Enzymes in Food and Beverage Processing

10 Enzymes Involved in Fruit Softening RUSSELL PRESSEY

Downloaded by UNIV OF MINNESOTA on October 1, 2013 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch010

R. B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Ga. 30604

Softening o f the f l e s h i s one of the most dramatic changes accompanying the r i p e n i n g o f many fruits. Although other parame t e r s o f q u a l i t y are important, the peak o f fruit ripeness i s u s u a l l y a s s o c i a t e d with a fairly narrow range of firmness. Furthermore, texture that i s considered optimal f o r fruit consumed f r e s h may not be best f o r fruit that i s processed. S o f t e n ing undoubtedly r e f l e c t s changes i n c e l l walls of fruit t i s s u e s as the fruit progresses through r i p e n i n g i n t o senescence. Once the process i s initiated i n mature fruit, the period o f acceptable texture may be s h o r t , even with r e f r i g e r a t i o n and c o n t r o l l e d atmosphere s t o r a g e . A p r e r e q u i s i t e to improved methods o f c o n t r o l l i n g s o f t e n i n g , whether to initiate and a c c e l e r a t e it or to prevent s o f t e n i n g beyond the optimum stage, i s an understanding o f c e l l wall s t r u c t u r e , i t s changes, and the enzymes involved in its degradation. I.

H i s t o l o g y of Fleshy

Fruits.

A p p r e c i a t i o n o f the biochemical aspects o f fruit softening r e q u i r e s some knowledge o f the development and h i s t o l o g y o f fruits. F r u i t growth may be considered to begin i n the f l o r a l primordium (1_). The ovary wall becomes the p e r i c a r p o f the fruit. During development, the ground t i s s u e o f the p e r i c a r p remains r e l a t i v e l y homogeneous and p a r e n c h y m a l c . Initial development occurs mainly through c e l l m u l t i p l i c a t i o n . The p o s t f e r t i l i z a t i o n period i s marked by c e l l enlargement, although cell division a l s o continues i n ovaries o f l a r g e - f r u i t e d plants (2j. The p e r i c a r p may become d i f f e r e n t i a t e d i n t o three d i s t i n c t parts: the exocarp, the mesocarp, and the endocarp. F r u i t s are h i g h l y d i v e r s i f i e d i n t h e i r morphology, and f r u i t development i s not r e s t r i c t e d to the ovary but often involves n o n c a r p e l l a r y parts of the flower ( 2 ) . I f the e n t i r e ground t i s s u e develops i n t o f l e s h y t i s s u e , the f r u i t i s a b e r r y . A l l the f l e s h y t i s s u e of a berry may o r i g i n a t e from the ovary w a l l , 172

In Enzymes in Food and Beverage Processing; Ory, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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as in the grape. In c o n t r a s t , a considerable part of the tomato f r u i t c o n s i s t s of p l a c e n t a . The body of the tomato i s p e r i c a r p developed from the ovary wall and c o n s i s t s of o u t e r , r a d i a l , and inner w a l l s . During development, the placenta shows much more a c t i v e c e l l d i v i s i o n than the ovary w a l l . The r e s u l t i s l o c u l a r c a v i t i e s i n the p e r i c a r p that contain the seeds imbedded i n a j e l l y - l i k e tissue ( 2 J . Mohr and S t e i n (3) have described the f i n e s t r u c t u r a l changes in the outer p e r i c a r p of tomato f r u i t during development and r i p e n i n g . S h o r t l y a f t e r f e r t i l i z a t i o n , the vacuoles of each c e l l of the p e r i c a r p enlarge to form one l a r g e vacuole, so that a l a y e r of protoplasm l i n e s the inner surface of the c e l l w a l l s . I n t e r c e l l u l a r spaces develop subsequently, and c e l l walls par t i a l l y separate along the middle lamella r e g i o n , s t a r t i n g at the i n t e r c e l l u l a r spaces. As the f r u i t nears m a t u r i t y , the c e l l s become very l a r g e ( 1 0 0 - 5 0 0 μ diameter) and separation of c e l l w a l l s i n c r e a s e s . Degeneration of protoplasm begins, with the disappearance of membraneous s t r u c t u r e s . In the r i p e , red f r u i t , separation of adjacent c e l l s i s common, and degeneration of protoplasmic components i s pronounced. The development of many f r u i t s involves maturation of the ovary wall i n t o a p e r i c a r p with a conspicuous stony endocarp surrounded by a f l e s h y mesocarp. Important stone f r u i t s include the o l i v e , peach, plum, a p r i c o t , and c h e r r y . The f l e s h y t i s s u e of the peach c o n s i s t s of l o o s e l y packed parenchyma c e l l s that increase in s i z e from the periphery toward the i n t e r i o r . The mesocarp grows by r a p i d c e l l enlargement. I n t e r c e l l u l a r spaces are common in peach t i s s u e . C e l l wall thickness increases during peach development from 0 . 5 μ , a f t e r c e s s a t i o n of c e l l d i v i s i o n , to about l y , during the p i t hardening phase ( 4 ) . Increases in c e l l wall thickness are more marked in the l a t e phases of c e l l growth, and reach a maximum of about 2y in hard r i p e f r u i t . During subsequent r i p e n i n g , the c e l l w a l l s decrease in thickness. There are two d i s t i n c t types of peaches, the soc a l l e d "freestone" and " c l i n g s t o n e " v a r i e t i e s . E a r l y reports (5) described d i f f e r e n c e s in c e l l wall thickness between the two t y p e s , but Reeve ( 4 ) could not confirm t h i s and observed that the c e l l w a l l s decrease in thickenss to the same degree in c l i n g s t o n e as in freestone f r u i t . C e l l walls do not appear to rupture during r i p e n i n g ( 4 ) . The apple i s an example of another v a r i a t i o n in f r u i t development. The f l e s h of the apple i s derived from the f l o r a l tube as well as from the c a r p e l l a r y t i s s u e . The ovary region (the core) c o n s i s t s of f i v e c a r p e l s imbedded in f l e s h y paren c h y m a l c exocarp. The f l o r a l tube region of the a p p l e , which forms the bulk of the e d i b l e p a r t , a l s o c o n s i s t s of parenchyma cells. During growth, c e l l d i v i s i o n ceases at about 3 weeks a f t e r f u l l bloom (6j. Subsequent increase in f r u i t s i z e i s due mainly to c e l l enlargement and increase in the volume of i n t e r c e l l u l a r spaces, which are abundant i n apple t i s s u e ( 7 J . L


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According to Nelmes and Preston (7J, r a p i d wall synthesis during c e l l extension i s followed by r e a s s i m i l a t i o n o f the p e c t i c mater i a l s j u s t before m a t u r i t y . The c e l l walls decrease i n thickness at t h i s stage. Despite the d i v e r s i t i e s i n f r u i t s i n s i z e , shape, s t r u c t u r e , and development, patterns o f r i p e n i n g tend to be q u i t e s i m i l a r . Chlorophyll i n the c h l o r o p l a s t s o f the outermost c e l l s decreases and e v e n t u a l l y disappears. Carotenoids and anthocyanins develop and give f r u i t s t h e i r c h a r a c t e r i s t i c c o l o r s . Other changes are the development o f s p e c i f i c f l a v o r s , increases i n sweetness, and decreases i n a c i d content. The l o s s o f firmness associated with r i p e n i n g can be a t t r i b u t e d to changes i n the s t r u c t u r e o f c e l l w a l l s which w i l l be discussed next. II.

C e l l Wall S t r u c t u r e .

The f l e s h o f succulent f r u i t s c o n s i s t s l a r g e l y or e n t i r e l y o f parenchyma c e l l s (2J. These c e l l s are r e l a t i v e l y u n d i f f e r e n t i a t e d , but are h i g h l y complex p h y s i o l o g i c a l l y because they possess l i v i n g p r o t o p l a s t s . They are g e n e r a l l y polyhedral or elongated with t h i n primary w a l l s . The walls o f two contiguous c e l l s are separated by the i n t e r c e l l u l a r substance or middle l a m e l l a which i s r i c h i n p e c t i c substances (8). The d i s t i n c t i o n often i s not obvious between the middle lamella and the c e l l wall which appear as a u n i t . Mature parenchyma t i s s u e has abundant i n t e r c e l l u l a r spaces ( 9 ) . C e l l wall composition o f plants i s u s u a l l y determined on the e t h a n o l - i n s o l u b l e f r a c t i o n , which c o n s i s t s p r i m a r i l y of the c e l l wall and middle l a m e l l a . Jermyn and Isherwood (1_0) have found t h a t t h i s f r a c t i o n o f r i p e pears contains 21.4% glucosan, 3.5% g a l a c t a n , 1.1% mannan, 21% x y l a n , 10% araban, 11.5% p o l y g a l a c t u r o n i c a c i d , and 16.1% l i g n i n . Tomato c e l l w a l l s contain 17% c e l l u l o s e , 22% p e c t i n , 17% p r o t e i n , 21% araban-galactan, and 1323% xylose plus glucose (1J_). Knee (1_2) has reported the comp o s i t i o n o f a c e t o n e - i n s o l u b l e residues o f apples i n mg/g t i s s u e are: a r a b i n o s e , 2.58; x y l o s e , 0.52; g a l a c t o s e , 3.81; glucose 32.8; and g a l a c t u r o n i c a c i d , 3.53. The composition o f dates (percent, dry weight basis o f the whole f r u i t ) i s : 0.8% c e l l u l o s e , 1.5% h e m i c e l l u l o s e a , 0.8% hemicellulose b, 3.7% p e c t i n , and 0.3% l i g n i n ( 1 3 ) . C e l l u l o s e i s the s k e l e t a l substance o f the plant c e l l wall and, as the above data show, i t i s a major wall component i n most fruits. It i s a l i n e a r 3-1,4-glucosan. There have been occas i o n a l suggestions that other monosaccharides may be present (14), but c e l l u l o s e i s by f a r the most homogeneous polysaccharide i n the c e l l w a l l . The lengths o f i n d i v i d u a l c e l l u l o s e chains have been estimated a t 6000-8000 monomers (1_5). These chains are combined i n t o bundles, c a l l e d m i c r o f i b r i l s , that are up to 250°A wide and contain 2000 molecules i n a t r a n s e c t i o n (1_6). The



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arrangement of molecules apparently i s o r d e r l y because X-ray studies have demonstrated regions of c r y s t a l l i n i t y in the microfibrils. The f r i b r i l l a r system i s i n t e r p e n e t r a t e d by c a p i l l a r i e s of various s i z e s , which are occupied by water, p a r a c r y s t a l l i n e c e l l u l o s e , and n o n c e ! l u l o s i c components of the c e l l w a l l . In the newly formed primary w a l l , the o r i e n t a t i o n of m i c r o f i b r i l s i s predominately t r a n s v e r s e , but becomes more d i s p e r s e during c e l l enlargement. The bulk of the primary wall i s n o n c e l l u l o s i c polysacchar i d e s , forming a dense, amorphous gel in which the c e l l u l o s e m i c r o f i b r i l s are imbedded. Meristematic and parenchymous t i s s u e s are p a r t i c u l a r l y r i c h in polysaccharides c a l l e d the p e c t i c substances ( p e c t i n ) . Pectin i s the main component of the middle l a m e l l a , as was i n d i c a t e d e a r l i e r , but i t i s a l s o present in the primary c e l l w a l l . The s t r u c t u r e of pectin may vary with the source. For example, a pure homogalacturonan has been i s o l a t e d from sunflower heads (17) and J a c k f r u i t (18). But the basic s t r u c t u r e of p e c t i n from most sources c o n s i s t s of long blocks of l i n e a r «-l ,4-galacturonan i n t e r s p e r s e d by rhamnose through CI and C2 (19., 20, 21_, 22). Talmadge et a l . (23) suggested that the p e c t i n from suspension c u l t u r e d sycamore c e l l s c o n s i s t s of blocks of 8 u n i t s of g a l a c t u r o n i c a c i d i n t e r r u p t e d by u n i t s of rhamnose-galacturonic acid-rhamnose. Neutral sugars other than rhamnose may be present i n f r u i t pectins. For example, p u r i f i e d pectin from apples contained, in a d d i t i o n to 1.2% rhamnose, 9.3% arabinose, 1.4% g a l a c t o s e , 0.80% x y l o s e , and t r a c e s of f u c o s e , 2-0-methylxylose and 2-0-methylfucose (21). Galacturonosylgalactose and g a l a c t u r o n o s y l x y l o s e have been t e n t a t i v e l y i d e n t i f i e d in the hydrolyzates of apple p e c t i n (21_), but the linkages remain unknown. It i s not c l e a r whether the neutral sugars occur in the galacturonan chain or as branches on the c h a i n . Rees and Wight (24) estimated that about 60% of the p e c t i n s from mustard cotyledons c a r r y side c h a i n s . Branching of g a l a c t u r o n i c a c i d i s thought to occur mainly through C3. Pectins from f r u i t t i s s u e s are considered to be much l e s s branched, however (20., 21_, 22). The only g a l a c t u r o n o s y l - g a l a c t u r o n i c a c i d linkage i d e n t i f i e d in p e c t i n i s «-1,4. On t h i s b a s i s , i t i s u n l i k e l y that branches of galacturonan, i f they e x i s t , are attached d i r e c t l y to the galacturonan backbone. It i s p o s s i b l e , however, that such branches are attached through neutral sugars on the c h a i n . The carboxyl group in g a l a c t u r o n i c a c i d introduces another v a r i a b l e i n the s t r u c t u r e of p e c t i n . These groups are p a r t l y e s t e r i f i e d with methanol, but the d i s t r i b u t i o n of e s t e r groups i s not known. It has been suggested that the f r e e carboxyl groups may be involved i n i n t e r m o l e c u l a r linkages (25). Calcium i n t e r a c t s with f r e e carboxyl groups to form i n s o l u b l e s a l t s . By forming i n t e r m o l e c u l a r b r i d g e s , calcium could c o n t r i b u t e to the s t r u c t u r e and binding p r o p e r t i e s of p e c t i n in the c e l l w a l l .



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Doesburg (26) has proposed that movement of calcium i n c e l l walls may cause s o l u b i l i z a t i o n of p e c t i n during f r u i t r i p e n i n g . Because arabinan and galactan often accompany the g a l a c turonan i n preparation of f r u i t p e c t i n s , many e a r l y workers considered the p e c t i n substances i n terms of the three p o l y s a c charides. A p o s s i b l e reason f o r t h i s apparent a s s o c i a t i o n may be simply that t h e i r s o l u b i l i t i e s are s i m i l a r , although recent studies on p e c t i n from suspension-cultured sycamore c e l l s sug gested covalent i n t e r a c t i o n s (23). H i r s t and Jones (27) sep arated the arabinans from apple and c i t r u s pectins and found that they c o n s i s t mainly of arabinose i n a h i g h l y branched structure. Later workers (28) questioned the existence of a homoarabinan. B a r r e t t and Northcote (21) i s o l a t e d , from apple p e c t i n , an arabinan-galactan complex containing nearly equal q u a n t i t i e s o f the monomers, but could not separate the two components. In c o n t r a s t , Talmadge et a l . (23) showed that a branched arabinan and a l i n e a r galactan are released from syca more c e l l p e c t i n by endopolygalacturonase treatment. The galactan i n f r u i t pectins has not been c h a r a c t e r i z e d , but by analogy with Lupinus a!bus galactan (29), i s assumed to be a l i n e a r 3-1,4-polymer. In a d d i t i o n to the e s s e n t i a l l y homogeneous arabinans and g a l a c t a n s , h i g h l y branched polysaccharides c o n t a i n ing both arabinose and galactose are common i n plants (21_). The remaining n o n c e l l u l o s i c polysaccharides i n the c e l l wall can be s o l u b i l i z e d with i n c r e a s i n g concentrations of KOH. The hemicelluloses are c h a r a c t e r i z e d by d i v e r s i t y i n composition, l i n k a g e s , and branching. Mannose, g a l a c t o s e , arabinose, x y l o s e , g l u c o s e , and other monomers may be present. These a l k a l i - s o l u b l e polysaccharides are complex mixtures that vary with the source and method o f e x t r a c t i o n . The stumbling block i n c h a r a c t e r i z i n g them has been d i f f i c u l t y in separating the components. There have been a few reports of homogeneous polysaccharides obtained by d i f f e r e n t i a l e x t r a c t i o n and p r e c i p i t a t i o n . A 3-1,4-xylan with g l u c u r o n i c a c i d s i d e chains has been i s o l a t e d from pear c e l l walls (30). A complex glucan has been extracted from mango f r u i t ( 3 1 ) . C a l l o s e , a 3 - 1 , 3 - g l u c a n , may be present i n f r u i t tissues"T32). Bauer et a l . (33) obtained from sycamore c e l l s , a neutral f r a c t i o n c o n s i s t i n g almost e x c l u s i v e l y of x y l o g l u c a n . The s t r u c t u r e of t h i s polymer i s a repeating u n i t containing 4 residues of β-1,4-1 inked glucose and 3 residues of x y l o s e , with s i n g l e xylose branches on 3 of the glucosyl r e s i d u e s . Plant c e l l w a l l s contain a small amount of p r o t e i n that has r e l a t i v e l y high proportions of seryl and hydroxyprolyl residues (34, 3 5 ) . I t has been proposed that t h i s p r o t e i n serves a s t r u c t u r a l r o l e on the basis that c e l l wall polysaccharides form g l y c o s i d i c linkages with the hydroxy-acid residues (36). Knee (37) confirmed that c e l l walls from apples contain a low l e v e l of g l y c o p r o t e i n r i c h i n hydroxyproline. This g l y c o p r o t e i n i s r e l a t i v e l y s o l u b l e and can be separated from the galacturonan by ion exchange chromatography. Knee concluded that the g l y c o p r o t e i n



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and galacturonan are not l i n k e d c o v a l e n t l y , but he suggested that the two components may be p h y s i c a l l y a s s o c i a t e d in the c e l l wall. The current view on c e l l wall s t r u c t u r e i s that the p o l y saccharides and p r o t e i n form a polymeric network, i n v o l v i n g covalent linkages and hydrogen bonding (38). The model proposed f o r the s t r u c t u r e of suspension-cultured sycamore c e l l walls i s based on the premise that xyloglucan i s hydrogen-bonded to cellulose. The reducing ends of the xyloglucan side chains are connected to the rhamnogalacturonan through the l i n e a r g a l a c t a n . The rhamnogalacturonan, in t u r n , i s connected to the hydroxyp r o l i n e - r i c h p r o t e i n through a branched a r a b i n o g a l a c t a n . A second chain of rhamnogalacturonan chain i s a t t a c h e d , through a r a b i n o g a l a c t a n , to the same protein molecule and the order of components i s reversed to another c e l l u l o s e c h a i n . The s t r u c t u r a l component of the c e l l wall can thus be considered as a macromolecule. III.

Enzymes Involved in C e l l Wall Changes of Ripening

Fruits.

1. Cellulase. Because of i t s abundance in f r u i t t i s s u e and prominent r o l e in c e l l wall s t r u c t u r e , c e l l u l o s e should be an important f a c t o r in f r u i t t e x t u r e . Working with many v a r i e t i e s o f a p p l e s , Kertesz et a l . (39) found that the l e v e l of c e l l u l o s e c o r r e l a t e s with i n i t i a l firmness of f r e s h l y harvested f r u i t , but they concluded that subsequent softening of apples i s not due to changes in c e l l u l o s e . B a r t l e y (40) confirmed that the c e l l u l o s i c glucose content of apple c e l l w a l l s d i d not change in the r i p e n i n g f r u i t . In peaches, Nightingale et a l . (41) observed a decrease in c e l l u l o s e content during r i p e n i n g , but they used a method that i s now considered u n r e l i a b l e . Jermyn and Isherwood (10) found a s i m i l a r small decrease in c e l l u l o s e in r i p e n i n g pears. In a highly organized component l i k e c e l l u l o s e , changes in molecular o r i e n t a t i o n i n the microf i b r i l s could be more important than changes in c e l l u l o s e l e v e l s . Using t h i s approach, S t e r l i n g (42) examined the physical s t a t e of c e l l u l o s e in r i p e n i n g peaches with X-ray techniques. He found that the c r y s t a l l i n e m i c e l l e s enlarge in diameter during peach r i p e n i n g . S t e r l i n g a t t r i b u t e d the mi c e l l a r enlargement to c e l l u l o s e degradation r a t h e r than to formation of new m i c r o f i brils. He conceded, however, that the l i m i t e d degradation of c e l l u l o s e cannot c o n t r i b u t e g r e a t l y to peach s o f t e n i n g . The i n s o l u b i l i t y of c e l l u l o s e has hindered d e t e c t i o n of c e l l u l o l y t i c enzymes in f r u i t s . Many workers have circumvented the problem by using carboxymethylcellulose (CMC), a s o l u b l e d e r i v a t i v e of c e l l u l o s e , as the s u b s t r a t e . The high v i s c o s i t y of aqueous s o l u t i o n s of CMC allows the use of the very s e n s i t i v e v i s c o m e t r i c method f o r measurement of c e l l u l a s e a c t i v i t y which i s extremely low i n many plant t i s s u e s . The f i r s t report of c e l l u l a s e in higher plants was by Tracy (43) who found a c t i v i t y



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i n tobacco and other p l a n t s . Dickinson and McCollum (44J and Hall (45) detected s i m i l a r a c t i v i t y in e x t r a c t s of r i p e tomatoes. The long incubation periods (20 hr) required to measurably reduce the v i s c o s i t y of CMC i n d i c a t e the low a c t i v i t y in tomatoes. The c e l l u l a s e in the 1 o c u l a r material of tomatoes i s s o l u ble in water, whereas a high concentration of NaCl i s required to s o l u b i l i z e the enzyme from p e r i c a r p t i s s u e (45). Hall (46) found that c e l l u l a s e i s present in the outer p e r i c a r p of young, green tomatoes. The a c t i v i t y decreases during tomato maturation, and then increases at the onset of r i p e n i n g (46.). In c o n t r a s t , the c e l l u l a s e in the inner p e r i c a r p and the placental t i s s u e i s low i n young f r u i t and remains low u n t i l the turning stage, when the a c t i v i t y increases s h a r p l y , e s p e c i a l l y in the placental portion. Hall concluded that c e l l u l a s e may be involved not only i n tomato softening during r i p e n i n g but a l s o in c e l l enlargement during f r u i t development. Sobotka and Watada (47) confirmed that c e l l u l a s e i s present in mature green tomatoes and that i t increases sharply with ripening. They measured f r u i t firmness and c e l l u l a s e v i s c o m e t r i c a l l y in two commercial v a r i e t i e s and several breeding lines. The v a r i e t i e s with f i r m f r u i t e x h i b i t e d lower c e l l u l a s e a c t i v i t y than those with s o f t f r u i t . The r a t e of softening of each v a r i e t y was c l o s e l y a s s o c i a t e d with an increase in c e l l u l a s e , but a sharp decrease in tomato firmness e a r l y in the r i p e n i n g process was not accompanied by an increase in c e l l u l a s e . Hobson (48), who conducted a d e t a i l e d study on the r e l a t i o n ship between tomato softening and c e l l u l a s e , used acetone prec i p i t a t e s of tomato e x t r a c t s and a reductometric rather than a v i s c o m e t r i c assay. In agreement with other workers, he found high c e l l u l a s e in young tomatoes. The a c t i v i t y decreased s t e a d i l y , as f r u i t s i z e i n c r e a s e d , u n t i l the mature green stage and then began to i n c r e a s e . C e l l u l a s e continued to increase up to the red s t a g e , but not i n t o the o v e r r i p e stage. In c o n t r a s t to other workers, Hobson found higher c e l l u l a s e in the f i r m e r of two v a r i e t i e s , and firmness was not c o r r e l a t e d with c e l l u l a s e in f r u i t in the sub-genera E r i o p e r s i c o n and E u l y c o p e r s i c o n . He concluded that the c o n t r i b u t i o n of c e l l u l a s e to softening i s of minor importance in tomatoes. An explanation f o r the d i f f e r e n c e in opinion concerning the r o l e of c e l l u l a s e in tomato softening may l i e in the r e c e n t l y revealed complexity of tomato c e l l u l a s e . Pharr and Dickinson (49) i d e n t i f i e d two enzymes i n 1 o c u l a r contents of r i p e n i n g tomatoes. One enzyme reduced the v i s c o s i t y of CMC s o l u t i o n s and generated reducing groups, but i t d i d not attack i n s o l u b l e cellulose. The second enzyme hydrolyzed c e l l o b i o s e r a p i d l y , and the r a t e of cleavage decreased as the chain length of the substrate increased. Sobotka and S t e l z i g (50) provided evidence f o r four c e l l u l o y t i c enzymes in tomatoes. Two of the enzymes were e n d o c e l l u l a s e s that degraded both CMC and i n s o l u b l e c e l l u -



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lose. The e n d o c e l l u l a s e s d i f f e r e d in pH optimum, degradation of short chain c e l l o d e x t r i n s , and the s i z e of product released from i n s o l u b l e c e l l u l o s e . One of the other enzymes was c h a r a c t e r i z e d as a n o n s p e c i f i c 3-glucosidase. It hydrolyzed c e l l o b i o s e r a p i d l y ; the r a t e of cleavage decreased as the chain length i n c r e a s e d . The f o u r t h enzyme a l s o hydrolyzed c e l l o b i o s e , but the rate of r e a c t i o n decreased very slowly with i n c r e a s i n g chain l e n g t h . This enzyme produced glucose as the main product, and t h e r e f o r e can be c l a s s i f i e d as an e x o c e l l u l a s e . C e l l u l a s e , as measured v i s c o m e t r i c a l l y with CMC, has a l s o been studied in r e l a t i o n to the softening of dates and peaches. A c t i v i t y was absent in dates at the green stage but began to develop between the green and e a r l y red stages (51_). Activity increased sharply at the e a r l y red stage and reached a maximum at the l a t e red stage. The development of c e l l u l a s e c l o s e l y p a r a l l e l e d the l o s s of f i r m n e s s , suggesting a r o l e f o r t h i s enzyme in date s o f t e n i n g . S i m i l a r l y , c e l l u l a s e was not detected in green peaches, but a c t i v i t y developed and increased during r i p e n i n g (52). The increase in c e l l u l a s e was g r e a t e s t before peach firmness decreased s i g n i f i c a n t l y , suggesting that c e l l u l a s e may not only be involved in peach s o f t e n i n g , but a l s o in i n i t i a t i o n of the process. 2. P e c t i c Enzymes. The p e c t i c substances have received more a t t e n t i o n i n r e l a t i o n to f r u i t softening than any other c e l l wall component. The reasons f o r the concentration on p e c t i n are i t s predominance in the middle l a m e l l a , the r e l a t i v e l y high l e v e l of p e c t i n in most f r u i t s , and above a l l , the appearance of w a t e r - s o l u b l e p e c t i n that accompanies the softening of many f r u i t s . Pectin s o l u b i l i z a t i o n occurs in apples (40), pears (10), peaches (53, 54, 55), tomatoes (56), and other fruits. The s o l u b l e p e c t i n content of apples increased more than 3 - f o l d during a change in firmness of 4.8 to 3.4 kg (40). The p e c t i c changes in r i p e n i n g pears are q u i t e s i m i l a r to those in a p p l e s . The proportion of s o l u b l e p e c t i n i s very low in unripe pears, but increases markedly with r i p e n i n g (10). Some e a r l i e r workers (56) found a d i r e c t r e l a t i o n between r i p e n i n g and s o l u b l e p e c t i n i n pears and proposed the use of s o l u b l e p e c t i n as an index of m a t u r i t y . There i s controversy over whether p e c t i n i s s o l u b i l i z e d in tomatoes, however. Woodmansee et a l . (57) were not able to confirm e a r l i e r claims of p e c t i n s o l u b i l i z a t i o n in tomatoes. Changes in p e c t i n s o l u b i l i t y are most pronounced in f r e e stone peaches. Postlmayr et a l . (54) found that 5% of the dry matter of unripe Fay E l b e r t a peaches was p e c t i n , a f o u r t h of which was w a t e r - s o l u b l e . On r i p e n i n g , the p e c t i n content decreased to 4.1%, but the proportion of water-soluble p e c t i n increased to 71% o f the t o t a l . In c o n t r a s t to the changes in the freestone v a r i e t y , r i p e n i n g of H a l f o r d c l i n g s t o n e peaches was accompanied by a decrease in t o t a l p e c t i n from 4.4 to 3.8%,



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but the s o l u b l e p e c t i n content remained r e l a t i v e l y unchanged. Shewfelt (55) studied three pectin f r a c t i o n s ( w a t e r - s o l u b l e , V e r s e n e - s o l u b l e , and V e r s e n e - i n s o l u b l e ) in f i v e freestone and one c l i n g s t o n e v a r i e t i e s . In the c l i n g s t o n e peaches, the prop o r t i o n of the three p e c t i n f r a c t i o n s remained e s s e n t i a l l y constant. But in freestone peaches, the water-soluble p e c t i n increased r a p i d l y at the expense of the other two f r a c t i o n s . Only 20% of the p e c t i n in unripe peaches was w a t e r - s o l u b l e , whereas the l e v e l increased to 80% in r i p e peaches. Shewfelt et a l . (58) subsequently i s o l a t e d and c h a r a c t e r i z e d the three pectin f r a c t i o n s . They demonstrated that peach r i p e n i n g i s accompanied not only by p e c t i n s o l u b i l i z a t i o n but a l s o by reduct i o n i n molecular weights, as determined v i s c o m e t r i c a l l y . In y e t another study on peaches, Pressey et a l . (59) found that the t o t a l p e c t i n content of E l b e r t a and Red Haven peaches d i d not change s i g n i f i c a n t l y during f r u i t softening while the s o l u b l e p e c t i n increased s h a r p l y . They analyzed the s o l u b l e p e c t i n by gel f i l t r a t i o n and found that the molecular weights of the p e c t i n decreased p r o g r e s s i v e l y during r i p e n i n g . The complexity of p e c t i n seems to o f f e r a number of p o s s i b i l i t i e s f o r enzyme a c t i o n leading to s o l u b i l i z a t i o n . At one extreme, r e l e a s e of p e c t i n molecules may represent cleavage of i t s linkages to other c e l l wall components. Cleavage could i n v o l v e terminal residues of the galacturonan chains or hydrol y s i s of the polymers attached to the c h a i n s . Evidence i s accumulating to show that t h i s may be the case in apples because the pectin does not appear to be degraded during s o l u b i l i z a t i o n . On the other hand, p e c t i n may be s o l u b i l i z e d by h y d r o l y s i s of l o n g , i n s o l u b l e molecules. In t h i s c a s e , h y d r o l y s i s could occur in the galacturonan chains or at the linkages with rhamnose. Two enzymes that could be involved in the mechanism are p e c t i n esterase and polygalacturonase. a. Pectinesterase. P e c t i n e s t e r a s e c a t a l y z e s the h y d r o l y s i s of methyl e s t e r s in p e c t i n . It i s h i g h l y s p e c i f i c f o r the methyl e s t e r and the e s t e r as i t occurs in p e c t i n , and does not hydrolyze other e s t e r s and the methyl e s t e r in short chain galacturonans. The enzyme i s a c t i v a t e d by d i v a l e n t cations or monovalent c a t i o n s at high c o n c e n t r a t i o n s . The optimum pH range i s 5 to 8 and i s a f f e c t e d by c a t i o n s . The a c t i o n of p e c t i n esterase on p e c t i n produces blocks of f r e e carboxyl groups (60), i n d i c a t i n g that d e e s t e r i f i c a t i o n occurs in a l i n e a r manner. Solms and Deuel (61 ) suggested t h a t . e s t e r h y d r o l y s i s occurs only adjacent to f r e e carboxyl groups, in d e t a i l e d studies on the mode o f a c t i o n of tomato p e c t i n e s t e r a s e , Lee and Macmillan (62) demonstrated that i t acts a t both the reducing ends and i n t e r i o r l o c i on h i g h l y e s t e r i f i e d p e c t i n c h a i n s . P e c t i n e s t e r a s e occurs commonly in various parts of higher p l a n t s , i n c l u d i n g the f r u i t . Kertesz (63) found that tomatoes are a p a r t i c u l a r l y r i c h source of the enzyme. The a c t i v i t y i s



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high in green tomatoes and, according to Kertesz, increases about 4 - f o l d during r i p e n i n g . This suggests that softening may be r e l a t e d to high p e c t i n e s t e r a s e a c t i v i t y . However, Hamson (64) reported that p e c t i n e s t e r a s e i s higher in f i r m than in s o f t varieties. In a study on s i x breeding l i n e s and one v a r i e t y , Hall and Dennison (65) d i d not f i n d a s i g n i f i c a n t c o r r e l a t i o n between firmness and p e c t i n e s t e r a s e . Furthermore, Hobson (66) observed only a 40% increase in p e c t i n e s t e r a s e during r i p e n i n g of tomatoes. In c o n t r a s t to the f i n d i n g s of Hamson, the f i r m e r of the two v a r i e t i e s contained lower p e c t i n e s t e r a s e . Part of the confusion about p e c t i n e s t e r a s e in tomatoes may be due to the complexity of the enzyme. The a c t i v i t y can be resolved i n t o a number of components (67). The number of p e c t i n esterases and t h e i r r e l a t i v e l e v e l s vary with both tomato v a r i e t y and stage of r i p e n e s s . For example, green Marion tomatoes contained two p e c t i n e s t e r a s e s . On r i p e n i n g , one enzyme decreased while the other nearly t r i p l e d , and a t h i r d form of p e c t i n esterase appeared. One of the enzymes appears to be the major component in most v a r i e t i e s . The same three enzymes were present in Homestead tomatoes in the same p r o p o r t i o n s . The dwarf v a r i e t y P i x i e contained two of the above enzymes and a d i f f e r e n t enzyme. The four tomato p e c t i n e s t e r a s e s d i f f e r e d i n molecular weight, s t a b i l i t y to heat, pH optimum, and a c t i v a t i o n by c a t i o n s . An even greater heterogeneity of tomato p e c t i n e s t e r a s e has been demonstrated by t h i n - l a y e r i s o e l e c t r i c focusing (68). At l e a s t e i g h t enzymes were separated, although one component predominated in a l l samples. The pectinesterases appear to have s i m i l a r molecular weights, and to d i f f e r mainly with respect to charge p r o p e r t i e s . P e c t i n e s t e r a s e has been detected in many other f r u i t s , although the a c t i v i t i e s are u s u a l l y much lower than i n tomatoes. There have been c o n f l i c t i n g reports on the presence of the enzyme in a p p l e s . Apparently i t i s present in low amounts and does not change during apple r i p e n i n g (69). Nagel and Patterson (70) reported that p e c t i n e s t e r a s e in pears decreases during maturation. The l e v e l of p e c t i n e s t e r a s e in peaches v a r i e s with the v a r i e t y , and the changes in a c t i v i t y show no d i s t i n c t trend with advancing ripeness (55). The degree of e s t e r i f i c a t i o n of p e c t i n in avocados g r a d u a l l y decreases during r i p e n i n g (71). Pectinesterase i s high in young avocados (72). A pronounced decrease in a c t i v i t y was observed during the i n t e n s i v e growth stage, and s t i l l f u r t h e r reduction during f r u i t maturation. S i m i l a r l y , p e c t i n e s t e r a s e decreased sharply in stored avocados through the softening p e r i o d . The more mature the f r u i t at h a r v e s t , the more pronounced was the decrease in p e c t i n e s t e r a s e during storage (72). H u l t i n and Levine {73) separated the p e c t i n e s t e r a s e a c t i v i t y in bananas i n t o three f r a c t i o n s by d i f f e r e n t i a l e x t r a c t i o n . The enzymes d i f f e r e d in pH optimum, thermal s t a b i l i t y , and s e n s i t i v i t y to SDS, as well as in s o l u b i l i t y . In c o n t r a s t to the



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decrease in p e c t i n e s t e r a s e in avocados, the a c t i v i t y in bananas increased about 1 0 - f o l d during r i p e n i n g . One of the p e c t i n esterases increased c o n t i n u a l l y during r i p e n i n g , whereas the other two enzymes were maximal a f t e r about 2 days of r i p e n i n g . The g r e a t e s t increase in a l l three enzymes occurred as the bananas changed from green to y e l l o w . Thus, the t o t a l p e c t i n e s t e r a s e a c t i v i t y increases in bananas and decreases in avocados during r i p e n i n g , but in most f r u i t s the enzyme i s present at the immature stage and appears to remain r e l a t i v e l y constant during r i p e n i n g . A r o l e f o r t h i s enzyme in f r u i t s o f t e n i n g , t h e r e f o r e , i s not apparent. Multiple forms of p e c t i n e s t e r a s e are present in bananas and tomatoes, and a s p e c i f i c form of enzyme might be involved in s o f t e n i n g . A l s o , the d i r e c t r o l e of pectinesterase in p e c t i n s o l u b i l i z a t i o n i s not obvious because a decrease in e s t e r i f i c a t i o n alone would not be expected to increase s o l u b i l i t y . To the c o n t r a r y , an increase in f r e e carboxyl groups would lead to greater i n t e r a c t i o n with C a and to decreased s o l u b i l i t y . But p e c t i n e s t e r a s e a c t i o n must precede degradation of p e c t i n by polygalacturonase, and in t h i s way p e c t i n e s t e r a s e could exert r e g u l a t i o n on the process of f r u i t s o f t e n i n g . Judging from the lack of a c o n s i s tent pattern of f l u c t u a t i o n of the enzyme, r e g u l a t i o n of p e c t i n esterase probably involves a mechanism other than changes in enzyme c o n c e n t r a t i o n . The f a c t that pectin i s never completely d e e s t e r i f i e d in tomatoes, f o r example, even though p e c t i n e s t e r a s e i s always p r e s e n t , suggests that other r e g u l a t o r y mechanisms are active. 2 +

b. Polygalacturonase. P e c t o l y t i c enzymes are c l a s s i f i e d on the basis of mode of a c t i o n as e i t h e r hydrolases or l y a s e s . The l a t t e r degrade galacturonans by a t r a n s e l i m i n a t i o n mechanism i n a random or terminal manner with preference f o r low or high ester substrates. The hydrolases a l s o can f u n c t i o n in a random or uniform manner, but a l l known enzymes in t h i s group act only on d e e s t e r i f i e d substrates and t h e r e f o r e are c a l l e d p o l y g a l a c turonases. They are the only p e c t o l y t i c enzymes known to occur in f r u i t tissues. Polygalacturonase was f i r s t detected in r i p e tomatoes which remain the r i c h e s t plant source. The l i s t of f r u i t s c o n t a i n i n g the enzyme i s now f a i r l y l o n g , and the f o l l o w ing d i s c u s s i o n w i l l deal with each commodity s e p a r a t e l y . i. Tomatoes. In c o n t r a s t to p e c t i n e s t e r a s e and c e l l u l a s e , polygalacturonase i s not present in green, immature tomatoes (63, 66). It appears i n f r u i t near the onset of r i p e n i n g and increases as the f r u i t s o f t e n s . It i s t h i s apparent coincidence of polygalacturonase with softening that has suggested a key r o l e f o r t h i s enzyme in the process. Numerous studies have been d i r e c t e d at e s t a b l i s h i n g such a r e l a t i o n s h i p . One approach i s to study the enzyme l e v e l in r e l a t i o n to tomato firmness which d i f f e r s considerably with a v a r i e t y .



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Tomato l i n e s with high pigment c h a r a c t e r i s t i c s tend to be f i r m e r than other l i n e s (74). Foda (75) found that high pigment l i n e s contained more protopectin and l e s s polygalacturonase than a commençai v a r i e t y . Sobotka and Watada (76) a l s o observed that two l i n e s c o n t a i n i n g the high pigment c h a r a c t e r i s t i c had cons i d e r a b l y lower polygalacturonase than l i n e s without the chara c t e r i s t i c . The a c t i v i t y was not only lower, but i t increased at a slower rate than in other v a r i e t i e s . Hobson (77_) reported that r i p e f r u i t o f the v a r i e t y Potentate were both f i r m e r and lower in polygalacturonase than those of the v a r i e t y Immuna. A f u r t h e r study by Hobson (78) revealed a highly s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between polygalacturonase and the compression o f 6 d i f f e r e n t v a r i e t i e s at the commerical p i c k i n g stage. A f r u i t r i p e n i n g i n h i b i t o r ( r i n ) mutant of tomato can be produced by backcrossing (79). Tomatoes containing the r e c e s s i v e gene soften much more slowly than normal v a r i e t i e s . Hobson (80) observed that Potentate tomatoes backcrossed in t h i s manner were a s s o c i a t e d with markedly reduced s o l u b i l i z a t i o n of p e c t i n and c o n s i d e r a b l y lower l e v e l s of polygalacturonase. This was confirmed by Buescher and T i g c h e l a a r (81.) who found that backcrossed Rutgers tomatoes remained f i r m and did not develop p o l y g a l a c turonase. The p h y s i o l o g i c a l d i s o r d e r "blotchy" r i p e n i n g has a l s o been evaluated in terms of polygalacturonase a c t i v i t y . The red areas of blotchy f r u i t contained l e s s than two-thirds the a c t i v i t y in evenly red f r u i t , and the a c t i v i t y was even lower in non-red areas (77). Polygalacturonase a c t i v i t y i s highest in the outer l o c u l e wall of the p e r i c a r p t i s s u e followed by the inner l o c u l e walls and the placental t i s s u e (77). The enzyme i s not present in the l o c u l a r contents. The a c t i v i t y f i r s t appears i n the placenta which u s u a l l y shows i n c i p i e n t yellowness. It then develops in both the inner and outer l o c u l e walls as the change in c o l o r spreads to the p e r i c a r p . Hobson suggested that the p a r a l l e l appearance of polygalacturonase and c o l o r a t i o n i s f u r t h e r e v i dence f o r a r e l a t i o n s h i p between the enzyme and tomato r i p e n i n g . McColloch et a l . (82) a l s o noted that deep red c o l o r i n tomatoes was a s s o c i a t e d with high polygalacturonase. Tomato polygalacturonase has been p a r t i a l l y p u r i f i e d and c h a r a c t e r i z e d (83, 84). It cleaves pectate randomly f i r s t to o l i g o g a l a c t u r o n a t e s and u l t i m a t e l y to g a l a c t u r o n i c a c i d , but the r a t e of h y d r o l y s i s decreases r a p i d l y with decreasing chain length. I f the i n i t i a l r a t e o f pectate h y d r o l y s i s i s 100, the rates of h y d r o l y s i s of t e t r a - , t r i - , and d i - g a l a c t u r o n a t e are approximatley 7, 1.6, and 1, r e s p e c t i v e l y (84). Some of the p r o p e r t i e s of the a c t i v i t y in crude e x t r a c t s suggest that the a c t i v i t y i s produced by more than one polygalacturonase. McColloch and Kertesz (85) observed t h a t most of the a c t i v i t y was destroyed by heating at r e l a t i v e l y low temperatures, but part of i t survived heating to 90°. Patel and Phaff (84) examined the h e a t - s t a b l e component and concluded that i t was



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s i m i l a r to the heat-unstable enzyme. But they observed double pH optima f o r the enzyme a c t i n g on t r i - a n d t e t r a - g a l a c t u r o n a t e s and a t t r i b u t e d t h i s to the presence of two enzymes. The observat i o n that pectate i n h i b i t s the h y d r o l y s i s of small substrates at low pH, but only to the extent of 70%, i s f u r t h e r evidence f o r a complex system o f polygalacturonase in tomatoes (86). McColloch (87) separated two polygalacturonases from tomatoes by moving boundary e l e c t r o p h o r e s i s . One of the peaks was more a c t i v e than the o t h e r , but the enzymes were not c h a r a c t e r ized. Pressey and Avants (88) separated the a c t i v i t y i n tomato e x t r a c t s i n t o two peaks (PG I and PG II) by chromatography on DEAE-Sephadex A-50. The enzymes d i f f e r e d in molecular weight and s t a b i l i t y to temperature and pH, although the more s t a b l e component d i d not s u r v i v e heating to 90°. The pH optima f o r both enzymes were near 4 . 5 . The a c t i v i t i e s of both enzymes s h i f t e d to the a c i d side with decreasing substrate s i z e and i n c r e a s i n g NaCl c o n c e n t r a t i o n , but PG I was l e s s dependent on these f a c t o r s . At enzyme l e v e l s producing equal numbers of reducing groups, PG II was much more e f f e c t i v e than PG I in reducing the v i s c o s i t y of pectate. However, the e f f e c t of PG I on the v i s c o s i t y o f pectate i s too r a p i d f o r a mechanism of end group cleavage. ii. Peaches. The changes in f l e s h firmness and p e c t i n s o l u b i l i t y are p a r t i c u l a r l y pronounced in r i p e n i n g freestone peaches. Shewfelt et a l . (58) demonstrated that p e c t i n i s not only s o l u b i l i z e d , but that the molecular weights of the s o l u b l e f r a c t i o n g r a d u a l l y decrease, suggesting that peach r i p e n i n g i s accompanied by p e c t i n degradation. Pressey et a l . (59) confirmed t h a t reduction in p e c t i n molecular weights occurs during both t r e e and postharvest r i p e n i n g . Although e a r l i e r attempts to detect polygalacturonase in peaches were u n s u c c e s s f u l , the presence of t h i s enzyme was revealed by incubation of waterwashed residues of peach t i s s u e with polygalacturonate (59). Polygalacturonase a c t i v i t y was absent during the f r u i t e n l a r g e ment stage; i t appeared as the peaches began to ripen and then increased s h a r p l y . The appearance of the enzyme p a r a l l e l e d the formation o f s o l u b l e p e c t i n . Both processes were preceded by some f r u i t s o f t e n i n g , but they coincided with the most r a p i d l o s s of firmness. Pressey and Avants (89) subsequently s o l u b i l i z e d and chara c t e r i z e d the peach polygalacturonase. The procedure involved e x t r a c t i o n o f water-washed residues of r i p e freestone peaches with 0.2 M NaCl followed by u l t r a f i l t r a t i o n of the macromolecules. Attempts to p u r i f y the a c t i v i t y by chromatography on Sephadex G-100 revealed two enzymes (PG I and PG I I ) . The enzymes d i f f e r e d in a number o f ways, i n c l u d i n g pH optimum, c a t i o n a c t i v a t i o n , and e f f e c t of substrate s i z e . PG I functioned o p t i m a l l y at pH 5.5 and required Ca2+ f o r a c t i v i t y (89). It cleaved d i g a l a c t u r o n a t e very s l o w l y , but the r a t e of cleavage



10.

PRESSEY

185

Fruit Softening

increased with substrate chain length to a maximum f o r substrates with a degree of polymerization of about 20. This enzyme exh i b i t e d the highest a f f i n i t y f o r the l a r g e s t substrates (90). PG II was most r e a c t i v e with l a r g e s u b s t r a t e s ; i t s pH optimum was near 4.5 f o r the l a r g e s t substrate ( p e c t a t e ) , but i t s h i f t e d to the a c i d s i d e as the substrate s i z e decreased (89). The two peach polygalacturonases d i f f e r e d markedly in mode o f a c t i o n . PG II was a t y p i c a l endopolygalacturonase; i t r a p i d l y reduced the v i s c o s i t y of pectate r e l a t i v e to a slow r e l e a s e of reducing groups. T h i s enzyme was very e f f e c t i v e in s o l u b i l i z i n g p e c t i n from washed peach c e l l w a l l s . In c o n t r a s t , PG I had a very small e f f e c t on the v i s c o s i t y of pectate. The product of i t s r e a c t i o n was i d e n t i f i e d as g a l a c t u r o n i c a c i d . The evidence presented showed that cleavage occurs at the nonreducing ends of the substrate c h a i n s , and that a l l chains are p r o g r e s s i v e l y shortened. The polygalacturonase in peaches t h e r e f o r e , c o n s i s t s of endo- and exo-cleaving enzymes. T h i s was the f i r s t report of an exopolygalacturonase in f r u i t t i s s u e , but we now know that t h i s enzyme i s widespread in higher p l a n t s . i i i . Dates. Hasegawa et a l . (91) found a t r a c e of p o l y galacturonase, as measured by the r e l e a s e of reducing groups from p e c t a t e , in green dates. The a c t i v i t y remained low during r i p e n i n g o f dates u n t i l the l a t e red stage at which time the a c t i v i t y rose sharply to a maximum at the 50% s o f t stage. The greatest part of the increase in polygalacturonase, t h e r e f o r e , occurred immediately before date s o f t e n i n g . The development of a c t i v i t y a l s o followed softening w i t h i n s i n g l e d a t e s , which commences at the a p i c a l end and progresses toward the stem end. They concluded from the c l o s e r e l a t i o n between polygalacturonase and s o f t e n i n g t h a t t h i s enzyme may be involved in c o n t r o l l i n g the texture of d a t e s . P r o p e r t i e s of the date enzyme have not been determined. iv. Avocado. McCready and McComb (92) f i r s t reported that polygalacturonase i s absent in green avocados, but considerable a c t i v i t y develops during r i p e n i n g . Reymond and Phaff (93) found t h a t the enzyme f i r s t appears at the blossom end and spreads toward the stem end. In a more d e t a i l e d study, Zauberman and Schiffmann-Nadel (72) d i d not detect polygalacturonase in f r u i t at various stages of development immediately a f t e r harvest. In harvested f r u i t , the enzyme developed e a r l i e r in more mature f r u i t s , although the maximum l e v e l s of enzymes appeared to be independent of f r u i t maturity. Avocado polygalacturonase e x h i b i t s optimal a c t i v i t y at pH 5.5 and in the presence of 0.14 M Na (93). Phosphate and Calgon were i n h i b i t o r y , but Ca2+ at 0.1 M was without e f f e c t . The enzyme appears to be an endopolygalacturonase; i t hydrolyzes pectate to intermediate o l i g o g a l a c t u r o n a t e s which are then slowly hydrolyzed to g a l a c t u r o n i c a c i d (93, 9 4 ) . Reymond and +



186


Phaff [93_) concluded that avocados contain an i n h i b i t o r o f polygalacturonase because recovery of the enzyme during p u r i f i c a t i o n often exceeded 100%. Furthermore, the a d d i t i o n o f ext r a c t s o f immature avocados to p u r i f i e d polygalacturonase r e s u l t ed i n 48% i n h i b i t i o n , confirming the existence o f an i n h i b i t o r . P r e c i p i t a b i l i t y o f the i n h i b i t o r by 50% ammonium s u l f a t e suggests that i t i s a p r o t e i n . v. Pears. McCready and McComb (92J presented evidence showing that r i p e n i n g o f pears i s accompanied by degradation o f p e c t i c substances. During the r i p e n i n g o f pears, the s o l u b i l i t y of p e c t i n increased and p r e c i p i t a b i l i t y with alcohol decreased. They d i d not detect polygalacturonase i n unripe pears, but they found considerable a c t i v i t y i n r i p e pears. Pressey and Avants (95) have demonstrated that the a c t i v i t y c o n s i s t s o f endo- and exo-polygalacturonases, s i m i l a r to the system o f p o l y g a l a c turonases found i n peaches. The exopolygalacturonase was o p t i m a l l y a c t i v e at pH 5.5 and was a c t i v a t e d by Ca2+ and Sr2+. It was most r e a c t i v e with polygalacturonate possessing a chain length o f about 12 u n i t s . The endopolygalacturonase had a pH optimum a t 4.5; i t was most r e a c t i v e with a substrate possessing a chain length o f about 80. vi. Cucumbers. B e l l (96) detected p e c t o l y t i c a c t i v i t y i n cucumbers by using p e c t i n at pH 4 as the substrate i n the v i s c o metric assay. The choice o f p e c t i n over pectate f o r the subs t r a t e may have been the reason f o r the low l e v e l o f a c t i v i t y found. When Pressey and Avants (97) reexamined the p o l y g a l a c turonase i n cucumbers, they found that the a c t i v i t y c o n s i s t s s o l e l y o f an e x o s p l i t t i n g enzyme. This enzyme i s s i m i l a r to peach exopolygalacturonase i n pH optimum (5.5), molecular weight (59,000), and mode o f a c t i o n . In c o n t r a s t to the exopolygalacturonases, the cucumber enzyme was r e a d i l y extracted by water. It was a c t i v a t e d by C a , but not by S r . Substrates with chain lengths from about 6 to 12 were cleaved most r a p i d l y . 2 +

2 +

v i i . Cranberries. Patterson et a l . (98) found p o l y g a l a c turonase i n c r a n b e r r i e s that were breaking down i n storage and softened by b r u i s i n g , but not i n sound b e r r i e s . It i s p o s s i b l e that the a c t i v i t y detected was of microbial o r i g i n . However, A r a k j i and Yang (99) i s o l a t e d and c h a r a c t e r i z e d a p o l y g a l a c turonase from McFarlin c r a n b e r r i e s . They found that phenol binding agents increased the y i e l d s of a c t i v i t y . The enzyme was endo-cleaving with a pH optimum near 5. c. Other Enzymes. Enzymatic cleavage o f any linkage involved i n maintaining the s t r u c t u r e and r i g i d i t y o f the middle lamella and c e l l wall could a f f e c t f r u i t firmness. Even the phenomenon o f p e c t i n s o l u b i l i z a t i o n may be due to enzymatic cleavage of linkages between pectin and other c e l l wall components



10.

PRESSEY

Fruit Softening

187

r a t h e r than to degradation of the p e c t i n c h a i n . T h i s may be the case in a p p l e s . Attempts to f i n d polygalacturonase in r i p e apples have been unsuccessful (100). Furthermore, s o l u b l e p e c t i n i s formed during apple r i p e n i n g , but the p e c t i n chains are not shortened (100). The s o f t e n i n g of apples i s accompanied by not only p e c t i n s o l u b i l i z a t i o n but also by the loss of galactose residues from the c e l l wall (101). A small decrease i n arabinose a l s o o c c u r s , but the x y l a n , c e l l u l o s e , and n o n c e l l u l o s i c glucans are not hydrolyzed. The l o s s of galactose i s s u b s t a n t i a l , decreasing to about a t h i r d of the i n i t i a l l e v e l , and precedes the increase in s o l u b l e p e c t i n and the decrease in apple firmness. This has prompted searches f o r galactan-degrading enzymes in apples. B a r t l e y (102) has found that both s o l u b l e and c e l l wall preparat i o n s from apple cortex hydrolyze 3-galactan and p-nitrophenyl d e r i v a t i v e s of «- and 3 - g a l a c t o s i d e . The 3-galactosidase a c t i v i t y was present at a high l e v e l in f i r m apples and increased l e s s than 2 - f o l d during r i p e n i n g . B a r t l e y concluded that 3g a l a c t o s i d a s e may control p e c t i n s o l u b i l i z a t i o n , but the f a c t that the l o s s of galactose precedes the r e l e a s e of p e c t i n led him to suggest that the h y d r o l y s i s of other bonds may be i n volved (102). Wallner and Walker (103) measured the enzymatic a c t i v i t y a g a i n s t a number of g l y c o s i d e s and polysaccharides to determine whether the cleavage of c e r t a i n linkages v a r i e s with the stage of ripeness of tomatoes. E x t r a c t s of tomatoes hydrolyzed glucomannan, galactomannan, l a m i n a r i n , a r a b i n o g a l a c t a n , x y l a n , and p-nitrophenyl d e r i v a t i v e s of «-D and 3 - D - g a l a c t o s i d e , «-Dg l u c o s i d e , «-D-and 3 - D - x y l o s i d e , as well as c e l l u l o s e and p o l y galacturonate. The major enzymes, in terms of reducing group f o r m a t i o n , were l a m i n a r i n a s e , polygalacturonase, and 3 - g a l actosidase. With the exception of glucomannanase, xylanase, and p o l y g a l a c t u r o n a s e , the enzymes were present at a l l stages of ripeness. During r i p e n i n g , the 3-galactosidase increased 4 - f o l d in the p l a c e n t a , but l e s s in other parts of the f r u i t . The other enzymes did not change s i g n i f i c a n t l y . However, on the basis of s u s c e p t i b i l i t y of c e l l walls to degradation by the mixture of tomato enzymes, Wallner and Walker concluded that changes in the c e l l wall occur before the appearance of p o l y galacturonase. An enzyme other than polygalacturonase may t h e r e f o r e be i n v o l v e d , but has not been i d e n t i f i e d . Conclusions F r u i t s o f t e n i n g probably i s due to breakdown of the middle lamellae and c e l l w a l l s . T h i s d é s i n t é g r a t i o n may i n v o l v e movement of calcium in the c e l l walls (26) or a l t e r a t i o n of hydrogen bonding between c e l l u l o s e and xyloglucan (38) in response to changes in pH. But evidence of covalent bond cleavage in c e l l wall components points to r o l e s f o r several of the h y d r o l y t i c


188


enzymes found in f r u i t t i s s u e s . There has been a tendency to emphasize the p e c t i c enzymes because of the apparent r e l a t i o n s h i p between p e c t i n s o l u b i l i z a t i o n and f r u i t s o f t e n i n g . The impor tance of polygalacturonase i s supported by i t s widespread occur rence i n f r u i t s and i t s appearance during r i p e n i n g . The d i s covery of exopolygalacturonase in f r u i t s suggests that cleavage of terminal linkages in c e l l wall macromolecules may be involved in s o f t e n i n g . But the process i s complex and undoubtedly r e q u i r e s numerous enzymes, some of which remain to be i d e n t i f i e d .


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