INTRODUCTION

Top Previous Next References

This review summarizes our current knowledge on the different classes of enzymes involved in plant cell wall polysaccharide degradation produced by Aspergilli, the genes encoding these enzymes, and the regulation of these genes. The data from literature is presented in tables as much as possible to provide easy comparisons of the enzymes and genes reported so far. Only enzymes for which a detailed characterisation was published are presented this way, requiring at least a MW and one of three other characteristics (pI, pH optimum or T optimum). Enzymes that have been characterised in less detail are mentioned in the text when this provided additional information valuable to this review. The tables of genes list the assignment of the corresponding enzymes to the different glycosidase, polysaccharide lyase and carbohydrate esterase families (69, 145-147) as described by B. Henrissat at URL: http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html.

Plant cell wall polysaccharides are the most abundant organic compounds found in nature. They make up 90% of the plant cell wall and can be divided into three groups: cellulose, hemicellulose, and pectin (256). Cellulose represents the major constituent of cell wall polysaccharides and consists of a linear polymer of -1,4-linked D-glucose residues. The cellulose polymers are present as ordered structures (fibers), and their main function is to ensure the rigidity of the plant cell wall.

Hemicelluloses are more heterogeneous polysaccharides and are the second most abundant organic structure in the plant cell wall. The major hemicellulose polymer in cereals and hardwood is xylan. Xylan consists of a -1,4-linked D-xylose backbone and can be substituted by different side groups such as L-arabinose, D-galactose, acetyl, feruloyl, p-coumaroyl, and glucuronic acid residues (400). A second hemicellulose structure commonly found in soft- and hardwoods is (galacto)glucomannan (369), which consists of a backbone of -1,4-linked D-mannose and D-glucose residues with D-galactose side groups (see "Structural features of galacto(gluco)mannan" below). Softwoods contain mainly galactoglucomannan, whereas in hardwoods glucomannan is the most common form. Xyloglucans are present in the cell walls of dicotyledonae and some monocotylodonae (e.g., onion). Xyloglucans consist of a -1,4-linked D-glucose backbone substituted by D-xylose. L-Arabinose and D-galactose residues can be attached to the xylose residues, and L-fucose has been detected attached to galactose residues in xyloglucan. Xyloglucans interact with cellulose microfibrils by the formation of hydrogen bonds, thus contributing to the structural integrity of the cellulose network (56).

Pectins form another group of heteropolysaccharides and consist of a backbone of -1,4-linked D-galacturonic acid residues (see "Structural features of pectin" below). In specific "hairy" regions the galacturonic acid backbone is interrupted by -1,2-linked L-rhamnose residues. Long side chains consisting mainly of L-arabinose and D-galactose residues can be attached to these rhamnose residues. In pectins of certain origins (e.g., sugar beet and apple), ferulic acid can be present as terminal residues attached to O-5 of the arabinose residues or O-2 of the galactose residues.

The hemicellulose and pectin polysaccharides, as well as the aromatic polymer lignin, interact with the cellulose fibrils, creating a rigid structure strengthening the plant cell wall. They also form covalent cross-links, which are thought to be involved in limiting cell growth and reducing cell wall biodegradability. Two types of covalent cross-links have been identified between plant cell wall polysaccharides and lignin (117). The cross-link formed by diferulic acid bridges is studied in most detail. Diferulic acid bridges between polysaccharides and lignin have been identified in many plants. They have been shown to occur between arabinoxylans from bamboo shoot cell walls (162), between pectin polymers in sugar beet (275), and between lignin and xylan in wheat (22). A second type of cross-link is the ester linkage between lignin and glucuronic acid attached to xylan, which was identified in beech wood (160, 359). Recently, indications of a third type of cross-linking have been reported involving a protein- and pH-dependent binding of pectin and glucuronoarabinoxylan to xyloglucan (311). This not yet fully characterized binding is dependent on the presence of fucose on the xyloglucan.

Cellulose consists of linear -1,4-linked D-glucopyranose chains that are condensed by hydrogen bonds into crystalline structures, called microfibrils (205). These microfibrils consist of up to 250 glucose chains and are linked by hemicelluloses (56). In addition to this crystalline structure, cellulose contains noncrystalline (amorphous) regions within the microfibrils. The relative amounts of crystalline and noncrystalline cellulose vary depending on the origin (232).

Two major types of xyloglucans have been identified in the plant cell wall (Fig. 1). Xyloglucan type XXXG consists of repeating units of three -1,4-linked D-glucopyranose residues, substituted with D-xylopyranose via an -1,6-linkage, which are separated by an unsubstituted glucose residue. In xyloglucan type XXGG, two xylose-substituted glucose residues are separated by two unsubstituted glucose residues. The structural features of these, as well as some other types of xyloglucans, have been discussed in detail by Vincken et al. (391). The xylose residues in xyloglucan can be substituted with -1,2-L-fucopyranose--1,2-D-galactopyranose and -1,2-L-galactopyranose--1,2-D-galactopyranose disaccharides (138, 391). L-Arabinofuranose has been detected -1,2-linked to main-chain glucose residues or xylose side groups (151, 157, 308, 409). In addition, xyloglucans can contain O-linked acetyl groups (243, 339). Xyloglucans are strongly associated with cellulose and thus add to the structural integrity of the cell wall. They are thought to play an important role in regulating cell wall extension. The length of the xyloglucan polymers enables them to cross-link many cellulose microfibrils, creating a rigid structure (248).

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Schematic presentation of the repeating units of the two major xyloglucan structures.

The structure of xylans found in cell walls of plants can differ greatly depending on their origin, but they always contain a -1,4-linked D-xylose backbone (101, 399). The schematic representation of xylan (Fig. 2) also lists the different structures which can be attached to the xylan backbone and which result in the large variety of xylan structures found in plants. Although most xylans are branched structures, some linear polysaccharides have been isolated (102, 261). Cereal xylans contain large quantities of L-arabinose and are therefore often referred to as arabinoxylans, whereas hardwood xylans are often referred to as glucuronoxylans due to the large amount of D-glucuronic acid attached to the backbone.

View larger version (9K): [in this window] [in a new window]   FIG. 2.   Schematic presentation of xylan.

Arabinose is connected to the backbone of xylan via an -1,2- or -1,3-linkage either as single residues or as short side chains. These side chains can also contain xylose -1,2-linked to arabinose, and galactose, which can be either -1,5-linked to arabinose or -1,4-linked to xylose. Acetyl residues are attached to O-2 or O-3 of xylose in the backbone of xylan, but the degree of acetylation differs greatly amongst xylans from different origin. Glucuronic acid and its 4-O-methyl ether are attached to the xylan backbone via an -1,2-linkage, whereas aromatic (feruloyl and p-coumaroyl) residues have so far been found attached only to O-5 of terminal arabinose residues (324, 343, 397). As a consequence of all these features, the xylans form a very heterogeneous group of polysaccharides (27, 47, 156, 328).

Galactomannans and galactoglucomannans form a second group of hemicellulolytic structures present in plant cell walls. They are the major hemicellulose fraction of gymnosperms (20), in which they represent 12 to 15% of the cell wall biomass. Galactomannans are most commonly found in the family of Leguminoseae, in which they represent 1 to 38% of seed dry weight, but have also been identified in species of other plants such as Ebenaceae and Palmae (75, 97). They consist of a backbone of -1,4-linked D-mannose residues, which can be substituted by D-galactose residues via an -1,6-linkage (Fig. 3). Depending on the source of the polysaccharide, mannose/galactose ratios can vary from 1.0 to 5.3 (75, 97).

View larger version (6K): [in this window] [in a new window]   FIG. 3.   Schematic presentation of galactomannan.

Galactoglucomannan is the major hemicellulolytic component of softwood. Two different structures can be identified within this group of polysaccharides (Fig. 4) (369). Both consist of a -1,4-linked D-mannose backbone, which can be substituted by -1,6-linked D-galactose. The galactoglucomannan backbone also contains -1,4-linked D-glucose residues. Water-soluble galactoglucomannan has a higher galactose content than does water-insoluble galactoglucomannan and in addition contains acetyl residues attached to the main chain (369). Approximately 20 to 30% of the backbone glucose and/or mannose residues are esterified with acetyl groups at C-2 or C-3 (233). Recently, the structure of a galactoglucomannan from Nicotiana plumbaginifolia was analyzed (338). Apart from side chains consisting of single -1,6-linked galactose residues, this polysaccharide also contained a disaccharide consisting of a galactose residue -1,2-linked to a galactose residue that is -1,6-linked to the main chain.

View larger version (9K): [in this window] [in a new window]   FIG. 4.   Schematic presentation of the two galactoglucomannan structures.

Pectins are complex heteropolysaccharides which contain two different defined regions (85, 290). The "smooth" regions consist of a backbone of -1,4-linked D-galacturonic acid residues, which can be acetylated at O-2 or O-3 or methylated at O-6. In the "hairy" regions, two different structures can be identified, a xylogalacturonan consisting of a D-xylose-substituted galacturonan backbone and rhamnogalacturonan I. In rhamnogalacturonan I (Fig. 5), the D-galacturonic acid residues in the backbone are interrupted by -1,2-linked L-rhamnose residues, to which long arabinan and galactan chains can be attached at O-4. The arabinan chains consist of a main chain of -1,5-linked L-arabinose residues that can be substituted by -1,3-linked L-arabinose and by feruloyl residues attached terminally to O-2 of the arabinose residues (66, 134). The galactan side chains contain a main chain of -1,4-linked D-galactose residues, which can be substituted by feruloyl residues at O-6 (66, 134). Approximately 20 to 30% of the feruloyl residues in sugar beet pectin are attached to arabinan side chains, whereas the other feruloyl residues are attached to galactan side chains (134). Rhamnogalacturonan I also contains acetyl groups ester-linked to O-2 or O-3 of galacturonic acid residues of the backbone (326, 327). Rhamnogalacturonan II is a polysaccharide of approximately 30 monosaccharide units with a backbone of galacturonic acid residues that is substituted by four side chains. The structures of these side chains have been determined and have been shown to contain several uncommon sugars such as 2-O-methyl-L-fucose and 3-deoxy-D-manno-2-octulosonic acid (247). The structural arrangement in which these side chains are attached to the backbone of rhamnogalacturonan II have also been determined and have demonstrated two possible arrangements for this oligosaccharide (148). Whether rhamnogalacturonan II is covalently linked to the pectin main chain is not known.

View larger version (14K): [in this window] [in a new window]   FIG. 5.   Schematic presentation of the hairy region of pectin.

Aromatic compounds are thought to play an important role in the structure and function of the plant cell wall. Ferulic acid can be linked to both the hemicellulose (343) and the pectin (314) fractions of plant cell walls and is able to cross-link these polysaccharides to each other as well as to the aromatic polymeric compound lignin (163, 230). This cross-linked structure results in an increased rigidity of the cell wall. An increase in ferulic acid cross-links during ageing of the plant cell suggests a function for these cross-links in limiting cell growth (118, 395). A role for these cross-links in preventing biodegradability of the plant cell wall by microorganisms has also been suggested. Indications for a limited enzymatic degradation of arabinoxylan due to ferulate cross-links have been obtained (104, 131). Additionally, the antimicrobial effects of these aromatic compounds (21) may contribute to to the plant defense mechanism against phytopathogenic microorganisms.

In cereals, cinnamic acids (mainly ferulic acid) are ester-linked to arabinose residues in arabinoxylan in the primary cell wall. Ferulic acid was detected both as terminal residues and as ferulate dimers linked in several ways (Fig. 6), such as 5,5' or 5,8' carbon-carbon bonds (163, 217).

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Schematical presentation of ferulic acid and diferulic acid structures identified in plant cell walls.

BIODEGRADATION OF PLANT CELL WALL POLYSACCHARIDES

Top Previous Next References

Aspergillus

The genus Aspergillus is group of filamentous fungi with a large number of species. The first record of this fungus can be found in Micheli's Nova Plantarum Genera (258), but a more detailed description of the aspergilli did not appear until the middle of the 19th century. In 1926 a first classification of these fungi was proposed describing 11 groups within the genus (366). A reexamination of the genus was published by Thom and Raper (367), identifying 14 distinct groups. Some of these groups consist of pathogenic fungi (e.g., A. fumigatus, A. flavus, and A. parasiticus), but most important for industrial applications are some members of the group of black aspergilli (A. niger and A. tubingensis). In addition to the morphological techniques traditionally applied, new molecular and biochemical techniques have been used in the reclassification of this group of aspergilli (137, 226, 257, 271, 283, 386). These analyses resulted in the clear distinction of eight groups of black aspergilli (A. niger, A. tubingensis, A. foetidus, A. carbonarius, A. japonicus, A. aculeatus, A. heteromorphus, and A. ellipticus) (283). Products of several of these species have obtained a GRAS (Generally Regarded As Safe) status, which allows them to be used in food and feed applications. The black aspergilli have a number of characteristics which make them ideal organisms for industrial applications, such as good fermentation capabilities and high levels of protein secretion. In particular, the wide range of enzymes produced by Aspergillus for the degradation of plant cell wall polysaccharides are of major importance to the food and feed industry. Recently, several Aspergillus spp. have received increased interest as hosts for heterologous protein production (74).

Four classes of enzymes are involved in the biodegradation of cellulose. Endoglucanases (EC 3.2.1.4) (Table 1) hydrolyze cellulose to glucooligosaccharides. Cellobiohydrolases (EC 3.2.1.91) release cellobiose from crystalline cellulose. -Glucosidases (EC 3.2.1.21) (Table 1) degrade the oligosaccharides to glucose. Exoglucanases (Table 1) release glucose from cellulose and glucooligosaccharides. The distinction between exoglucanases and cellobiohydrolases is not always clear due to differences in the methods used to study these enzymes. All four classes of enzymes have been identified in aspergilli, although the number of isozymes produced by different species or even strains of the same species can differ. An analysis of the production of endoglucanases by 45 A. terreus isolates not only revealed different electrophoretic mobilities for the enzymes of the different isolates but also indicated the absence of endoglucanase I in a number of the isolates (341). Endoglucanases and -glucosidases are also able to degrade the backbone of xyloglucan. From A. aculeatus an endoglucanase has been purified that is specific for the substituted xyloglucan backbone (287). This enzyme was not able to hydrolyze cellulose, and treatment of plant cell walls with the enzyme liberated only xyloglucan oligosaccharides.

View this table: [in this window] [in a new window]   TABLE 1.   Physical properties of endo- and exoglucanases and -glucosidases from Aspergillus

Three exoglucanases have been purified from A. nidulans (24) but only exoglucanase I (Exo-I) was studied in detail. Exo-I, Exo-II, and Exo-III differed significantly in their molecular mass (29, 72.5, and 138 kDa, respectively). Exo-II and Exo-III had a had a higher affinity for cellulose than did Exo-I (24). Two exoglucanases have been identified in A. terreus (170). Two cellobiohydrolases have been purified from A. ficuum (143) and A. terreus (170). The two enzymes from A. ficuum have very different molecular masses (128 and 50 kDa, respectively), whereas the molecular masses of the A. terreus cellobiohydrolases are nearly identical (28.5 and 29.5 kDa, respectively).

Production of cellulolytic enzymes by aspergilli has been observed using the following carbon sources: cellulose (164, 285, 301), sophorose and 2-O--D-glucopyranosyl-D-xylose (155), and cellobiose, glucose and xylose (8). However, other factors were important as well. Production of both endo- and exoglucanases in A. fumigatus was much higher when ammonia was used as a nitrogen source instead of nitrate (347), whereas the production of -glucosidase in A. terreus was higher on nitrate than on ammonia (301). In A. nidulans, an endoglucanase was identified that was developmentally regulated and that was produced only during cleisthothecial development (25). For several -glucosidases, transglycosylation activity has been observed using cellobiose (33, 396), cellotriose, methyl--glucoside, and ethyl--glucoside (407) as substrates.

Based on the derived amino acid sequences, the gene products have been assigned to different glycosidase families. Endoglucanases are assigned mainly to families 5 and 12 (Table 2), with the exception of CelB from A. oryzae. This enzyme was assigned to family 7, which also contains the Aspergillus cellobiohydrolases (Table 2). The only exoglucanase gene cloned so far was assigned to family 74 (Table 2). All -glucosidases from Aspergillus have been assigned to family 3 of the glycosidases (Table 2). All cellulose-degrading enzymes have a retaining mechanism. The exoglucanase from A. aculeatus (family 74) is the only enzyme for which the catalytic mechanism has not yet been determined.

View this table: [in this window] [in a new window]   TABLE 2.   Genes encoding endoglucanases, exoglucanases, cellobiohydrolases, and -glucosidases from Aspergillus and their assignment to the glycosidase families

The biodegradation of the xylan backbone depends on two classes of enzymes. Endoxylanases (EC 3.2.1.8) are able to cleave the xylan backbone into smaller oligosaccharides, which can then be degraded further to xylose by -xylosidases (EC 3.2.1.37). Both classes of enzymes, as well as their encoding genes, have been characterized from many organisms. Various endoxylanases have been identified in Aspergillus (Table 3). Although variation is detected in their molecular mass or pH optimum, the major difference between the enzymes is in their pI, which ranges from 3.5 (168) to 9.0 (119). Endoxylanases also differ in their specificity toward the xylan polymer. Some enzymes cut randomly between unsubstituted xylose residues, whereas the activity of other endoxylanases strongly depends on the substituents on the xylose residues neighboring the attacked residues.

View this table: [in this window] [in a new window]   TABLE 3.   Physical properties of Aspergillus endoxylanases and -xylosidases

In several aspergilli, three different endoxylanases have been identified (119, 168, 210). The best-studied Aspergillus endoxylanases, with respect to substrate specificity, are the three enzymes from A. awamori (210). Counting from the reducing end, A. awamori endoxylanase I is unable to remove one unsubstituted xylose residue adjacent to singly substituted xylose residues or two unsubstituted xylose residues adjacent to doubly substituted xylose residues (206). A. awamori endoxylanase III was not able to remove two unsubstituted xylose residues adjacent to singly or doubly substituted xylose residues toward the reducing end (206).

Hydrolysis of a glucuronoxylan by an endoxylanase from A. niger (130) resulted mainly in xylobiose, xylotriose, and xylose, but hydrolysis of an arabinoxylan by the same enzyme resulted mainly in oligosaccharides with a degree of polymerization of more than 3. This suggests that the action of this endoxylanase is reduced by the presence of arabinose residues on the xylan backbone. All xylanases that have been purified to date are produced when Aspergillus is grown on xylan. Most of these enzymes are also produced when xylose was used as a carbon source, but all at lower levels than on xylan. This is discussed in more detail below (see "Carbon catabolite repression").

Several genes encoding endoxylanases from aspergilli have been cloned. The encoded enzymes have been assigned to glycosidase families 10 and 11 (Table 4), and they all work via a retaining mechanism. Based on the data of the A. kawachii endoxylanases, it would appear that the acidic endoxylanases belong to family 11 whereas the neutral endoxylanases belong to group 10. However, more data on other neutral and acidic endoxylanases are needed to verify this. Recently, a method was developed to experimentally determine whether an endoxylanase belongs to family 10 or 11 (272). This method is based on the irriversible inhibition of family 11 endoxylanases by epoxyl glycosides of D-xylose and xylooligosaccharides, whereas family 10 endoxylanases are unaffected (272).

View this table: [in this window] [in a new window]   TABLE 4.   Genes encoding Aspergillus endoxylanases and -xylosidases and their assignment to the glycosidase families

-Xylosidases have been identified in several aspergilli (Table 3). These enzymes are highly specific for small unsubstituted xylose oligosaccharides (degree of polymerization of up to 4), and their action results in the production of xylose. Although this activity is of major importance for the complete degradation of xylan, absence of the enzyme does not interfere with the induction of the xylanolytic system (382). The ability of an A. awamori -xylosidase to release xylose from xylooligosaccharides was studied to determine its substrate specificity (206). This enzyme was able to release xylose from the nonreducing end of branched oligosaccharides only when two contiguous unsubstituted xylose residues were present adjacent to singly or doubly substituted xylose residues.

Based on the sequence of the corresponding genes, -xylosidases from Aspergillus spp. have all been assigned to glycosidase family 3 (Table 4) and have a retaining mechanism.

For some -xylosidases, transxylosylation activity has been detected (26, 200, 335, 350), allowing the production of novel xylose containing oligosaccharides using these enzymes. Production of xylooligosaccharides from xylose using -xylosidase in a condensation reaction was also demonstrated (159), suggesting a possible application for these enzymes in the synthesis of specific oligosaccharides.

The degradation of the galacto(gluco)mannan backbone depends on the action of -endomannanases (EC 3.2.1.78) and -mannosidases (EC 3.2.1.25), which are commonly produced by aspergilli (Table 5). -Endomannanases, generally referred to as -mannanases, hydrolyze the backbone of galacto(gluco)mannans, resulting in mannooligosaccharides. The ability of -mannanases to degrade the mannan backbone depends on several factors, such as the number and distribution of the substituents on the backbone and the ratio of glucose to mannose (250). -Mannanase is most active on galactomannans with a low substitution of the backbone (64). The presence of galactose residues on the mannan backbone significantly hinders the activity of -mannanase (252), but this effect is small if the galactose residues in the vicinity of the cleavage point are all on the same side of the main chain (251). -Mannanases release predominantly mannobiose and mannotriose from mannan, confirming that they are true endohydrolases (4, 64, 105, 306). It has been shown that A. niger -mannanase binds to four mannose residues during catalysis (249).

View this table: [in this window] [in a new window]   TABLE 5.   Physical properties of Aspergillus -D-mannanases and -D-mannosidases

-Mannosidases (EC 3.2.1.25) are exo-acting enzymes, which release mannose from the nonreducing end of mannooligosaccharides. The substrate specificity of A. niger -mannosidase has recently been studied (3). The enzyme is able to completely release terminal mannose residues when one or more adjacent unsubstituted mannose residues are present. The presence of a galactose-substituted mannose residue adjacent to the terminal mannose residue reduces the activity of -mannosidase to 18 to 43%, compared to unsubstituted substrates, depending on the size of the oligosaccharide (3). Both -mannanase and -mannosidase have transglycosylation activity (153, 169, 252) and can therefore be used for the synthesis of specific oligosaccharides.

Complete degradation of the galacto(gluco)mannan backbone to mannose by -mannanase and -mannosidase also depends on the action of -glucosidase and -galactosidase (see "- and -D-galactosidases" below). Galactomannan-degrading enzymes are produced when Aspergillus is grown on milled soybean (59), locust bean gum (4), galactomannan (64), and mannose (270). So far, only one -mannanase-encoding gene (A. aculeatus man1 [accession no. L35487]) (59) and two -mannosidase -encoding genes (A. aculeatus manB [accession no. AB015509] and A. niger mndA [accession no. AJ251874]) (1, 356) have been reported. Based on the sequences of these genes, the Aspergillus -mannanase and -mannosidases are assigned to glycosidase families 5 and 2, respectively. Both types of enzymes use a retaining mechanism for catalysis.

The structural differences between the main chain of the hairy and smooth regions of pectin have implications for the enzymes involved in the degradation of these regions. The backbone of the smooth region can be hydrolyzed by pectin lyases (EC 4.2.2.10), pectate lyases (EC 4.2.2.2), and polygalacturonases (EC 3.2.1.15 and EC 3.2.1.67). In Aspergillus, families of genes encoding these types of enzymes have been identified (51, 140). Several classes of enzymes are involved in the degradation of the hairy-region backbone.

The pectin main-chain-degrading enzymes can be divided into hydrolases (Table 6) and lyases (Table 7). Six types of hydrolases have been identified in aspergilli. Several endopolygalacturonases are produced that all cleave within the pectin smooth region (10, 185, 284), whereas exopolygalacturonases cleave at the nonreducing terminal end of this region (35, 139, 182, 259). The A. aculeatus and A. tubingensis exopolygalacturonases were able to release galacturonic acid from polygalacturonic acid, sugar beet pectin, and xylogalacturonan (35, 180, 182). It also released the dimer -Xyl-(1,3)-GalA from xylogalacturonan, indicating that the action of the enzyme is not hindered by the presence of xylose on the terminal galacturonic acid residue. The seven endopolygalacturonases produced by A. niger differ in their specific activity (varying from 25 to 4,000 U/mg), sensitivity to methylation of the substrate (36, 183, 281, 284), and mode of action. Four of the enzymes (endopolygalacturonases I, A, C, and D) show processive behavior, also known as multiple attack on a single chain (36, 281, 284), whereas the other three enzymes (endopolygalacturonases II, B, and E) work via a single-attack mechanism (36, 281, 284).

Endorhamnogalacturonan hydrolases cleave within the main chain of rhamnogalacturonan and have been identified in several aspergilli (203, 325, 352). These enzymes are severely hindered in their activity by the presence of acetyl residues on the main chain and require the presence of rhamnogalacturonan acetyl esterase (see "Acetyl- and methylesterases" below) for efficient hydrolysis of the rhamnogalacturonan backbone (90). Also characterized are two exo-acting enzymes, rhamnogalacturonan rhamnohydrolase (265) and rhamnogalacturonan galacturonohydrolase (264), that further degrade the oligosaccharides from the nonreducing end. Recently the activity of an endo-acting xylogalacturonase has been characterized (377) that is specific for a xylose-substituted galacturonic acid backbone. The stereochemical course of hydrolysis of several enzymes involved in the degradation of the main chain of the pectin hairy regions was studied recently (39, 297). Enzymes acting on the hairy regions of pectin, exogalacturonase, rhamnogalacturonan hydrolase, rhamnogalacturonan rhamnohydrolase, and -rhamnosidase (297), as well as enzymes acting on the smooth regions, endopolygalacturonase I and II (39), all hydrolyzed the substrate via an inverting mechanism.

Sequencing of the corresponding genes grouped all Aspergillus endo- and exopolygalacturonases and rhamnogalacturonases in the same glycosidase family, which has an inverting mechanism of hydrolysis (Table 8).

Pectin, pectate, and rhamnogalacturonan lyases cleave the pectin backbone by -elimination, which results in the formation of a 4,5-unsaturated nonreducing end. Pectin lyases prefer substrates with a high degree of methylesterification, whereas pectate lyases prefer those with a low degree of esterification. A clearer distinction between these two types of enzymes can be made based on the absolute requirement of Ca²⁺ ions for catalysis by pectate lyases versus the lack of Ca²⁺ ion requirement by pectin lyases (173). Six pectin lyase genes have been identified in A. niger (37), but so far no indications have been obtained for the presence of more than one pectate lyase (38, 76). The A. niger pectin lyases characterized (A, B, and II) prefer substrates with a high degree of esterification.

Only one rhamnogalacturonan lyase has been identified in aspergilli (203, 266). This enzyme has a higher molecular mass than the pectin and pectate lyases and was positively influenced by Ca²⁺ but did not require Ca²⁺ ions for catalysis (266). The activity of the enzyme was positively affected by the presence of galactose side chains and negatively affected by the presence of arabinose side chains and acetyl residues (266).

Lyases working on the smooth regions (pectin and pectate lyases) and on the hairy regions (rhamnogalacturonan lyases) of pectin have been assigned to two different glycosidase families (Table 8), indicating that the differences in the structure of the substrates require a different enzyme structure as well. In this respect, the lyases are different from the galacturonan hydrolases, which all belong to the same glycosidase family.

Crystal structures have been obtained for endopolygalacturonase II (385) and pectin lyases A and B (246, 393) from A. niger and for rhamnogalacturonan hydrolase A from A. aculeatus (293). All enzymes have the same -helical topology.

-D-Xylosidases. -D-Xylosidases can release -linked xylose residues from xyloglucan. Only a limited number of -xylosidases has been characterized from Aspergillus (Table 9). These enzymes are all highly specific for -linked xylose residues (410, 411) but differ with respect to the type of glycoside they can hydrolyze. Both enzymes from A. niger were able to act on p-nitrophenyl--D-xylanopyranoside, isoprimeverose, and oligosaccharides derived from xyloglucan (244, 245). -Xylosidase I from A. flavus is also able to act on all three types of substrates (410), but -xylosidase II from this fungus is active only on p-nitrophenyl--D-xylanopyranoside and to a small extent on isoprimeverose (411). -Xylosidase I from A. flavus is produced constitutively, whereas -xylosidase II from this fungus is specifically induced by xylose (411).

View this table: [in this window] [in a new window]   TABLE 9.   Physical properties of Aspergillus -D-xylosidases

-L-Arabinofuranosidases and arabinoxylan arabinofuranohydrolases. Arabinose residues can be removed by -L-arabinofuranosidases (EC 3.2.1.55) and arabinoxylan arabinofuranohydrolases. These enzymes and their corresponding genes from many different microorganisms have been studied and have been shown to differ strongly in substrate specificity. Several arabinofuranosidases and arabinoxylan arabinofuranohydrolases have been purified from Aspergillus spp. (Table 10) and studied with respect to their activity on polymeric and oligomeric substrates. The A. niger arabinofuranosidase purified by Kaneko et al. (174) was able to release only terminal -1,3-linked arabinose residues, whereas arabinofuranosidase B from A. niger was able to release terminal -1,2-, -1,3- and -1,5-linked arabinose residues (34). Unlike some of the arabinofuranosidases, the arabinoxylan arabinofuranohydrolase (AXH) from A. awamori was not able to release arabinose from pectin or pectin-derived oligosaccharides but is highly specific for arabinose residues linked to xylan (209). Wood and McCrae (403) reported the ability of an A. awamori arabinofuranosidase to release feruloylated arabinose residues from wheat straw arabinoxylan. Large differences can be observed when the molecular mass and pI of the arabinofuranosidases characterized are compared (Table 10).

Endo- and exoarabinases. Endoarabinases (EC 3.2.1.99) hydrolyze the -1,5-linkages of arabinan polysaccharides, which are present as side chains of pectin. Although some arabinofuranosidases are also able to hydrolyze polymeric arabinan (see the previous section), endoarabinases strongly enhance the efficiency of arabinan degradation and positively influence the action of arabinofuranosidases. So far, no indications have been obtained for the presence of more than one endoarabinase in any Aspergillus sp. (Table 10). The production of endoarabinases by Aspergillus spp. was observed on sugar beet pulp (376) and L-arabinose and L-arabitol (303, 375). In A. niger, induction of AbnA seems to occur simultaneously with the induction of AbfA and AbfB (111).

- and -D-galactosidases. The removal of D-galactose residues from plant cell wall polysaccharides requires the action of -galactosidases (EC 3.2.1.22) and -galactosidases (EC 3.2.1.23) (Table 12). -Galactosidases release terminal galactose residues from the galactan side chains of pectins. -Galactosidases are involved in the degradation of galacto(gluco)mannan, removing galactose from the mannose residues of the backbone. The presence of terminal -linked galactose residues in certain galactoglucomannans (338) suggest that both - and -galactosidases may play a role in the degradation of these polysaccharides. Studies addressing the activity of - and -galactosidases on xylan have not been reported. However, the production of - and -galactosidases on crude substrates containing xylan indicates a putative role for these enzymes in the degradation of xylan. Production of -galactosidases has been reported on arabinoxylan (242), glucose (412), locust bean gum (81), wheat and rice bran (345), lactose and galactose (309), galactomannan (64), and guar flour (5). Aspergillus spp. produce -galactosidase during growth on arabinoxylan (242), polygalacturonic acid (254), wheat bran (129), and lactose (305). Several different -galactosidases have been purified from Aspergillus spp. (Table 12), but there are no indications for the production of more than one -galactosidase by any Aspergillus sp. The differences in molecular mass observed for the purified -galactosidases (Table 12) are most probably due to strain differences and differences in glycosylation of the enzymes.

View this table: [in this window] [in a new window]   TABLE 12.   Physical properties of Aspergillus - and -galactosidases and endo- and exogalactanases

View this table: [in this window] [in a new window]   TABLE 13.   Genes encoding Aspergillus -galactosidases, -galactosidase, and -1,4-endogalactanases and their assignment to the glycosidase families

Endo- and exogalactanases. The galactan side chains of pectin are hydrolyzed by endogalactanases (EC 3.2.1.89), exogalactanases, and -galactosidases (see the previous section). Endogalactanases are able to hydrolyze the galactan polysaccharides, resulting in the liberation of galactobiose and galactose. Production of endogalactanases was observed on beet pulp (190), soybean (61), and locust bean gum (15). Differences between the enzymes exist with respect to their ability to hydrolyze -1,3-, -1,4- or -1,6 linkages between galactose residues. Two types of arabinogalactans are present as side chains of pectins. Type I consists of a backbone of -1,4-linked galactopyranose residues, while type II consists of a backbone of -1,3-linked galactopyranose residues that can be branched by -1,6-linked galactopyranose residues. For the complete degradation of these polysaccharides, all three types of endogalactanases would be required, but so far mainly -1,4-endogalactanases have been reported (Table 12). Two exogalactanases have been purified from A. niger. The -1,4-exogalactanase (43) was able to release galactose from galactooligosaccharides and potato galactan (44, 45). Additionally, this exogalactanase possessed galactose transferase activity (43-45), indicating a possible application for this enzyme in the production of specific galactooligosaccharides and a retaining mechanism of hydrolysis. The -1,3-exogalactanase (288) was not active against native plant polysaccharides but had a high activity against a -1,3-galactan obtained from gum arabic by partial acid hydrolysis and two Smith degradations. The enzyme was capable of releasing the -1,6-side chains of type II arabinogalactans by hydrolyzing the -1,3-linkages in the main chain adjacent to the branching point (288).

-Glucuronidases. Glucuronic acid residues and their 4-O-methyl ethers can be removed from the xylan backbone by -glucuronidases (EC 3.2.1.131). The activity of this enzyme has been detected in a large number of fungal and bacterial culture filtrates, but -glucuronidases have been purified from only a small number of organisms. -Glucuronidases have been isolated from A. niger and A. tubingensis (Table 14). The enzyme is active mainly on small xylooligomers and therefore is dependent on the action of endoxylanases. -Glucuronidases have the highest activity against oligosaccharides, whereas only low or no activity is observed against polymeric substrates (40, 93). Synergy between -glucuronidases and endoxylanases and between -glucuronidases and -xylosidase has been reported (90, 93).

View this table: [in this window] [in a new window]   TABLE 14.   Physical properties of Aspergillus -glucuronidases

Feruloyl and p-coumaroyl esterases. Several types of feruloyl and p-coumaroyl esterases can be identified based on their physical properties as well as by substrate specificity (Table 15). All enzymes (except A. awamori p-coumaroyl esterase) are active on methylferulate, which is a synthetic substrate commonly used for feruloyl esterase assays. Studies of the activities of feruloyl esterases against natural substrates have focused mainly on xylan and xylan-derived oligosaccharides, from which most enzymes were able to release ferulic acid. Only two of these enzymes, FaeA (90, 92) and CinnAE (217), have been shown to release ferulic acid from pectin. A comparative study using A. niger FaeA and CinnAE (216) demonstrated a preference of FaeA for substrates with a methoxy group at position 3 of the aromatic ring, and an increase in activity was observed when the number of methoxy groups on the aromatic ring increased. The activity of CinnAE was low or absent on substrates containing a methoxy group at position 3 of the aromatic ring, whereas additional methoxy groups at other positions of the aromatic ring reduced CinnAE activity compared to unsubstituted compounds. Hydroxy substitutions on the aromatic ring increased the activity of CinnAE but reduced FaeA activity. These two enzymes were also studied with respect to their ability to release ferulic acid from oligosaccharides derived from sugar beet pulp and wheat bran (302). FaeA was able to release ferulic acid, which was linked to O-5 of arabinose (as present in wheat arabinoxylan). FaeA was not able to release ferulic acid linked to O-2 of arabinose (as present in sugar beet pectin) but did release ferulic acid linked to O-6 of galactose (also present in sugar beet pectin), suggesting a specificity for the linkage rather than the polymeric compound. CinnAE (FAE-I) was able to release ferulic acid from all oligosaccharides tested but was more active against arabinose-linked ferulic acid (302). These data suggest that the different feruloyl esterases from A. niger have complementary functions in the degradation of cell wall polysaccharides. Although this has not been studied in detail for other organisms, differences in substrate specificity have been identified for other feruloyl esterases. A. awamori produces a coumaroyl esterase, which is unable to hydrolyze feruloyl esters (253). A similar enzyme has not been reported for other organisms, but in nearly all purifications feruloyl esterase activity was monitored using methylferulate as a substrate. Coumaroyl esterase activity would therefore not be detected.

Acetyl- and methylesterases. Acetylesterases and methylesterases release acetyl and methyl residues from the backbone of cell wall polysaccharides (Table 15). Acetylxylan esterases (EC 3.1.1.72) remove acetyl from O-2 or O-3 of xylose in the xylan-main chain. Although acetylxylan esterase activity has been detected in several aspergilli, such as A. niger, A. japonicus, and A. nidulans (41, 186, 342), only a limited number of acetylxylan esterases have been purified from Aspergillus spp. (208, 213, 351). Unlike most other accessory enzymes, acetylxylan esterases are highly active on the polymeric substrate and are thought to be important for efficient degradation of the xylan backbone by endoxylanases. The presence of the A. niger acetylxylan esterase enabled degradation of steamed birchwood xylan by three types of endoxylanase and a -xylosidase, which could not degrade this substrate in the absence of the esterase (208), indicating the importance of this enzyme in xylan degradation.