Chapter 7
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Pectin-Degrading Enzymes in Fruit and Vegetable Processing A. G. J. Voragen and W. Pilnik Department of Food Science, Agricultural University,Bomenweg2, 6703HDWageningen,Netherlands Pectic substances are important structural components of the middle lamellea and the primary cell walls of higher plants, in particular fruits and vegetables. The extent and mode of their degradation by enzymes affect many aspects of the processing of fruits and vegetables and many quality attributes of fruit and vegetable products. Pectic enzymes occur as endogenous enzymes in higher plants; they are also added as processing aids during processing. Technical pectinase preparations are mainly derived from Aspergillus niqer and contain next to pectic enzymes many other carbohydrases. The classification of pectic enzymes in general, their occurrence in higher plants and micro-organisms and the properties of pectic enzymes from some plants and food grade micro-organisms are described with special emphasis on their substrate specificity. Their technological roles and applications, also in combination with (hemi-)cellulases, in a variety of processes are discussed. Evidence is presented for the existence of a new type of pectic enzyme which acts specifically in the hairy regions of pectic substances. Pectins are important constituents of the c e l l walls of edible parts of f r u i t s and vegetables. They are the main components of the middle lamellea which has led to the c l a s s i c a l view that they function as i n t e r c e l l u l a r adhesive. The primary walls are considered as a firmgel composed of cellulose m i c r o f i b r i l s embedded i n a matrix of pectins, hemi-cellulose and protein with l i g n i n v i r t u a l l y absent. In the secondary walls, which usually occur, cellulose and hemicellulose prevail (1-4). The processing of f r u i t s and vegetables cause changes i n pectic substances. Changes i n texture of f r u i t s and vegetables during β
0097-6156/89/0389-0093$06.75/0 1989 American Chemical Society
In Biocatalysis in Agricultural Biotechnology; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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r i p e n i n g and post h a r v e s t s t o r a g e , and changes i n c o n s i s t e n c y and c l o u d b e h a v i o r of p r o d u c t s d e r i v e d from f r u i t s and v e g e t a b l e s d u r i n g p r o c e s s i n g or s t o r a g e are i n g e n e r a l a s c r i b e d t o changes brought about by endogenous p e c t i c enzymes ( 5 , 6 ) . T e c h n i c a l p e c t i n a s e p r e p a r a t i o n s , commonly d e r i v e d from A s p e r g i l l u s n i q e r or r e l a t e d f u n g i , are produced i n d u s t r i a l l y as p r o c e s s i n g a i d s , m a i n l y f o r f r u i t j u i c e e x t r a c t i o n and c l a r i f i c a t i o n ( 7 - 9 ) . With i n c r e a s i n g knowledge of the f i n e s t r u c t u r e of p e c t i n s as c e l l w a l l components and of the p r o p e r t i e s of p e c t i c enzymes and t h e i r t e c h n o l o g i c a l r o l e s , the management of endogenous enzyme systems and the c o n v e n t i o n a l a p p l i c a t i o n s of t e c h n i c a l p e c t i n a s e p r e p a r a t i o n s have been improved and new a p p l i c a t i o n s have been developed. Pectin structure P e c t i n s are composed of a rhamno-galacturonan backbone i n which 1,4l i n k e d α-D-galacturonan c h a i n s are i n t e r r u p t e d at i n t e r v a l s by the i n s e r t i o n of 1 , 2 - l i n k e d α-L-rhamnopyranosyl r e s i d u e s . Other c o n s t i t u e n t sugars are a t t a c h e d i n s i d e c h a i n s and i n c l u d e p r e d o m i n a n t l y D - g a l a c t o s e , L - a r a b i n o s e and D-xylose. P a r t of t h e s e sugars occur i n s h o r t s i d e c h a i n s , one t o t h r e e u n i t s l o n g , g l y c o s i d i c a l l y l i n k e d t o C-4 and/or C-3 of L-rhamnose or C-2 or C-3 of some of the g a l a c t u r o n a t e r e s i d u e s . The major sugars Dg a l a c t o s e and L - a r a b i n o s e are p r e s e n t i n more complex c h a i n s of c o n s i d e r a b l e l e n g t h w i t h s t r u c t u r e s s i m i l a r t o a r a b i n a n s and ( a r a b i n o - ) g a l a c t a n s . P a r t 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 c a r r y m e t h y l e s t e r groups; a c e t y l groups may o c c u r as s u b s t i t u e n t s t o the h y d r o x y l groups at C-2 and C-3 ( 1 , 2 ) . P e c t i n s from sugar beet and s p i n a c h have been found t o c a r r y e s t e r l i n k e d f e r u l o y l - g r o u p s at t e r m i n a l a r a b i n o s y l and g a l a c t o s y l r e s i d u e s of the s i d e c h a i n s (1012). By d e g r a d a t i o n of e x t r a c t e d , p u r i f i e d p e c t i n s from v a r i o u s s o u r c e s s p e c i f i c a l l y i n the g a l a c t u r o n a n backbone by β-elimination or e n z y m a t i c a l l y w i t h depolymerases, e s s e n t i a l l y pure, degraded g a l a c t u r o n a n f r a c t i o n s and p e c t i n f r a c t i o n s of h i g h e r m o l e c u l a r weight i n which v i r t u a l l y a l l of the n e u t r a l sugars were c o n c e n t r a t e d have been o b t a i n e d (13-18). From these r e s u l t s i t was concluded t h a t t h e r e i s an 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 i n which the n e u t r a l sugar r e s i d u e s are c o n c e n t r a t e d i n b l o c k s of more h i g h l y s u b s t i t u t e d rhamno-galacturonan r e g i o n s ( " h a i r y " ) , s e p a r a t e d by u n s u b s t i t u t e d ("smooth") r e g i o n s c o n t a i n i n g almost e x c l u s i v e l y D - g a l a c t o s y l u r o n i c r e s i d u e s (16) ( F i g . l ) . The i s o l a t i o n of rhamnogalacturonans r i c h i n n e u t r a l sugars from c e l l w a l l s u s i n g p e c t o l y t i c enzymes i s i n agreement w i t h t h i s concept (19). For apple p e c t i n f r a c t i o n s De V r i e s et a l . (20) e s t a b l i s h e d t h a t the g a l a c t o s y l u r o n i c r e s i d u e s i n the h a i r y r e g i o n s were almost c o m p l e t e l y m e t h y l a t e d ; i n the smooth r e g i o n s 70% of them were m e t h y l a t e d . P e c t i c enzymes; c l a s s i f i c a t i o n and s u b s t r a t e
specificity
P e c t i c enzymes are d e f i n e d and c l a s s i f i e d on the b a s i s of t h e i r a c t i o n toward the g a l a c t u r o n a n p a r t of the p e c t i n s . Two main groups are d i s t i n g u i s h e d : p e c t i n m e t h y l e s t e r a s e s ( p e c t i n e s t e r a s e , PE;EC 3.1.11.1), which s p l i t o f f methoxy groups from p e c t i n , t r a n s f o r m i n g
In Biocatalysis in Agricultural Biotechnology; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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7.
V O R A G E N AND PILNIK
Pectin-Degrading Enzymes
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i t g r a d u a l l y i n t o low e s t e r p e c t i n s and p e c t i c a c i d , and p e c t i n depolymerases, which s p l i t the g l y c o s i d i c l i n k a g e s i n the g a l a c t u r o n a n backbone. Three groups o f p e c t i n depolymerases e x i s t : p o l y g a l a c t u r o n a s e s ( g a l a c t u r o n a s e s , PG;EC 3.2.2.15 and 3.2.1.67) s p l i t g l y c o s i d i c l i n k a g e s next t o f r e e c a r b o x y l groups i n the g a l a c t u r o n a n c h a i n by h y d r o l y s i s ; p e c t a t e l y a s e s (PAL;EC 4.2.2.2 and 4.2.2.9) degrade g l y c o s i d i c l i n k a g e s next t o f r e e c a r b o x y l groups by β-elimination ( F i g . 2 ) ; they have an a b s o l u t e requirement f o r c a l c i u m i o n s . Both endo and exo types o f PGs and PALs are known. The endo types (EC 3.2.1.15, EC 4.2.2.2) s p l i t the p e c t i n c h a i n a t random; a s t r o n g r e d u c t i o n i n v i s c o s i t y o f the s u b s t r a t e s o l u t i o n w i l l be accompanied by a s m a l l i n c r e a s e i n r e d u c i n g end groups. Exo-PGs (EC 3.2.1.67) s p l i t o f f mono- o r dimers from the n o n - r e d u c i n g end o f t h e c h a i n ; exo-PALs s p l i t o f f u n s a t u r a t e d dimers from the r e d u c i n g end. T h i s means t h a t the v i s c o s i t y o f a s u b s t r a t e s o l u t i o n i s reduced o n l y v e r y s l o w l y . For p o l y g a l a c t u r o n a s e s , as w e l l as f o r p e c t a t e l y a s e s , p e c t a t e and low methoxyl p e c t i n s are the p r e f e r r e d s u b s t r a t e s , w h i l e they are h a r d l y a c t i v e on h i g h l y m e t h y l a t e d p e c t i n s . I n c o n t r a s t p e c t i n l y a s e s (PL;EC 4 . 2 . 2 . 1 0 ) , another group o f e l i m i n a t i v e s p l i t t i n g enzymes, are s p e c i f i c f o r h i g h l y methylated p e c t i n s . Of t h i s group o n l y the endo type has been d e s c r i b e d . N a t u r a l l y o c c u r r i n g p e c t i n s can be e f f i c i e n t l y degraded by the c o m b i n a t i o n o f p e c t i n e s t e r a s e and p o l y g a l a c t u r o n a s e o r p e c t a t e l y a s e , o r e l s e , i f they art s u f f i c i e n t l y h i g h l y m e t h y l a t e d by p e c t i n l y a s e alone ( 1 9 , 2 6 ) . A t h i r d group o f h y d r o l y t i c enzymes s p e c i f i c f o r h i g h l y m e t h y l a t e d p e c t i n s ( p o l y m e t h y l g a l a c t u r o n a s e , EC 3.2.1.41) has been d e s c r i b e d p a r t i c u l a r l y i n the o l d e r l i t e r a t u r e p r i o r t o the d i s c o v e r y o f the p e c t i n l y a s e by A l b e r s h e i m e t a l . ( 2 1 ) . We have never been a b l e t o f i n d e i t h e r exo-PL nor endo- and exo-PMG i n o u r e x t e n s i v e s c r e e n i n g o f commercial p r e p a r a t i o n s and m i c r o - o r g a n i s m s . Many a u t h o r s now agree t h a t enzymes d e s c r i b e d as endo-or exo-PMG may v e r y w e l l have been a combined PE-PG a c t i v i t y , o r a PL a c t i v i t y o r even PG a c t i v i t y alone i n cases where o n l y i n i t i a l v e l o c i t i e s were measured. E x t e n s i v e reviews o f p e c t i c enzymes have been p u b l i s h e d (22-26).
P i l n i k e t a l . (27) and Rombouts (28) have determined the k i n e t i c parameters Κα and Vnax and d e g r a d a t i o n l i m i t s o f p u r i f i e d endo-PG from Kluyveromvces f r a q i l i s , endo-PL from A s p e r g i l l u s n i g e r and v a r i o u s endo-PALs on p e c t i n s w i t h d i f f e r e n t c o n t e n t s o f methoxyl e s t e r groups which were randomly d i s t r i b u t e d a l o n g the g a l a c t u r o n a n c h a i n . These p e c t i n s were prepared by p a r t i a l a l k a l i n e s a p o n i f i c a t i o n of a h i g h l y ( 9 5 % ) methylated p e c t i n a t low temperature t o a v o i d c h e m i c a l t r a n s - e l i m i n a t i v e d e g r a d a t i o n . F i g u r e 3 shows the r e l a t i o n found between degree o f m e t h y l a t i o n and i n i t i a l a c t i v i t y f o r f u n g a l endo-PL and endo-PAL from B a c i l l u s polvmyxa, Pseudomonas f l u o r e s c e n s and A r t h r o b a c t e r 370 under c o n d i t i o n s o f s a t u r a t i o n w i t h s u b s t r a t e . For endo-PAL, low methoxyl p e c t i n s a r e , s u r p r i s i n g l y , the best s u b s t r a t e s and optima have been found a t m e t h y l a t i o n l e v e l s o f 2 6 % , 3 5 - 4 0 % and 4 4 % , r e s p e c t i v e l y . A l s o the d e g r a d a t i o n l i m i t s and the a f f i n i t y (measured by 1/Km) o f the enzymes f o r the s u b s t r a t e s showed s i m i l a r p a t t e r n s . R e s u l t s o b t a i n e d by Rombouts (28) f o r PAL from Pseudomonas f l u o r e s c e n s i n d i c a t e t h a t the a f f i n i t y i s not o n l y i n f l u e n c e d by the degree o f e s t e r i f i c a t i o n but a l s o by the c a l c i u m c o n c e n t r a t i o n . For exo-PAL the best s u b s t r a t e i s p e c t a t e .
In Biocatalysis in Agricultural Biotechnology; Whitaker, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.
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BIOCATALYSIS IN AGRICULTURAL BIOTECHNOLOGY
Figure 1. Schematic structure of apple pectin; rhamnogalacturonan backbone with regions r i c h i n neutral sugar side chains (hairy regions).
^pectinesterase COOCH3
COOCH3
COIOCH3
COOH
COOCH
3
OOOCH
3
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