h a t the degree of e s t e r i f i c a t i o n of the " h a i r y " r e g i o n s i s almost 100%. The homogalacturonan r e g i o n s have an a v e r age degree of e s t e r i f i c a t i o n of about 70%. The d i s t r i b u t i o n o f the m e t h o x y l a t e d and f r e e c a r b o x y l groups was, however, unknown. The i n t e r m o l e c u l a r d i s t r i b u t i o n was e s t a b l i s h e d by e s t i m a t i n g the degree of e s t e r i f i c a t i o n i n the many f r a c t i o n s o b t a i n e d by e x t e n s i v e f r a c t i o n a t i o n o f many p e c t i n f r a c t i o n s by g e l f i l t r a t i o n and DEAEc e l l u l o s e chromatography. These r e s u l t s a r e summarized i n F i g . 7 where the amounts of f r a c t i o n s w i t h a c e r t a i n degree o f e s t e r i f i c a t i o n ( e x p r e s s e d i n p r o p o r t i o n o f t o t a l AUA p r e s e n t i n a l l f r a c t i o n s ) are p l o t t e d v e r s u s degree o f e s t e r i f i c a t i o n (DE). I t can be seen t h a t almost a l l f r a c t i o n s were found t o have a DE of about 70-80%. M i n o r amounts of p e c t i n a r e p r e s e n t i n f r a c t i o n s w i t h a DE of 50% and i n f r a c t i o n s w i t h a DE of about 95%. I n f o r m a t i o n about the i n t r a m o l e c u l a r d i s t r i b u t i o n was d e r i v e d from s t u d i e s of t h e o l i g o m e r i c , p a r t i a l l y e s t e r i f i e d g a l a c t u r o n i d e s o b t a i n e d by e x t e n s i v e d e g r a d a t i o n of p e c t i n f r a c t i o n s by p e c t a t e l y a s e and p e c t i n l y a s e . The o l i g o m e r s were f r a c t i o n a t e d by HPLC u s i n g an amino-bonded s i l i c a column ( 1 5 ) . In F i g . 8 the chromatograms f o r p e c t i n s w i t h 8% o f the g l y c o s i d i c l i n k a g e s s p l i t by p e c t i n l y a s e and f o r p e c t i n s w i t h 7% o f the g l y c o s i d i c l i n k a g e s s p l i t by p e c t a t e l y a s e a r e shown. The p a r t i a l l y e s t e r i f i e d g a l a c t u r o n i d e s a r e s e p a r a t e d a c c o r d i n g t o the number of f r e e c a r b o x y l groups and not a c c o r d i n g t o c h a i n l e n g t h . Peaks a, b and c r e p r e s e n t o l i g o g a l a c t u r o n i d e s w i t h z e r o , one and two f r e e c a r b o x y l g r o u p s . The o l i g o m e r c o m p o s i t i o n o f a peak c o u l d be e s t a b l i s h e d by c o l d a l k a l i s a p o n i f i c a t i o n o f the m e t h y l a t e d g a l a c t u r o n i d e s i n the p o o l e d f r a c t i o n s . F i g . 9 shows the d i s t r i b u t i o n o f the degree of p o l y m e r i z a t i o n of the m e t h y l o l i g o g a l a c t u r o n i d e s c o n t a i n i n g one f r e e c a r b o x y l group (peak b) o b t a i n e d from a n a t i v e p e c t i n and a l s o the d i s t r i b u t i o n found f o r peak b o f t r a n s - e s t e r i f i e d p e c t i n w i t h the same DE as the n a t i v e p e c t i n . T h i s t r a n s - e s t e r i f i e d p e c t i n was o b t a i n e d by e s t e r i f i c a t i o n of t h e n a t i v e p e c t i n f r a c t i o n t o a DE of about 95% and subsequent s a p o n i f i c a t i o n i n the c o l d w i t h a c a l c u l a t e d amount of 0,1 M p o t a s s i u m h y d r o x i d e t o the o r i g i n a l DE of the n a t i v e p e c t i n . T h i s p r o c e d u r e i s supposed t o g i v e p e c t i n s w i t h a random d i s t r i b u t i o n p a t t e r n of the m e t h y l e s t e r groups i n c o n t r a s t to s a p o n i f i c a t i o n with c i t r u s p e c t i n e s t e r a s e which i s supposed to g i v e a b l o c k w i s e d i s t r i b u t i o n (16, 17). The d i s t r i b u t i o n p a t t e r n s o b t a i n e d showed o n l y s m a l l d i f f e r e n c e s , s u g g e s t i n g t h a t the n a t i v e d i s t r i b u t i o n and the m o d i f i e d d i s t r i b u t i o n a r e s i m i l a r . T h i s c o u l d be f u r t h e r s u b s t a n t i a t e d by t h e o r e t i c a l c o n s i d e r a t i o n s based on assumptions c o n c e r n i n g the mode o f a c t i o n of the enzymes (number of a d j a c e n t e s t e r i f i e d g a l a c t u r o n i d e r e s i d u e s n e c e s s a r y f o r bond s p l i t t i n g ) and the i n t r a m o l e c u l a r d i s t r i b u t i o n of methoxyl groups (DE,



DE VRIES ET AL.


Figure 6. Gel f i l t r a t i o n of apple pectin "hairy" regions a f t e r β-elimination on Sephacryl S-300. (Reproduced with permission from reference 3. Copyright 1983 Carbohydrate Polymers).

proportion of total AUA

A U Λ 40

60

100 80 10 DE(%)

Figure 7. O c c u r r e n c e o f p e c t i n s w i t h a c e r t a i n degree e s t e r i f i c a t i o n i n p e c t i n e x t r a c t s o f a p p l e AIS.


of


46


Figure 8. High-pressure l i q u i d chromatograms of pectin fractions degraded with pectin lyase (a) and pectate lyase (b) to degradation l i m i t s , r e s p e c t i v e l y , of 18> and 7%. (Reproduced with permission from reference 4. Copyright 1983 Carbohydrate Polymer).

of total AUA

•

modified distribution

^2

native distribution

5

6 7 degree of polymerization

Figure 9. D i s t r i b u t i o n of the degree of polymerization of methyloligogalacturonides containing one free carboxyl group obtained from a native and from a t r a n s e s t e r i f i e d p e c t i n . Methyloligogalacturonides were completely d e e s t e r i f i e d ( i n a l k a l i ) and the degree of polymerization of oligogalacturonides was determined by HPLC. (Reproduced with permission from reference 4. Copyright 1983 Carbohydrate Polymer).



47


4. DE VRIES ET AL.

i n f i n i t e c h a i n l e n g t h and random d i s t r i b u t i o n ) . W i t h h e l p o f s t a t i s t i c s t h e f r e q u e n c i e s o f o c c u r r e n c e o f c e r t a i n sequences i n a p e c t i n m o l e c u l e and t h e o c c u r r e n c e o f m e t h y l o l i g o g a l a c t u r o n i d e s w i t h z e r o , one e t c . f r e e c a r b o x y l groups c a n be c a l c u l a t e d . The d i f f e r e n c e s b e tween c a l c u l a t e d and e x p e r i m e n t a l d a t a were found t o be r e l a t i v e l y small. The e f f e c t o f d i f f e r e n t i n t r a m o l e c u l a r d i s t r i b u t i o n s o f methoxyl a t e d and f r e e c a r b o x y l groups on t h e d i s t r i b u t i o n o f t h e degree o f p o l y m e r i z a t i o n o f o l i g o g a l a c t u r o n i d e s i n enzymatic d i g e s t s o f p e c t i n s i s i l l u s t r a t e d i n F i g . 10. These d a t a were o b t a i n e d by s a p o n i f y i n g 95% e s t e r i f i e d p e c t i n t o 60% e s t e r i f i c a t i o n w i t h c i t r u s p e c t i n e s t e r a s e (PE, s a p o n i f i e d ) o r w i t h a l k a l i ( a l k a l i s a p o n i f i e d ) and deg r a d i n g r e s p e c t i v e l y 12% and 8% o f t h e g l y c o s i d i c l i n k a g e s o f t h e two p e c t i n s w i t h p e c t a t e l y a s e . The d i g e s t s were s a p o n i f i e d and a n a l y z e d f o r o l i g o g a l a c t u r o n i d e c o m p o s i t i o n by HPLC. I n t h e PE s a p o n i f i e d p e c t i n d i g e s t t h e lower o l i g o g a l a c t u r o n i d e s were p r e s e n t i n l a r g e r amounts. T h i s i s due t o t h e p r e f e r e n t i a l a t t a c k o f p e c t a t e l y a s e on d e - e s t e r i f i e d r e g i o n s . These r e s u l t s a l s o i n d i c a t e a random distribution. Conclusion Chromatographic t e c h n i q u e s i n c o m b i n a t i o n w i t h pure enzymes have been used s u c c e s s f u l l y as t o o l s i n t h e e l u c i d a t i o n o f t h e f i n e s t r u c t u r e of a p p l e p e c t i n s . A p p l e p e c t i n s were found t o c o n s i s t o f " h a i r y r e g i o n s , h a v i n g a backbone o f rhamnogalacturonan c a r r y i n g a r a b i n o g a l a c t a n s i d e c h a i n s and x y l o g a l a c t u r o n a n and "smooth" r e g i o n s , t h e l a t t e r b e i n g homogalacturonans w i t h a degree o f e s t e r i f i c a t i o n o f 70-80%. The i n t r a m o l e c u l a r d i s t r i b u t i o n o f t h e methoxyl groups c o u l d not be d i s t i n g u i s h e d from a random d i s t r i b u t i o n . F u t u r e r e s e a r c h e f f o r t s s h o u l d be d i r e c t e d t o t h e problem o f p o s s i b l e c o n n e c t i o n s between t h e s e s t r u c t u r a l f e a t u r e s o f p e c t i n and i t s p r o p e r t i e s as g e l l i n g agent and as d i e t a r y f i b r e . 1 1

%

of total AUA

3

Structural Studies of Apple Pectins with Pectolytic Enzymes - American

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