Institute for Microbial and Biochemical Technology USDA, Forest Service, Forest Products Laboratory and Department of Bacteriology University of Wisconsin, Madison, Wisconsin
Xylanases are classified into two major families (F or 10 and G or 11) of glycosyl hydrolases. Both use ion pair catalytic mechanisms and both retain anomeric configuration following hydrolysis. Family 10 xylanases are larger, more complex and produce smaller oligosaccharides; Family 11 are more specific for xylan. Alkaline active and extreme thermophilic enzymes are of particular interest. Such xylanases are being commercialized for bleaching pulps and other applications.
Hemicelluloses are widely distributed heteropolysaccharides. The enzymes that degrade them are ubiquitous and diverse. In nature, xylans have L-arabinose, acetyl, glucuronic, 4-O-methylglucuronic, and p-coumaric side chains, and ferulic acid cross linkages.[1] Intra-chain hydrogen bonding occurs through the O-3 position giving unsubstituted xylan a helical twist. Acetylation and substitution, however, disrupt and complicate that structure. Xylans are complexed with cellulose and pectin and are bound to lignin. As esterification and substitution increases, digestibility of the hemicellulose decreases.[2] Removal of the side chains is carried out by acetyl esterase[3] ferulic esterase,[4] glucuronosidase,[5] or arabinosidase,[6] but discussion of such enzymes is beyond the scope of this review.
Native xylans present a formidable substrate for degradation, but commercial xylans recovered by alkaline extraction have a low degree of substitution. Alkaline extraction increases the yield, but it also de-esterifies the substrate, removes acetyl groups, breaks most of the cross linkages, and increases enzymatic degradability. Most assays and screens use commercial xylans. Therefore, despite the large number of xylanases now known, they represent only a small fraction of the enzymatic repertoire involved in hemicellulose degradation. Screens using substrates closer to native polymers will enable the discovery of new enzymes with novel activities or substrate specificities.
Xylanases are drawing increased attention because of their usefulness in facilitating the bleaching of kraft pulp.[7] [8] They increase the extractablity of lignin[9] and release chromophores[10] from pulp. Xylanases also improve the quality of dough and help bread to rise.[11] Xylanases could be used in the bioconversion of lignocellulosic materials to fuels and chemicals.
Xylanases can be divided into two major families of glycosyl hydrolases: Family 10 (F) and 11 (G).[12] The relatedness of enzymes within these families can be demonstrated by pairwise alignments of the protein sequences or by the basic local alignment search tool (BLAST) to discern sequence similarity. BLAST searches using recognized Family 10 (F10) or Family 11 (F11) xylanase protein sequences identify sets of enzymes that are mutually exclusive. By BLAST searches, listings of 77 F10 and 88 F11 xylanases were retrieved at the end of 1995. Only one sequence - that of a bifunctional enzyme possessing both F10 and F11 catalytic regions[13] - was identified using sequences of either F10 or F11 enzymes.
BLAST searches with Family F sequences turn up similarities to [[beta]]-(1->3) and [[beta]]-(1->4) glucanases. Recently, crystallographic studies have shown that the largest cellulase family (Family 1 or A) has a protein fold and an active site similar to those of F10 xylanases.[14] Members of F10 will act on both PNP-xylobiose and PNP-cellobiose, however, the overall catalytic efficiency on PNP-xylobioside is about 50 times higher.[15] This suggests that F10 enzymes act mainly on xylan. However, the relatively greater solubility of the xylan substrate and the higher reactivity of the xylan glycosidic linkage can increase the hydrolytic rate for xylan as compared to cellulose.
For a contemporary listing of the various members of the glycosyl hydrolase family (including xylanase families 10 and 11) visit Amos Bairoch's site listing the SWISS-PROT entries for glycosyl hydrolases.
The F10 catalytic domain is a cylindrical [[alpha]]/[[beta]] barrel resembling a salad bowl with the catalytic site at the narrower end, near the C-terminus of the [[beta]]-barrel.[16,17] There are five xylopyranose binding sites. Catalytic domains of these enzymes belong to a "superfamily" that includes Family A cellulases, [[beta]]-glucosidase, [[beta]]-galactosidase, [[beta]]-(1-3)-glucanases, and [[beta]]-(1->3, 1->4)-glucanases.[18] F10 xylanases have relatively high molecular weights, and they tend to form low DP oligosaccharides. Based on Cex from C. fimi, the overall structure resembles a tadpole[19] with a catalytic (N-terminus) "head" and a cellulose binding domain (C-terminus) "tail."
F11 catalytic domains consist principally of [[beta]]-pleated sheets formed into a two layered trough that surrounds the catalytic site.[20,21] Protruding down into the trough, and located toward one side of the protein is a long loop terminating in an isoleucine (Figure 1). Törrönen and Rouvinden have likened the trough to the palm and fingers and the loop to the thumb of a right hand.[22] The positions of many amino acids are essentially identical in the F11 xylanases from bacterial (Bacillus circulans) and fungal (Trichoderma harzianum) origins. The Trichoderma enzyme, however is more complex. It has at least one extra course of [[beta]]-pleated sheets making up the palm and fingers of the groove (cf. Tyr174 and Asn63 in the Bacillus enzyme and Tyr79 and Asn171 of the Trichoderma enzyme).
Family 11 xylanase from Bacillus circulans (1XNB)
Family 11 xylanase from Trichoderma harzianum (1XND)
Figure 1a. The overall three-dimensional structure of the Family 11 xylanases from Bacillus circulans (1XNB) and Trichoderma harzianum (1XND) are highly similar despite the great genetic distance that separates them.
Two F11 xylanases are produced by Trichoderma. Xyn1 has an acidic pI (5.5), possesses a smaller, tighter groove than Xyn2, and a lower pH optimum[23] It also exhibits a fifteen-fold higher turnover number[24] and a three-fold lower Km. Xyn2 has a basic pI (9.0), a more open structure, and a wider pH range. Xyn2 tends to produce larger oligosaccharides. Both Xyn1 and Xyn2 release xylobiose in the retained [[beta]]-configuration.[25] The difference in pI between Xyn1 and Xyn2 is attributable to the presence of more lysine an arginine residues. There are four in Xyn1, and, ten in Xyn2. These are found on both sides of the "thumb" of the enzyme, and they possibly interact with the glucuronic acid side chains of xylan.[22] Binding of Trichoderma xylanases to polysaccharides is affected by the pH and the ionic strength.[26] Enzymes are totally bound to xylan when the pH is below their pI, but are mainly unbound at pH values above the pI.
The pH optimum depends on properties of the acid/base catalyst.[27] In Xyn1, this is Glu164; in Xyn2, it is Glu177. In Xyn1, Asp33 makes a strong hydrogen bond (2.9 Å) to Glu164, thereby lowering the pKa.[22] In Xyn2, an asparagine residue (Asn44) takes the place of Asp33; the hydrogen bond is much longer (3.7 Å), and the interaction is weaker. All acidic pI xylanases of F11 have an aspartic residue in this position; all basic xylanases have an asparagine residue. The exception that makes the rule is the xylanase of Schizophyllum commune.[28] It has an acidic pI but based on amino acid sequence is grouped with the basic enzymes; it has an asparagine interacting with the acid/base catalyst. In the Bacillus circulans xylanase, Arg112 interacts with Glu78 and Glu172 (which corresponds to Trichoderma Xyn2 Glu177) to raise the pKa of Glu172 to 6.8.[29]
Xylanases possess three to five subsites for binding the xylopyranose rings in the vicinity of the catalytic site. T. reesei Xyn2 has five pyranose binding sites; three are found in Xyn1. Xyn2 also tends to be more transglycosylating. the binding sites are numbered in either direction from the catalytic site and are assigned positive numbers in the direction of the reducing end of the substrate, which constitutes the leaving group (the aglycone), and negative numbers in the direction of the non-reducing end, which remains bound to the catalytic site. The subsites for binding xylopyranose residues are defined by the presence of tyrosine as opposed to tryptophan.[28],[30] Pyranose rings with axial hydroxyls present a hydrophobic surface that interacts with aromatic side chains. Tryptophan is essential for substrate binding in most glycosides but is not reported to play a role in xylanases.
Xylanases do not usually have xylan-specific binding domains,[31,32] but one is present in xylanase (XylD) of Cellulomonas fimi.[33] Substrate binding domains are more common in F10 than in F11 xylanases. They may play an important role in determining specificity and reactivity in pulp bleaching operations (see below). [26] Cellulose binding domains are found in xylanase, arabinofuranosidase [34] and acetylxylan esterase.[35] The only F11 xylanases known to have substrate binding domains are Thermomonospora fusca TfxA and Streptomyces lividans XylB.[36] TfxA binds to both cellulose and insoluble xylan, but the enzyme has activity only against xylan. The complete enzyme has a low Km (1.1 mg/ml); the Km of the catalytic fragment is higher (2.3 mg/ml), indicating that the binding site helps the enzyme "scavenge" for substrate. The S. lividans XylB has 64% identity with T. fusca TfxA. It has been shown to bind to insoluble xylan, but not to cellulose. T. fusca TfxA also has a 21 amino acid glycine-proline rich hinge region that separates the catalytic domain from a xylan/cellulose binding region. Xylanases from C. thermocellum and T saccharolyticum contain conserved domains that are responsible for the ability of these enzymes to resist thermal denaturation. Xylanase Y (XylY) from C. thermocellum also has a C-terminal protein docking sequence.[37] Xylanase B from the fungus Neocallimastix patriciarum has a F10 catalytic subunit and a non-catalytic linker sequence that consists of 45 tandem repeats of an octapeptide rich in hydroxyamino acids and proline.[38] Such linker sequences are common in modular polysaccharidases but infrequent in fungi. The segment does not bear any similarity to cellulose binding domains, but Neocallimastix is known to form large cellulase complexes, and this might also constitute a docking sequence.
All xylanases retain the anomeric configuration of the glycosidic oxygen following hydrolysis. This indicates that they use double displacement mechanism in which the reactive intermediate is bound to the enzyme (Fig. 2). This enables them to carry out transglycosylation reactions. In "retaining" glycosidases, distances between the nucleophile and the acid base catalyst are 5.4 to 5.5 Å.[12] In "inverting" glycosidases, the corresponding distances are greater. This is because for inversion to come about it is necessary for water to come between the aglycone and enzyme.
Figure 2. With reference to 1XNB of Fig. 1, A) The helical xylan substrate is positioned in the trough formed between Tyr65 and Tyr69. Glu172 is the acid/base catalyst and Glu78 is the nucleophile.
B) The glycone is bound to Glu78. This intermediate is retained during transglycosylating reactions.
C) Water displaces the nucleophile.
D) Dissociation and diffusion of the glycone (xylobiose) allows for movement of the enzyme to a new position on the substrate. Because the aglycone is released in step B and the glycone is released in D, many xylanases of Family 11 exhibit a random endo mechanism rather than progressivity.
The use of xylanases in bleaching kraft pulps has spurred an interest in the identification of enzymes with alkaline pH optima. Xylanase J from the alkaliphilic Bacillus sp. Strain 41M-1[39] has a pH optimum of 9, making it one of the most extreme. Other alkaline-active Bacillus xylanases include those from strains N-137,[40] TAR-1,[41] and V1-4.[42] In most instances, alkaline activity results from a broad pH optimum that extends from 5 to 9.5.
Thermotoga maritima is a hyperthermophilic, heterotorophic bacterium that is able to grow at 90deg. C. It produces two thermostable xylanases XynA and XynB.[43] XynA has a molecular mass of 120 kDa and appears to belong to F10. XynB has an apparent molecular mass of 40 kDa and probably belongs to F11. XynA and XynB exhibit optimal activity at 92deg. and 105deg. C, respectively. Both enzymes have acidic pH optima. A F10 xylanase from another Thermotoga species has been used to bleach kraft pulps.[44] Thermostabilization of the B. circulans xylanase has been achieved through the introduction of disulfide bonds.[45]
Xylanases with several different properties have been demonstrated in recent years. For example, they are often known to produce xylobiose, but an enzyme from Aeromonas has been shown to produce xylobiose exclusively.[46] Another Aeromonas xylanase produces only xylotetraose.[47]
Interaction of xylanases with their substrates depends upon the substitution of the xylan moiety. If xylan is substituted with arabinose, hydrolytic products with Streptomyces xylanase are slightly different than when glucuronic or 4-O-methylglucuronic acid substituents are present. This difference apparently does not depend on the sugar charge because when glucuronoxylan is reduced chemically to glucoxylan, the products are similar to those obtained with glucuronoxylan.[48] Products obtained from enzymatic digests of hardwood xylans suggest that this substrate may have (1->2) and (1->3) xylopyranosyl branches,[49] but it is also possible that such products arise from transglycosylation reactions.
Kraft cooking converts 4-O-methylglucuronic acid (MeGlcA) side groups into 4-deoxyhex-4-enuronic acid (HexA) groups almost completely. The HexA side groups are degraded by ozone or chlorine dioxide, but MeGlcA and arabinose side groups are relatively stable toward the bleaching chemicals.[50] The interaction of enzymes with kraft pulp depends on the presence of ionized side chains (see above) and the metal counter ions that may be present.[51] Metal-free pulp is poorly hydrolyzed. Xylanases exhibit different abilities to facilitate bleaching, but bleaching enhancement generally correlates with the release of chromophores from pulp.10
Elucidation of the tertiary structure and catalytic mechanisms of the two major families of microbial xylanases has enabled a better understanding of what determines the pH optimum and substrate binding sites. Genetic engineering has enhanced thermostability but not always with the desired activity at elevated temperature. Further engineering should improve the enzyme properties for commercial bleaching applications. The nature of the xylan binding and the details of enzyme interaction with side chains needs to better understood.
For comments or further information write to Tom Jeffries: twjeffri@facstaff.wisc.edu
Figure 1. The overall three-dimensional structure of the Family 11 xylanases from Bacillus circulans (1XNB) and Trichoderma harzianum (1XND) are highly similar despite the great genetic distance that separates them.
Figure 2. With reference to 1XNB of Fig. 1, A) The helical xylan substrate is positioned in the trough formed between Tyr65 and Tyr69. Glu172 is the acid/base catalyst and Glu78 is the nucleophile. B) the glycone is bound to Glu78. This intermediate is retained during transglycosylating reactions. C) Water displaces the nucleophile. D) Dissociation and diffusion of the glycone (xylobiose) allows for movement of the enzyme to a new position on the substrate. Because the aglycone is released in step B and the glycone is released in D, many xylanases of Family 11 exhibit a random endo mechanism rather than progressivity.