69 Polygalacturonases

Jacques A. E. Benen
Wageningen University, Wageningen, The Netherlands

Jaap Visser
Fungal Genetics and Technology Consultancy, Wageningen, The Netherlands

I.

INTRODUCTION

Polygalacturonases cleave the -1,4-D-galacturonosidic linkage by hydrolysis. Whereas endopolygalacturonases (EC 3.2.1.15) hydrolyze the polymer substrate randomly, the exopolygalacturonases are conned to cleave off galacturonic acid monomers (EC 3.2.1.67) or digalacturonides (EC 3.2.1.82) from the nonreducing end. The names of these enzymes suggest that polygalacturonic acid is their sole substrate. However, as found for the pectate lyases, endopolygalacturonases are also active on pectins with a lower or moderate degree of esterication. Recently it was shown that endopolygalacturonases A and B from Aspergillus niger even prefer a low degree of esterication (1). The polygalacturonases have all been grouped into family 28 of the general glycosyl hydrolase classication (2). In addition to the endopolygalacturonases, this family also comprises the rhamnogalacturonases and the recently cloned xylogalacturonase (3). Threedimensional structures of two endopolygalacturonases (4, 5) and a rhamnogalacturonase have been solved (6), and their close resemblance indeed justies the grouping in one family. By far the largest group of potential polygalacturonase genes (52 open reading frames) is formed by those from Arabidopsis thaliana. This large amount originates from the recent Arabidopsis genome-sequencing project. Such an abun-

dance of genes indicates a profound developmental role of the corresponding enzymes. Contrary to plant polygalacturonases, microbial polygalacturonases serve to mobilize nutrients from pectin to support growth. For several yeasts such as Kluyveromyces marxianus (7) and Saccharomyces cerevisiae (8), polygalacturonase production has been documented and respective genes have been cloned, whereas no obvious physiological function is served since those yeasts can not utilize galacturonic acid.

II.

ENDOGENOUS POLYGALACTURONASES IN PLANTS

In view of the important role of pectin in the primary cell wall of plants, it is obvious that endogenous polygalacturonases play important roles in the development of plants. Polygalacturonase activity has been demonstrated in almost any plant tissue. Based on mRNA expression studies in Arabidopsis thaliana (9), tomato [Lycopersicon esculentum (10)], and lemon [Cucumus melon (11)] among others, it was shown that there are at least three types of polygalacturonases: the pollen-specic polygalacturonases, the fruit-specic enzymes, and the abscission-specic polygalacturonases. For tomato it was established that the specic expression, i.e., auxin repression and ethylene and abscisic acid induction, resides in the regulatory

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elements within the individual promoters of the polygalacturonase-encoding genes (10). A phylogenetic analysis by Hadeld and coworkers (11) revealed that the plant polygalacturonases belong to three clades. Clade A comprises fruit- and abscisionspecic polygalacturonases without pro-sequence, clade B consists of fruit- and abscision-specic polygalacturonases with a pro-sequence, and clade C contains all the pollen-specic polygalacturonases. It has been hypothesized that all clade C enzymes are exopolygalacturonases, as those enzymes characterized were of the exolytic type. In an excellent review Hadeld and Bennet (12) have compiled the major ndings on endogenous plant polygalacturonases of the past two decades. In a more recent review Lang and Dornenburg (13) further elaborate on this and also pay attention to the role of polygalacturonases in wounding of plants, the interaction of phytopathogenic fungi and plants, and the plant response to this. To the food enzymologist the most interesting endogenous polygalacturonases are those present in the part of the plant that is used as or in food. One of the best-characterized subjects in this respect is the tomato. As a major food, either as the complete fruit or as puree, catsup, or juice, the inuence of endogenous polygalacturonases on fruit ripening and processing has been studied extensively. To this end genesilencing techniques have been employed that have revealed that endopolygalacturonases are not necessary for fruit ripening (reviewed in 1214). However, owing to the gene silencing it turned out that the shelf life of the tomato increased profoundly as a result of reduced cracking of the tomato. Upon homogenization of nongenetically modied tomatoes, the deleterious effect of endogenous polygalacturonases becomes evidenti.e., a decrease in viscosity of the homogenate upon aging. To prevent the action of these enzymes, tomatoes are homogenized after heat pretreatment to inactive the enzymes and yield the so-called hot break pastes.

A.

Three-Dimensional Structures of Polygalacturonases

Currently, 3-D structures are only available for the Erwinia carotovora endopolygalacturonase [PDB code: 1 BHE (4)] and the Aspergillus niger endopolygalacturonase II [PDB code: 1CZF (5)]. Despite limited sequence identity between the two enzymes (19%), a striking similarity of the two -helical structures is observed at the C-alpha trace level although certain differences can be observed as well, notably in the loop regions. Details are discussed by Van Santen et al. (5). Unfortunately, no structure is available for any enzyme-substrate complex. However, Van Santen et al. (5) and Armand et al. (15) present evidence that the substrate binds to the enzyme at the observed cleft with the reducing end of the substrate facing toward the C-terminal part of the enzyme. This orientation was also observed for the topologically highly similar pectate lyase C in complex with the substrate (16). B. Isoforms of Polygalacturonases

III.

BIOPHYSICAL PROPERTIES OF POLYGALACTURONASES

Although all polygalacturonases belong to family 28 of glycosyl hydrolases, considerable variation in molecular mass (3075 kDa) and isoelectric point (pI 3.88.8) can still be obtained from the databases going from enzyme to enzyme. Currently no relation has been established between molecular mass and/or isoelectric point and/or specicity.

The rapidly expanding family 28 of glycosyl hydrolases clearly demonstrates that many species analyzed have at least two genes encoding polygalacturonases. Thus, as for the pectic lyases, isoforms at the genetic level are plentiful. Wubben et al. (17) constructed a phylogenetic tree of 35 endopolygalacturonases of yeast and fungi which appeared to group into ve monophylogenetic groups. Hadeld et al. (11) analyzed the plant polygalacturonases in this respect which appeared to group in three clades. For eukaryotic polygalacturonases, many reports describe heterogeneity of polygalacturonase preparations as a result of glycosylation. To date, the N-glycosylation patterns of only two polygalacturonases have been analyzed in detail, viz., endopolygalacturonases I and II from Aspergillus niger (18, 19). The single N-glycosylation site in endopolygalacturonase II appeared to be heterogeneously glycosylated with glycans of the high mannose type (18), whereas of the two N-glycosylation sites of endopolygalacturonase I only one was similarly decorated as the one of polygalacturonase II and the other site was not glycosylated (19). For neither enzyme was O-glycosylation found. In contrast to A. niger pectic lyases, the A. niger polygalacturonases all contain a prosequence in addition to the presequence normally found in secreted enzymes. The role of the prosequence is not clear but

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is removed by a KEX-like protease in most cases (20, 21). Also, in Botrytis cinerea the prosequence was reported in four of the six endopolygalacturonase genes cloned (17). Sheehy et al. 922) and DellaPenna et al. (23) were among the rst to analyze processing of a tomato polygalacturonase. In addition to glycosylation, evidence for proteolytic processing was obtained. In addition to a presequence, a prosequence was reported as well. Moreover, Sheehy et al. (22) also observed C-terminal processing. It has been proposed that the prosequence is necessary for the targeting of the particular enzyme to a certain part of the cell wall (23), but it has also been hypothesized that the prosequence keeps the enzyme in an inactive state until the prosequence is processed at the proper location (12). As mentioned in Section II, the enzymes of clade B all have a prosequence in addition to the presequence.

IV.

BIOCHEMICAL PROPERTIES OF POLYGALACTURONASES

In the past, several polygalacturonases have been characterized biochemically. However, the most comprehensive and in-depth analyses have been carried out for polygalacturonases from the genus Aspergillus and notably for the seven-membered endopolygalacturonase family from A. niger N400 and for exopolygalacturonase from A. tubingensis NW752. The variety of biochemical properties of these enzymes is such that it covers essentially most other endo- and exopolygalacturonases. The discussion of biochemical properties will therefore be conned to this group of enzymes and will be complemented with additional data for other polygalacturonases where appropriate. A. A. niger endopolygalacturonases

pressed in view of their potential industrial application and to facilitate production, purication, and characterization (1, 20, 27, 28). Endopolygalac turonase D appeared quite different from the other A. niger endopolygalacturonases as it carries an 136 amino acid N-terminal extension whose function is not clear but which is not processed as evidenced by sequencing the N-terminus by Edman degradation (27). Several basic biochemical properties of the seven enzymes are listed in Table 1. As can be seen, there is only little variation in pH optima. However, there is an enormous difference in maximal turnover, ranging from 25 U/mg for endopolygalacturonase C to 4000 U/mg for endopolygalacturonase II. For the three enzymes with the lowest turnover rates, endopolygalacturonase C, D, and E, it was proposed that polygalacturonic acid was not the natural substrate. All three enzymes still retain some activity on pectins with a low degree of esterication (DE), making it unlikely that these are the preferred substrates (Table 2). This in contrast to endopolygalacturonases A and B, which both prefer low-DE pectin. For these latter two enzymes that is quite understandable since these enzymes are constitutively expressed, thus making their role as scouting enzymes which encounter primarily medium DE pectin quite likely (1). They are supposed to be involved in producing the inducers required to obtain the complete pectinase spectrum (1). The natural substrates for endopolygalacturonases C and E have yet to be identied. For endopolygalacturonase D,

Table 1 General Properties and Kinetics of Aspergillus niger Endopolygalacturonases Enzyme PG PG PG PG PG PG PG I II A B C D E Mr (kDa) 34.9 34.9 35.5 34.9 36.2 50.8 35.6 pH
a

S.A.b Km b Vmax b (U/mg) (mg/mL) (U/mg) 800 4000 1030 520 25 90 38 < 0:15 < 0:15 < 0:15 0.9 < 0:15 0.2 2.5 800 4000 1200 900 25 96 80

An early investigation of a commercial A. niger pectinase preparation by Kester and Visser (24) revealed the presence of six different endopolygalacturonases with different specic activities and different modes of action (cleaving patterns) on oligomeric substrates. Using a reversegenetics approach, genes encoding endopolygalacturonases I and II were cloned (21, 25), and, using the endopolygalacturonase II gene as a probe, genes encoding endopolygalacturonases A to E (1, 20, 26, 27) were cloned. All the endopolygalacturonase-encoding genes were individually overex-

4.2 4.2 4.0 5.0 4.1 4.2 3.8

a Determined in McIlvaine buffers using 0.25% polygalacturonic acid at 30 C. b Conditions: 50 mM sodium acetate, pH 4.2, 30 C. PG B: 50 mM sodium acetate, pH 5.0. S.A.: specic activity using 0.25% polygalacturonic acid.

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Table 2 Performance of Aspergillus niger Endopolygalacturonases on Pectins with Various Degrees of Esterication Degree of esterication (%) Endopolygalacturonase 0 7 22 45 60 75

Relative specic activity I II A B C D E 100 100 100 100 100 100 100 97 68 125 150 102 97 103 87 50 135 168 86 93 71 43 24 52 131 36 71 38 18 9 7 62 16 44 16 3 2 6 27 5 25 4

Enzymes were incubated with 0.25% (w/v) substrate in 50 mM sodium acetate buffer, pH 4.2, at 30 C.

it has been proposed that the enzyme is actually an oligogalacturonan hydrolase (27) based on the estimated number of subsites and other features (see below). 1. Effect of Environmental Factors Endopolygalacturonases are generally active between pH 3.5 and pH 6.0. Some enzymes have a quite narrow pH optimum whereas others have a relatively broad pH optimum. As the majority of endopolygalacturonases have been isolated from mesophilic organisms, it is not surprising that the enzymes are also mesophilic. Many polygalacturonases tested in this respect have their highest activity between 40 C and 55 C. However, it should be noted that at these temperatures denaturation sets in. Generally, assays are performed between 25 C and 37 C. Low-molecular-mass inhibitors for endopolygalacturonases have not been reported, although some inhibitory effect can be expected from divalent ions that interact with the substrate. The known inhibitors of polygalacturonases are the polygalacturonase inhibitor proteins (PGIPs) produced by plants. Polygalacturonases have long been implicated in the infection process of plants by phytopathogenic microorganisms and not until recently has direct evidence of this been obtained via gene knock-out studies in A. avus (29) and B. cinerea (30). PGIPs belong to the leucine-rich repeat proteins and are produced by nongraminaceous monocotyledonous and dicotyledonous plants. They have been

proposed to form part of the plants immune system (31). It had been known for quite some time that oligogalacturonides with degree of polymerization > 10 could effectively induce phytoalexin accumulation in plants (32). Cervone and coworkers proposed that inhibition of endopolygalacturonase activity by PGIP would increase the amount of larger oligogalacturonides and hence stimulate the phytoalexin accumulation which in turn would trigger the PGIP production (33). The effect of PGIP on endopolygalacturonase activity was demonstrated in vitro (34). Bergmann et al. (35) showed that the oligogalacturonides indeed exerted such an elicitor function by analyzing PGIP mRNA accumulation. Not only were the oligogalacturonides active in this respect, but wounding as well as infection resulted in a similar observation. Devoto et al. (36) demonstrated by using a PGIP1 promoter glucuronidase (GUS) reporter construct that of the minimally ve PGIP genes present in Phaseolus vulgaris L. at least the PGIP1 gene is only induced by wounding and not by elicitor molecules or infection. Since phytopathogenic fungi generally have a family of endopolygalacturonases at their disposal, different specicities of PGIPs are expected. Indeed, Desiderio et al. (37), Cook et al. (38) and Stotz et al. (39) demonstrated that the inhibition depends on the type of PGIP and the type of endopolygalacturonase. The most comprehensive study in this respect so far was carried out by Leckie and coworkers (40). They studied P. vulgaris PGIP1 and PGIP2 of which only the former cannot interact with the Fusarium moniliforme endopolygalacturonase. In a loss-of-function approach they determined that Q253 of PGIP2 was important for binding to the F. moniliforme endopolygalacturonase. By mutagenizing K253 of PGIP1 into Q253, PGIP1 acquired inhibitory properties toward F. moniliforme endopolygalacturonase. By modeling studies it was shown that K253 is exposed to the surface of the protein (40). Stotz et al. (39) not only demonstrated the differences in specicity of PGIPs and endopolygalacturonases, they also analyzed the evolution of 22 PGIP genes and 19 fungal endopolygalacturonase genes of different origin. It was demonstrated that advantageous amino acid substitutions dominate the molecular evolution of both PGIP and endopolygalacturonases and that the proteins are likely to evolve adaptively in response to natural selection. Unlike the demonstration of Q253 to be critical for F. moniliforme recognition (40), this residue was not signicant in the evolutionary analysis (39).

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2.

Specic Mechanism of Action

Endo- and exopolygalacturonases hydrolyze the glycosidic bond between two adjacent 1,4-linked Dgalacturonic acid residues. Biely et al. (41) demonstrated that for both exoPG and endopolygalacturonase, hydrolysis proceeds with inversion of the anomeric conguration. According to the general concept of glycosyl hydrolytic action inverting hydrolases should have two acidic residues, of which one activates the water that acts as a nucleophile and the other serves as the base, spaced 99.5 A apart. In neither the E. carotovora endopolygalacturonase (4) and A. niger endopolygalacturonase II 3D structures (5) nor in the A. aculeatus rhamnogalacturonase 3D structure (6) are acidic residues spaced at such a distance that they could play the proposed role. By sitedirected mutagenesis studies of A. niger endopolygalacturonase II and by comparison with the phage 22 tailspike rhamnosidase, Van Santen et al. (5) and Armand et al. (15) were able to propose the catalytic mechanism. A triad of three strictly conserved Asp residues at the bottom of the substrate-binding cleft of endopolygalacturonases forms the catalytic machinery. Two Asp residues, Asp180 and Asp202 (A. niger endopolygalacturonase II numbering), together activate the water that acts as the nucleophile whereas Asp201 serves as the base that protonates the leaving group (15). Asp201 is assisted in its role by an adjacent His residue (His223). A His residue has long been thought to be critical for catalysis (4244). By chemical modication Stratilova et al. (45) demonstrated the involvement of a tyrosine in catalysis of an endopolygalacturonase. Recently, ` Pages et al. (46) indeed demonstrated the indirect involvement in catalysis of a strictly conserved Tyr residue (Tyr291, A. niger endopolygalacturonase II numbering). Replacing Tyr291 by Phe reduced the activity to 7% of the wild-type enzyme (46).

3.

Subsites in Binding to Substrate

The A. niger endopolygalacturonase II was the rst enzyme to be characterized with respect to number of subsites, which was estimated at four (47). More recently the entire A. niger endopolygalacturonase family was characterized in this respect (1, 20, 27, 28). For endopolygalacturonase E the number of subsites was estimated to be at least ve, stretching from 4 to 1 (20) and subsite afnities were calculated according to the method outlined by Suganuma et al. (48). The calculation revealed that the total

contribution to the binding of the substrate by subsites 2, 1, and 1 was only small (1.1 kJ/mole). The major contribution of the substrate-binding energy, 14.4 kJ/mole, originated from subsite 3. The low afnity at subsites 2, 1, and 1 can be due to the fact that the substrates used, oligogalacturonates, are not the natural substrates for endopolygalacturonase E. For endopolygalacturonases I, II, A, B, and C, the number of subsites was estimated to be at least seven, from 5 to 2 (1, 28) and for endopolygalacturonase D the number of subsites is four, from 3 to 1 (27). For endopolygalacturonases I and II subsite energy maps were calculated as well (28). A major difference was observed for subsite 5. At this particular subsite, quite remote from the active site, the contribution to binding of the substrate was 6 kJ/mole higher for endopolygalacturonase I than for endopolygalacturonase II. It was proposed that this higher afnity at subsite 5 is the underlying principle for the processive (or multiple attack on a single chain) behavior of endopolygalacturonase I (28). Owing to the high afnity after hydrolysis, the product remains bound from subsites 5 to 1 instead of diffusing away, and subsequently shifts to subsites 4 to 1 for another hydrolytic event. Thus, the processivity appears as an exolytic action from the reducing end and continues until the degree of polymerization of the substrate has reduced so far (n 5 for endopolygalacturonase I) that it does not cover the remote high-afnity subsite anymore. For endopolygalacturonases A, C, and D, processive behavior was observed as well (1, 27, 28). For endopolygalacturonases A and C, the minimum chain length is 6 and for endopolygalacturonase D this is 4. Upon hydrolysis of polymeric substrates the product progression of typical endoacting enzymes such as endopolygalacturonases II, B, and E is characterized by a transient increase of oligomers with n > 6, which are gradually converting into smaller products whereas for the processive enzymes the product progression typically shows a strong increase of monomers from the beginning of the reaction with hardly any transient accumulation of longer oligomers. Of the processive A. niger endopolygalacturonases, endopolygalacturonase D is the most extreme in this respect. Furthermore, endopolygalacturonase D is the only A. niger endopolygalacturonase capable of hydrolyzing dimers. This, in conjunction with only four sub` sites, prompted Par enicova et al. (27) to propose endopolygalacturonase D as an oligogalacturonan hydrolase.

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B.

A. tubingensis Exopolygalacturonase

The A. tubingensis exopolygalacturonase (EC 3.2.1.67) hydrolyzes monomers from the nonreducing end of the substrate (49). Like the majority of A. niger endopolygalacturonases, exopolygalacturonase is optimally active between pH 4 and 5. The calculated subsite map reveals four subsites, stretching from 1 to 3. Subsite 1 has a very high afnity for a galacturonic acid unit (24.5 kJ/mole) whereas subsite 1 even has some repelling force (1:6 kJ/mole). The high afnity at subsite 1 and negative afnity at subsite 1 are probably why nonproductive complex formation occurs readily and why therefore the actual turnover, 220 sec1 , is much lower than the potential intrinsic rate (716 sec1 ) (49). Also, galacturonic acid appeared to be quite a strong inhibitor (Ki 0.3 mM). Both the A. tubingensis and A. aculeatus exopolygalacturonase are able to release xylogalacturonic acid dimers (-xylose-1,3-galacturonic acid) from apple pectin (50) or soy bean pectin (51). This ability may be a reection of the low afnity at subsite 1. It is not known whether xylose-substituted galacturonic acid may be present at subsite 1. In view of the high afnity for galacturonic acid it is likely that this may not be the case. Korner and coworkers (52) have shown by analysis of reaction products of partially methylated galacturonic acid oligomers by mass spectrometry that A. tubingensis exopolygalacturonase does not hydrolyze a nonmethylated galacturonic acid from the reducing end when the following residue is methylated. However, using dened monomethylated dimers and trimers, Kester et al. (53) showed that exopolygalacturonase can accommodate such residues at subsite 1 and 1 albeit with reduced efciency (< 20% of nonmethylated trimer and < 36% of nonmethylated dimer). The discrepancy between these two studies originates from the presence of galacturonic acid monomers, potent inhibitors of exopolygalacturonase, in the oligomer mixture used by Korner et al. (52). In prac tice, during pectin hydrolysis, exopolygalacturonase will cease action when a methylated galacturonic acid has to bind at subsite 1 thus leaving a nonmethylated residue at the nonreducing end.

Erwinia chrysanthemi (54, 55), Clostridium thermosaccharolyticum (56), and Ralstonia solancearum (57). By using a mixture of 4,5-unsaturated oligomers (generated by pectate lyase action) and saturated oligomers, Collmer et al. (54) were able to deduce that the Erwinia enzyme preferred hydrolysis of 4,5-unsaturated dimers, attacked the nonreducing end, and was a single-attack enzyme. The preferred action of this enzyme on unsaturated substrates is understandable in view of the enormous amount of different pectate lyases produced by this organism (see Chapter 80). Shevchik et al. (55) demonstrated that hydrolysis of saturated oligomers indeed occurs at the nonreducing end by using reduced hexagalacturonate. In addition, Kester et al. (53) showed that on puried 4,5-unsaturated trigalacturonate the enzyme is slightly more active than on the saturated counterpart (0.32 U/mL vs. 0.2 U/mL) and that the enzyme can not accommodate a methylated galacturonic acid residue at subsites 2, 1, or 1. For C. thermosaccharolyticum, Van Rijssel et al. (56) were able to show that the exopolygalacturonase activity is associated with a pectin methylesterase activity in a very large complex (1200 kDa). Although this enzyme primarily hydrolyzed dimers from the nonreducing end, the formation of trimers was observed as well. It is not known whether the formation of trimers is a result of a specic binding mode or is due to condensation or tranglycosylation. The latter is highly unlikely to invert. It is therefore interesting that yet another D-galacturonan, digalacturonohydrolase, has been studied that does transglycosylate (58). This enzyme, puried from Selenomonas ruminatium, released dimers from tri-, tetra-, and pentagalacturonates, but at high trimer concentrations bimolecular reactions were observed. This demonstrates that the particular exopolygalacturonase acts via retention of the anomeric conguration and thus should not be included in family 28 of glycosyl hydrolases. D. Synthetic Substrates

C.

Exo-Poly--D-Galacturonosidase

Exo-poly--D-galacturonosidase (EC 3.2.1.82) catalyzes the hydrolysis of digalacturonate from the nonreducing end of polygalacturonic acid (pectate). So far, the activity has only been detected in bacteria like

Using monomethylesteried di- and trigalacturonides carrying the monomethyl group at a dened position (59), Kester et al. (53) studied the specicity of Aspergillus polygalacturonases. The data obtained for the A. tubingensis exopolygalacturonase were discussed above, as were the data for endopolygalacturonase D (27). For all six remaining endopolygalacturonases (I, II, A, B, C, and E) it was shown that at subsite 1 a nonmethylated galacturonate residue is mandatory

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(53). At subsite 1 only endopolygalacturonase I, II, and B were able to accommodate a methylated galacturonate albeit very poorly (activities < 0:6% compared to nonmethylated trimer). At subsite 2 all six enzymes tolerated fairly well a methylated galacturonate (activities between 6% and 38%). This was also shown by Korner et al. (52), who used partially methy lated oligomers mixtures as substrate and analyzed the products of endopolygalacturonase II action by mass spectrometry. Strictly speaking, almost all commercially available pectin compounds are synthetic substrates, as they all have undergone some chemical treatment. This holds even more for pectin series of various degree of methylation that have been either methylated by chemical treatment (acidic methanol) or demethylated by sodium hydroxide treatment or pectin methylesterase action. Especially the pectin methylesterase-treated pectins can give valuable information about substrate preference of pectic depolymerases as a blockwise distribution of methyl groups can be obtained by plant pectin methylesterase action and a random distribution by fungal pectin methylesterase. By using a series of demethylated pectins which were differently prepared, ` Pages et al. (46) demonstrated that endopolygalacturonase II prefers blockwise demethylated substrate but that a single mutation (Glu252Ala) can change the preference profoundly toward less block specicity or to even more block specicity (Gln186Glu). Although these studies are based on kinetic studies (initial rates), valuable information on substrate specicity is also obtained by analyzing the products after various times of hydrolysis of pectins with various degree of methylation (52, 60, 61).

V.

APPLICATION OF POLYGALACTURONASES

Polygalacturonases have found widespread application in many industrial processes. The enzymes applied are mostly obtained from the lamentous fungi of the genus Aspergillus. These preparations generally contain a whole range of cell walldegrading and modifying enzymes and are predominantly applied in the fruit juice industry. Together with pectin methylesterase, endopolygalacturonases are essential in most of these applications e.g., clarication of fruit juices, enzymatic juice extraction, liquefaction of plant tissues (Chap. 67). Fungal pectinase preparations containing predominantly endopolygalacturonases and which are free of

pectin methylesterase activity are used successfully for the production of pulpy nectars. These comminuted fruit juices are viscous, pulpy drinks and are usually prepared by a mechanical-thermal dispersion process. However, products prepared with enzymes are superior in cloud stability and smooth consistency and have higher contents of soluble solids and pigments. These suspensions of loose cells from fruit and vegetable tissue are obtained by weakening the cell cohesion by a limited pectin breakdown, particularly in the middle lamella. Nutrients like vitamin A or proteins are protected inside the cells. Enzyme preparations, which have only one depolymerase system (commonly Endopolygalactronase(s)), are chosen. When endogenous pectin methylesterase is present in the fruit, blanching is indicated. Maceration can also be achieved with endopectin lyase or exopectate lyase. These enzymes might have potential application for processing of vegetables with a higher pH (62). Endopolygalacturonase(s) added to citrus concentrates will reduce the viscosity of these concentrates and contribute(s) to extend the cloud stability of citrus juices. Endopolygalacturonases can also be used for the manufacture of less calcium-sensitive pectin preparations that can be used in systems where the viscosity aspect is not important. Endopolygalacturonases will hydrolyze the galacturonan backbone preferentially in the nonesteried, calcium-sensitive regions. Polygalacturonases are widely distributed in higher plants. They are believed to contribute to fruit softening of pears, peaches, and avocado (62, 64). Polygalacturonases and also pectin methylesterase activities are abundant in tomatoes. Ingenious systems are used in the tomato processing industry to heatinactivate instantaneously (hot break process) to obtain the highly viscous juice preferred by the consumer and the high-consistency concentrated juice (tomato paste) used for sauces, soups, catsup, and similar products. When tomatoes are used for color and avor only, and consistency is provided by other ingredients such as starch, a more easily handled, thin, cold break juice is the starting material for paste production. A holding time is introduced between crushing and heat treatment to ensure breakdown of the pectins by combined pectin methylesterase/polygalacturonase action (64).

VI.

QUALITATIVE AND QUANTITATIVE DETERMINATION OF ACTIVITY

See Chapter 66 on pectic polysaccharides.

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REFERENCES
1. ` L Par enicova, JAE Benen, HCM Kester, J Visser. pgaA and pgaB encode constitutively expressed members of the Aspergillus niger endopolygalacturonase gene family. Biochem J 345:637644, 2000. PM Coutinho, B Henrissat. Carbohydrate-active enzymes: an integrated database approach. In: HJ Gilbert, GJ Davies, B Henrissat, B Svensson, eds. Recent Advances in Carbohydrate Bioengineering. Cambridge: Royal Society of Chemistry, 199, pp 3 12. CJB Van der Vlugt-Bergmans, PJA Meeuwsen, AGJ Voragen, AJJ van Ooyen. Endo-xylogalacturonan hydrolase, a novel pectinolytic enzyme. Appl Environ Microbiol 66:3641, 2000. R Pickersgill, D Smith, K Worboys, J Jenkins. Crystal structure of polygalacturonase from Erwinia carotovora spp. carotovora. J Biol Chem 273:2466024664, 1998. Y van Santen, JAE Benen, K-H Schroter, KH Kalk, S Armand, J Visser, BW Dijkstra. 1.69-A Crystal structure of endopolygalacturonase II from Aspergillus niger and identication of active site residues by sitedirected mutagenesis. J Biol Chem 274:3047430480, 1999. TN Petersen, S Kauppinen, S Larsen. The crystal structure of rhamnogalacturonase A from Aspergillus aculeatus: a right-handed parallel beta helix. Structure 5:533544, 1997. RF Schwan, AH Rose. Polygalacturonase production of Kluyveromyces marxianus: effect of medium composition. J Appl Microbiol 76:6267, 1994. P Blanco, C Sieiro, NM Reboredo, TG Villa. Genetic determination of polygalacturonase production in wild type and laboratory strains of Saccharomyces cerevisiae. Arch Microbiol 167:284288, 1997. M Torki, P Manadaron, F Thomas, F Quigley, R Mache, D Falconet. Differential expression of a polygalacturonase gene family in Arabidopsis thaliana. Mol Gen Genet 261:948952, 1999. SB Hong, ML Tucker. Genomic organization of six tomato polygalacturonases and 5 0 upstream sequence identity with tap1 and win2 genes. Mol Gen Genet 258:479487, 1998. KA Hadeld, JKC Rose, DS Yaver, RM Berka, AB Bennett. Polygalacturonase gene expression in ripe melon fruit supports a role for polygalacturonase in ripening-associated pectin disassembly. Plant Physiol 117:363373, 1998. KA Hadeld, AB Bennett. Polygalacturonases: many genes in search of a function. Plant Physiol 117:337 343, 1998. C Lang, H Dornenburg. Perspectives in the biological function and the technological application of polyga14.

2.

15.

3.

16.

4.

17.

5.

18.

19.

6.

7.

20.

8.

21.

9.

22.

10.

23.

11.

24.

25.

12.

13.

26.

lacturonases. Appl Microbiol Biotechnol 53:366375, 2000. GA Tucker, H Simons, N Errington. Transgenic tomato technology: enzymic modication of pectin pastes. Biotechnol Genet Eng Rev 16:293307, 1999. S Armand, MJM Wagemaker, HCM Kester, P Sanchez-Torres, Y van Santen, BW Dijkstra, J Visser, JAE Benen. The active-site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis. J Biol Chem 275:691 696, 2000. RD Scavetta, SR Herron, AT Hotchkiss, N Kita, NT Keen, JAE Benen, HCM Kester, J Visser, F Jurnak. Structure of plant cell wall fragment complexed to PelC. Plant Cell 11:10811092, 1999. JP Wubben, W Mulder, A Ten Have, JAL van Kan, J Visser. Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea. Appl Environ Microbiol 65:15961602, 1999. Y Yang, C Bergmann, J Benen, R Orlando. Identication of the glycosylation site and glycan structures of recombinant Endopolygalacturonase II (EPG-II) by mass spectrometry. Rapid Commun Mass Spec 11:12571262, 1997. J Colangelo, V Licon, J Benen, J Visser, C Bergmann, R Orlando. Characterization of the glycosylation of recombinant endopolygalacturonase I from Aspergillus niger. Rapid Commun Mass Spec 13:14481453, 1999. ` L Par enicova, JAE Benen, HCM Kester, J Visser. pgaE encodes a fourth member of the endopolygalacturonase gene family from Aspergillus niger. Eur J Biochem 251:7280, 1998. HJD Bussink, HCM Kester, J Visser. Molecular cloning, nucleotide sequence and expression of the gene encoding prepro-polygalacturonase II of Aspergillus niger. FEBS Lett 273:127130, 1990. RE Sheehy, J Pearson, CJ Brady, WR Hiatt. Molecular characterization of tomato fruit polygalacturonase. Mol Gen Genet 208:3036, 1987. D DellaPenna, DC Alexander, AB Bennett. Molecular cloning of tomato fruit polygalacturonase: analysis of polygalacturonase mRNA levels during ripening. Proc Natl Acad Sci USA 83:64206424, 1986. HCM Kester, J Visser. Purication and characterization of polygalacturonases produced by the hyphal fungus Aspergillus niger. Biotechnol Appl Biochem 12:150160, 1990. HJD Bussink, KB Brouwer, LH de Graaff, HCM Kester, J Visser. Identication and characterization of a second polygalacturonase gene of Aspergillus niger. Curr Genet 20:301307, 1991. HJD Bussink, FP Buxton, BA Fraaye, LH de Graaff, J Visser. The polygalacturonases of Aspergillus niger

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

are encoded by a family of diverged genes. Eur J Biochem 208:8390, 1992. ` L Par enicova, HCM Kester, JAE Benen, J Visser. pgaD encodes a new type of endopolygalacturonase from Aspergillus niger. FEBS Lett 467:333336, 2000. JAE Benen, HCM Kester, J Visser. Kinetic characterization of Aspergillus niger N400 endopolygalacturonases I, II and C. Eur J Biochem 259:577585, 1999. M Shieh, RL Brown, MP Whitehead, JW Cary, PJ Cotty, TE Cleveland, RA Dean. Molecular genetic evidence for the involvement of a specic polygalacturonase, P2c, in the invasion and spread of Aspergillus avus in cotton balls. Appl Environ Microbiol 63:35483552, 1997. A Ten Have, W Mulder, J Visser, JAL van Kan. The endopolygalacturonase gene BcPG1 is required for full virulence of Botrytis cinerea. Mol Plant Microbe Interact 11:10091016, 1998. G De Lorenzo, F Cervone. Polygalacturonase-inhibiting proteins (PGIPs): their role in specicity and defense against pathogenic fungi. In: G Stacey, NT Keen, eds. Plant-Microbe Interactions. New York; Chapman & Hall, 1997, Vol 3, pp 7693. MG Hahn, AG Darvill, P Albersheim. Host-pathogen interactions. XIX. The endogenous elicitor, a fragment of a plant cell wall polysaccharide that elicits phytoalexin accumulation in soybeans. Plant Physiol 68:11611169, 1982. F Cervone, G De Lorenzo, L Degra, G Salvi, M Bergami. Purication and characterization of a polygalacturonase inhibiting protein from Phaseolus vulgaris L. Plant Physiol 85:631637, 1987. F Cervone, MG Hahn, G De Lorenzo, A Darvill, P Albersheim. Host-pathogen interactions. XXXIII. A plant protein converts a fungal pathogenesis factor into an elicitor of plant defense responses. Plant Physiol 90:542548, 1989. C. Bergmann, Y Ito, D Singer, P Albersheim, AG Darvill, N Benhamou, L Nuss, G Salvi, F Cervone, G De Lorenzo. Polygalacturonase-inhibiting protein accumulates in Phaseolus vulgaris L. in response to wounding, elicitors and fungal infection. Plant J 5:625634, 1994. A Devoto, F Leckie, E Lupotto, F Cervone, G De Lorenzo. The promoter of a gene encoding a polygalacturonase-inhibiting protein of Phaseolus vulgaris L. is activated by wounding but not by elicitors or pathogen infection. Planta 205:165174, 1998. A Desiderio, B Aracri, F Leckie, B Mattei, G Salvi, H Tigelaar, JSC van Roekel, DC Baulcombe, LS Melchers, G De Lorenzo, F Cervone. Polygalacturonase-inhibiting proteins (PGIPs) with different specicities are expressed in Phaseolus vulgaris. Mol Plant Microbe Interact 10:852860, 1997. BJ Cook, RP Clay, CW Bergmann, P Albersheim, AG Darvill. Fungal polygalacturonases exhibit different

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

substrate degradation patterns and differ in their susceptibilities to polygalacturonase inhibiting proteins. Mol Plant Microbe Interact 12:703711, 1999. HU Stotz, JG Bishop, CW Bergmann, M Koch, P Albersheim, AG Darvill, JM Labavitch. Identication of target amino acids that affect interactions of fungal polygalacturonases and their plant inhibitors. Physiol Mol Plant Pathol 56:117130, 2000. F Leckie, B Mattei, C Capodicasa, A Hemmings, L Nuss, B Aracri, G De Lorenzo, F Cervone. The specicity of polygalacturonase-inhibiting protein (PGIP): a single amino acid substitution in the solvent exposed -strand/-turn region of the leucine rich repeats (LRRs) confers a new recognition capability. EMBO J 18:23522363, 1999. P Biely, JAE Benen, K Heinrichova, HCM Kester, J Visser. Inversion of conguration during hydrolysis of alpha-1,4-galacturonidic linkage by three Aspergillus polygalacturonases. FEBS Lett 382:249255, 1996. L Rexova-Benkova, M Mrackova. Active groups of extracellular endo-D-galacturonase of Aspergillus niger derived from pH effect on kinetic data. Biochim Biophys Acta 523:162169, 1978. R Cooke, C Ferber, L Kanagasabapathy. Purication and characterization of polygalacturonases from a commercial Aspergillus niger preparation. Biochim Biophys Acta 452:440451, 1976. C Caprari, B Mattei, ML Basile, G Slavi, V Crescenzi, G de Lorenzo, F Cervone. Mutagenesis of endopolygalacturonase from Fusarium moniliforme: histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonaseinhibiting protein (PGIP). Mol Plant Microbe Interact 9:617624, 1996. E Stratilova, M Dzurova, O Markovic, H Jornvall. An essential tyrosine residue of Aspergillus polygalacturonase. FEBS Lett 382:164166, 1996. ` S Pages, WHM Heijne, HCM Kester, J Visser, JAE Benen. Subsite mapping of Aspergillus niger endopolygalacturonase II by site-directed mutagenesis. J Biol Chem 275:2934829353, 2000. L Rexova-Benkova. The size of the substrate-binding site of an Aspergillus niger extracellular endopolygalacturonase. Eur J Biochem 39:109115, 1973. T Suganuma, R Matsuno, M Ohnishi, K Hiromi. A study of the mechanism of action of Taka-amylase A1 on linear oligosaccharides by product analysis and computer simulation. J Biochem 84:293316, 1978. HCM Kester, MA KustersVan Someren, Y Muller, J Visser. Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis. Eur J Biochem 240:739746, 1996. HCM Kester, JAE Benen, J Visser. The exopolygalacturonase from Aspergillus tubingensis is also active on

Copyright 2003 by Marcel Dekker, Inc. All Rights Reserved.

51.

52.

53.

54.

55.

56.

57.

xylogalacturonan. Biotechnol Appl Biochem 30:53 57, 1999. G Beldman, LAM van den Broek, HA Schols, MJF SearleVan Leeuwen, KMJ van Laere, AGJ Voragen. An exopolygalacturonase from Aspergillus aculeatus able to degrade xylogalacturonan. Biotechnol Lett 18:707712, 1996. R Korner, G Limberg, TMIE Christensen, JD Mikkelsen, P Roepstorff. Sequencing of partially methyl-esteried oligogalacturonates by tandem mass spectrometry and its use to determine pectinase specicities. Anal Chem 71:14211427, 1999. HCM Kester, D Magaud, C Roy, D Anker, A Doutheau, V Shevchik, N Hugouvieux-Cotte-Pattat, JAE Benen, J Visser. Performance of selected microbial pectinases on synthetic monomethyl-esteried diand trigalacturonates. J Biol Chem 274:3705337059, 1999. A Collmer, CH Whalen, SV Beer, DF Bateman, An exo-poly--D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi. J Bacteriol 149:626634, 1982. VE Shevchik, HCM Kester, JAE Benen, J Visser, J Robert-Baudouy, N Hugouvieux-Cotte-Pattat. Characterization of the exo-pectate lyase PelX of Erwinia chrysanthemi. J Bacteriol 181:16521663, 1999. M van Rijssel, GJ Gerwig, TA Hanse. Isolation and characterization of an extracellular glycosylated protein complex from Clostridium thermosaccharolyticum with pectin methylesterase and polygalacturonate hydrolase activity. Appl Environ Microbiol 59:828 836, 1993. O Huang, C Allen. An exo-poly-alpha-D-galacturonosidase, PeHB, is required for wild-type virulence of Ralstonia solanacearum. J Bacteriol 179:7369 7378, 1997.

58.

59.

60.

61.

62.

63.

64.

K Heinrichova, M Dzurova, L Rexova-Benkova. Mechanism of action of D-galacturonan digalacturonohydrolase of Selenomonas ruminantium on oligogalactosiduronic acids. Carbohydr Res 235:269280, 1992. D Magaud, C Grandjean, A Doutheau, D Anker, VE Shevchik, N Cotte-Pattat, J Robert-Baudouy. Synthesis of the two mono methyl esters of (1-4) linked D-galacturonic acid dimer and of precursors for the preparation of higher oligomers methyl esteried in denite positions. Carbohydr Res 314:189 199, 1998. AJ Mort, F Qiu, NO Maness. Determination of the pattern of methyl esterication in pectindistribution of contiguous nonesteried residues. Carbohydr Res 247:2135, 1993. PJH Daas, K Meyer-Hansen, HA Schols, GA De Ruiter, AGJ Voragen. Investigation of the non-esteried galacturonic acid distribution in pectin with endopolygalacturonase. Carbohydr Res 318:135145, 1999. AGJ Voragen, W Pilnik. Pectin-degrading enzymes in fruit and vegetable processing. In: JR Whitaker, PE Sonnet, eds. Biocatalysis in Agricultural Biotechnology. Washington: ACS Symp Ser No. 389, 1989, pp 93115. W Pilnik, AGJ Voragen. The signicance of endogenous and exogenous pectic enzymes in fruit and vegetable processing. In: PF Fox, ed. Food Enzymology. London: Elsevier Applied Science, 1991, pp 303336. W Pilnik, AGJ Voragen. Pectic enzymes in fruit and vegetable juice manufacture. In: T Nagodawithana, G Reed, eds. Enzymes in Fruit Processing. London: Academic Press, 1993, pp 363399.

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