Non-Specific GH30_7 Endo-β-1,4-xylanase from Talaromyces leycettanus

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-β-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear β-1,3-β-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched β-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear β-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the −2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.


Introduction
Xylan is one of the most plentiful polysaccharides on the Earth. It is a major hemicellulose of hardwoods and the second most abundant hemicellulose in softwoods. It also forms a substantial portion of cereals and herbaceous plant cell walls. In general, the xylan backbone is composed of 1,4-linked β-D-xylopyranosyl residues (Xylp). They may be decorated with α-D-glucuronic acid, 4-O-methyl-α-D-glucuronic acid and α-Larabinofuranosyl residues. Moreover, the Xylp residues may be acetylated. The pattern of xylan main chain substitution is species, tissue, and season specific; modulates the physico-chemical properties of the polymer; and renders the hemicellulose water soluble by preventing self-association of the xylan chains [1].
Xylan substitution also has a dramatic impact on its enzymatic digestibility. The depolymerization is catalyzed by endo-β-1,4-xylanases (EC 3.2.1.8). Most of the known xylanases belonging to GH10 and GH11 families act on unbranched or scarcely substituted regions of xylan [2]. Highly decorated xylans are attacked by some GH98 and GH5 xylanases capable of hydrolyzing a glycosidic linkage connecting two substituted Xylp residues [3]. Moreover, it seems that these enzymes not only tolerate but require a certain type of decoration, e.g. 3-O-arabinosylation, of Xylp residue accommodated in the −1 subsite [4]. There are also xylanases that require decoration with uronic acids for their action. The latter catalysts can be found in the GH30 family, specifically in subfamilies GH30_7 and GH30_8 [5].
A vast majority of prokaryotic GH30_8 members are (Me)GlcA appendage-dependent enzymes called glucuronoxylanases (EC 3.2.1.136), although some non-specific endoxylanases belong to the subfamily too [6]. The glucuronoxylan specificity is determined by a very strong interaction of the enzyme -2b subsite with the glucuronic acids, which results in the generation of a series of aldouronic acids of the general formula (Me)GlcA 2 Xyl n [7,8]. The same acidic products are formed by eukaryotic GH30_7 glucuronoxylanases although the recognition of (Me)GlcA residue is not entirely the same. However, the substrate and product specificity among GH30_7 members are more diverse since the subfamily also encompasses non-specific endo-β-1,4-xylanases, reducing-end xylose-releasing enzymes, and non-reducing end-acting xylobiohydrolases [6]. The rationale for the catalytic diversity among various GH30_7 enzymes is not fully understood. In this work, we studied the catalytic properties of an earlier patented but insufficiently characterized GH30 xylanase from Talaromyces leycettanus [9]. The enzyme shows unique properties with regard to tolerance of the main chain substitution and particularly to the decoration of Xylp residue accommodated in the −2 subsite, resulting in an exceptionally high degree of hydrolysis of different types of xylan. The enzyme thus shows a great potential in numerous biotechnological applications.

Optimum and Stability of the Enzyme
Optimal conditions for the enzyme action were tested using beechwood glucuronoxylan as a substrate. The enzyme has an acidic pH optimum 3.0-3.5, while at pH values 2.0 and 5.0 it showed about 75 and 60% of activity ( Figure S1). Approaching neutral pH values resulted in a rapid loss of enzyme activity, which was below 10% at pH 6.0. This acidophilic nature of the enzyme has already been reported [9]. Although the enzyme is most active at acidic pH, it remains active upon storage in a relatively broad pH range of 3.0-7.0 [9]. In the 15 min enzyme assay, the enzyme showed a sharp temperature optimum of 60 • C. At higher temperatures, the activity dropped (e.g. to 40% at 70 • C). About 50% of the maximum activity was determined at 40 • C; however, at room temperature (23 • C), the enzyme was almost inactive (approximately 1% activity) ( Figure S1). A similar temperature profile has been published [9]. We also examined the temperature stability of the enzyme for the first time. At the temperature optimum, 60 • C, the enzyme gradually lost activity, exhibiting about half activity after 3 h. At 50 • C, the enzyme was fully stable for 2 h and then the activity started to decline. At 40 • C, the enzyme retained activity for 3 h, but upon a longer incubation, the activity was reduced ( Figure S1).
The hydrolysis products were analyzed by TLC and MALDI-ToF MS. Glucuronoxylan was initially hydrolyzed to a series of products that migrated differently to linear β-1,4-xylooligosaccharides, suggesting that all the products are branched with MeGlcA ( Figure 1). Indeed, treatment of an early stage reaction mixture (after a heat-inactivation of TlXyn30A) with a GH3 β-xylosidase liberating xylose (Xyl) from the non-reducing end resulted in their conversion to a single oligosaccharide, aldotriouronic acid, having a structure MeGlcA 2 Xyl 2 ( Figure 2). This finding serves as evidence that the initial hydrolysis products generated by TlXyn30A are acidic oligosaccharides of the general structure MeGlcA 2 Xyl n , i.e., the fragments have a MeGlcA residue attached to the second Xylp residue from the reducing end.
The aldouronic acids MeGlcA 2 Xyl n did not accumulate in the reaction mixture, but their gradual degradation led to larger linear β-1,4-xylooligosaccharides of a degree of polymerization up to 12, which were further trimmed mainly to xylobiose (Xyl 2 ) accompanied by small amounts of Xyl and xylotriose (Xyl 3 ). Consequently, the limit hydrolyzate contained a single major aldouronic acid MeGlcA 2 Xyl 2 with traces of two larger homologs, MeGlcA 2 Xyl 3 and MeGlcA 2 Xyl 4 .
TLC monitoring of arabinoxylan hydrolysis indicated that after 5 min, when the acidic products generated from GX were clearly visible on the TLC plate, no detectable oligosaccharides were formed from AX. The initial products of AX hydrolysis were a series of larger arabinoxylooligosaccharides (Figure 1). Upon a longer incubation (24 h), the amount of these products decreased on account of the appearance of a broad palette of shorter products, mostly arabinoxylooligosaccharides resistant towards β-xylosidase. A small amount of Xyl 2 present in the hydrolysate disappeared after the application of β-xylosidase.  Figure 1. TLC analysis of the products generated by TlXyn30A xylanase from glucuronoxylan (GX), rhodymenan (Rho), and arabinoxylan (AX). After 24 h, TlXyn30A was heat-denatured and then the mixtures were treated with β-xylosidase for 24 h (lanes denoted by βX). Note a remarkable change in the products generated from GX and Rho upon the β-xylosidase treatment, in contrast to a resistance of the oligosaccharides generated from AX (marked by an asterisk). S-standards of linear β-1,4-xylooligosaccharides. . TLC analysis of the products generated by TlXyn30A xylanase from glucuronoxylan (GX), rhodymenan (Rho), and arabinoxylan (AX). After 24 h, TlXyn30A was heat-denatured and then the mixtures were treated with β-xylosidase for 24 h (lanes denoted by βX). Note a remarkable change in the products generated from GX and Rho upon the β-xylosidase treatment, in contrast to a resistance of the oligosaccharides generated from AX (marked by an asterisk). S-standards of linear β-1,4-xylooligosaccharides.
GX + TlXyn30A (early stage of hydrolysis) The above sample after ß-xylosidase treatment The aldouronic acids MeGlcA 2 Xyln did not accumulate in the reaction mixtur their gradual degradation led to larger linear β-1,4-xylooligosaccharides of a degr polymerization up to 12, which were further trimmed mainly to xylobiose (Xyl2) ac panied by small amounts of Xyl and xylotriose (Xyl3). Consequently, the limit hyd zate contained a single major aldouronic acid MeGlcA 2 Xyl2 with traces of two l homologs, MeGlcA 2 Xyl3 and MeGlcA 2 Xyl4.
TLC monitoring of arabinoxylan hydrolysis indicated that after 5 min, whe acidic products generated from GX were clearly visible on the TLC plate, no detec oligosaccharides were formed from AX. The initial products of AX hydrolysis were ries of larger arabinoxylooligosaccharides (Figure 1). Upon a longer incubation ( the amount of these products decreased on account of the appearance of a broad p of shorter products, mostly arabinoxylooligosaccharides resistant towards β-xylosi A small amount of Xyl2 present in the hydrolysate disappeared after the applicati β-xylosidase. Rhodymenan (β-1,3-β-1,4-xylan) was hydrolyzed at a rate comparable to AX was converted to at least two series of oligomeric products. A minor series consi linear β-1,4-xylooligosaccharides. The series of mixed lin β-1,3-β-1,4-xylooligosaccharides comprising a single β-1,3-linkage showed a sub tially higher chromatographic mobility than β-1,4-xylooligosaccharides. The sh mixed linkage xylooligosaccharide is an isomeric xylotriose having a stru Xylpβ3Xylpβ4Xyl, based on our previous publication [5]. After 24 h of incubation number as well as the quantity of the larger oligosaccharides was markedly reduc account of an increase in Xyl, Xyl2, and the isomeric xylotriose as the main final ucts, indicating an ability of TlXyn30A to hydrolyze certain β-1,3-xylosidic link New faint spots also appeared, which likely correspond to xylooligosaccharides h Rhodymenan (β-1,3-β-1,4-xylan) was hydrolyzed at a rate comparable to AX and was converted to at least two series of oligomeric products. A minor series consists of linear β-1,4-xylooligosaccharides. The series of mixed linkage β-1,3-β-1,4-xylooligosaccharides comprising a single β-1,3-linkage showed a substantially higher chromatographic mobility than β-1,4-xylooligosaccharides. The shortest mixed linkage xylooligosaccharide is an isomeric xylotriose having a structure Xylpβ3Xylpβ4Xyl, based on our previous publication [5]. After 24 h of incubation, the number as well as the quantity of the larger oligosaccharides was markedly reduced on account of an increase in Xyl, Xyl 2 , and the isomeric xylotriose as the main final products, indicating an ability of TlXyn30A to hydrolyze certain β-1,3-xylosidic linkages. New faint spots also appeared, which likely correspond to xylooligosaccharides having several β-1,3-linkages. All the products were converted to xylose upon the addition of β-xylosidase ( Figure 1).
The enzyme also catalyzed a considerably deeper hydrolysis of acetylglucuronoxylan (not shown) when compared to the performance of other types of endo-β-1,4-xylanases (GH10, GH11, and specific GH30 glucuronoxylanases). This result supported the conclusions obtained with other types of xylan that TlXyn30A unusually well tolerates xylan side residues.
Both acidic oligosaccharides were shortened by one xylose from the reducing end, yielding MeGlcA 2 Xyl 2 and MeGlcA 2 Xyl 3 , respectively (Figures 3 and 4A). The cleavage pattern of the short aldouronic acids confirms that the enzyme accommodates MeGlcA-substituted Xylp residue in the -2 subsite.
The hydrolysis rate of the short aldouronic acids was significantly faster than that of unbranched xylooligosaccharides ( Figure 3). It is in line with the glucuronoxylanase and endoxylanase activity observed on GX. Among linear xylooligosaccharides, only Xyl 2 was found to be resistant to the enzyme action, which is consistent with its accumulation in the GX and rhodymenan hydrolyzates. Xyl 3 (compound 5) served as a relatively poor substrate, requiring more than 4 h for its entire conversion to an equimolar mixture of Xyl 2 and Xyl. It is noteworthy that the conversion is not a simple hydrolysis, since larger oligosaccharides, mostly Xyl 5 , were detected in an early stage of the reaction, when a higher trisaccharide concentration was used. The transglycosylation pathway was also observed during Xyl 4 (compound 4) degradation, where the initial products were solely Xyl 2 and Xyl 3 . The tetrasaccharide was consumed within approximately 1 h and the later formation of monomeric Xyl coincided with the disappearance of Xyl 3 . Similarly, Xyl 5 and Xyl 6 were initially predominantly converted to an oligosaccharide mixture comprising from disaccharide to the oligosaccharide shorter by one Xyl residue than the substrate. The shortening continued to Xyl 2 as a final product accompanied with a small amount of Xyl. This mode of action on all linear xylooligosaccharides was not compatible with xylobiohydrolases acting from the non-reducing end and confirms that TlXyn30A behaves as a retaining endo-β-1,4-xylanase, exhibiting complex reaction pathways that include glycosyl transfer reactions.

Arabinofuranosyl Side Residues Tolerated in the −2 Subsite
We were eager to learn whether arabinosylation at position 2 or 3 of Xylp residue is tolerated by the enzyme. Therefore, singly and doubly decorated xylooligosaccharides

Arabinofuranosyl Side Residues Tolerated in the −2 Subsite
We were eager to learn whether arabinosylation at position 2 or 3 of Xylp residue is tolerated by the enzyme. Therefore, singly and doubly decorated xylooligosaccharides arabinosylated on the first or second Xylp residue from the non-reducing end were treated with the enzyme. 3 -Arabinosylated xylobiose (Araf α3Xylpβ4Xyl; compound 23) was left intact ( Figure 4A). Quite surprisingly, the enzyme was able to hydrolyze all other tested arabinoxylooligosaccharides comprising at least three xylosyl units (compounds 6-10). All of them were attacked at the second xylosidic linkage from the branch towards the reducing end ( Figure 4A), resulting in the liberation of Xyl regardless of the arabinose attached to position 2 and/or 3. A similar accommodation of arabinosylated Xylp residue in the −2 subsite has been reported for a non-specific Clostridium acetobutylicum GH30_8 endoxylanase CaXyn30A [10]. The latter enzyme hydrolyzed 2-O-singly and 2,3-doubly arabinosylated xylooligosaccharides faster than 3-arabinosylated, linear, and MeGlcAsubstituted counterparts.

Action of TlXyn30A on Artificial Compounds
The enzyme was also tested on several 4-nitrophenyl glycosides. Of them, 4-nitrophenyl glycosides of cellobioside and lactoside were not attacked. In contrast, xylobioside (compound 3) was readily hydrolyzed, predominantly to Xyl 2 ( Figure 4A), indicating that the aromatic aglycone was bound in the +1 subsite. The most interesting result was obtained with 4-nitrophenyl xylotrioside (compound 2), forming two productive enzyme-substrate complexes. In the first one, the aromatic aglycone was bound in the +1 subsite (similarly to nitrophenyl xylobioside), resulting in the liberation of the aglycone and Xyl 3 . In the second complex, Xyl 2 was liberated from the non-reducing end with a simultaneous release of 4-nitrophenyl xyloside. The first cleavage mode was, however, much more frequent ( Figure 4A), speaking for endoxylanase action and against xylobiohydrolase activity.

Catalytic Properties
The studied TlXyn30A xylanase behaves as a typical endo-acting enzyme, efficiently hydrolyzing several types of xylan. However, hardwood glucuronoxylan is still a preferred substrate since the enzyme recognizes to some extent the MeGlcA side residues. The polysaccharide is initially hydrolyzed to a mixture of aldouronic acids MeGlcA 2 Xyl n , suggesting the accommodation of MeGlcA-substituted Xylp residue in the −2 subsite. Such recognition of the MeGlcA side chain was also confirmed by the enzyme action on short aldouronic acids MeGlcA 3 Xyl 3 and MeGlcA 3 Xyl 4 . This mode of TlXyn30A initial action is identical to that of other GH30 glucuronoxylanases irrespective of their fungal (GH30_7) or bacterial (GH30_8) origin [6]. However, a distinct feature of TlXyn30A is that the aldouronic acids are further converted to shorter products and finally to aldotriouronic acid MeGlcA 2 Xyl 2 with a tiny admixture of aldotetraouronic acid MeGlcA 2 Xyl 3 ( Figure 5). This implies that aglycone-binding subsites +1 and +2 are capable of accommodating MeGlcA-decorated xylose residue. Such shortening of larger aldouronic acids is typical for GH30_7 xylobiohydrolases [5] and some eukaryotic glucuronoxylanases that exhibit xylobiohydrolase activity. The latter may be exemplified by Talaromyces cellulolyticus Xyn30B showing minor xylobiohydrolase activity [11] and Thermothelomyces thermophila Xyn30A, for which xylobiohydrolase action is roughly equal to glucuronoxylanase activity [12]. These two enzymes yield a roughly equimolar mixture of MeGlcA 2 Xyl 3 and MeGlcA 2 Xyl 2 as limit glucuronoxylan hydrolysis products, and the short aldouronic acids are a result of stepwise liberation of Xyl 2 from the non-reducing end of the initially formed MeGlcA 2 Xyl n . This is not the case of the glucuronoxylan hydrolyzate generated by TlXyn30A, where MeGlcA 2 Xyl 2 is much more abundant than MeGlcA 2 Xyl 3 . The reason for this difference is that TlXyn30A exhibits non-specific endoxylanase activity, instead of xylobiohydrolase activity. The non-specific endoxylanase-type mode of action, rather than xylobiohydrolase, is also observed during the cleavage of linear xylooligosaccharides. aldouronic acids are further converted to shorter products and finally to aldotriouronic acid MeGlcA 2 Xyl2 with a tiny admixture of aldotetraouronic acid MeGlcA 2 Xyl3 ( Figure 5). This implies that aglycone-binding subsites +1 and +2 are capable of accommodating MeGlcA-decorated xylose residue. Such shortening of larger aldouronic acids is typical for GH30_7 xylobiohydrolases [5] and some eukaryotic glucuronoxylanases that exhibit xylobiohydrolase activity. The latter may be exemplified by Talaromyces cellulolyticus Xyn30B showing minor xylobiohydrolase activity [11] and Thermothelomyces thermophila Xyn30A, for which xylobiohydrolase action is roughly equal to glucuronoxylanase activity [12]. These two enzymes yield a roughly equimolar mixture of MeGlcA 2 Xyl3 and MeGlcA 2 Xyl2 as limit glucuronoxylan hydrolysis products, and the short aldouronic acids are a result of stepwise liberation of Xyl2 from the non-reducing end of the initially formed MeGlcA 2 Xyln. This is not the case of the glucuronoxylan hydrolyzate generated by TlXyn30A, where MeGlcA 2 Xyl2 is much more abundant than MeGlcA 2 Xyl3. The reason for this difference is that TlXyn30A exhibits non-specific endoxylanase activity, instead of xylobiohydrolase activity. The non-specific endoxylanase-type mode of action, rather than xylobiohydrolase, is also observed during the cleavage of linear xylooligosaccharides. The recognition of MeGlcA-substituted Xylp residue in the -2 subsite of TlXyn30A is not the only substitution that the enzyme can tolerate. Hydrolysis of both 2-O-and 3-O-singly as well as 2,3-di-O-arabinosylated xylooligosaccharides showed that the arabinosylation at these positions does not abolish the enzyme action, and all types of arabinosylated xylose can also be accommodated in the −2 subsite ( Figure 4B). Based on the relatively fast chromatographic mobility of the shortest arabinoxylan fragments generated by TlXyn30A and their resistance to the GH3 β-xylosidases, which is unable to liberate the non-reducing end terminal Xylp residue linked to 3-O-arabinoxylated Xylp residue [13], we suggest that the arabinoxylooligosaccharides are of two types with regard of the non-reducing end. Those singly substituted at position 2 are terminated by the 2-O-arabinosylated Xylp residue, while those comprising 3-O-singly or 2,3-doubly arabinosylated Xylp residue (the vast majority of the fragments) may be terminated at the non-reducing end with an additional non-substituted xylose. Despite the uncertainty about the structure of arabinoxylooligosaccharides, efficient AX degradation to the short fragments is a unique and important capability of the enzyme in view of biotechnological applications.
The mode of cleavage of arabinosylated and glucuronylated xylooligosaccharides showed that a prerequisite for their hydrolysis is the presence of at least two unsubstituted Xylp residues from the branch towards the reducing end. In all cases, the decorated Xylp residue is accommodated in the −2 subsite and the pair of unsubstituted Xylp residues are bound to −1 and +1 subsites. The occupation of the +1 subsite by a short aliphatic aglycone, such as the methyl group, is not sufficient for the formation of a productive enzyme-substrate complex. This fact was documented by the resistance of me- Figure 5. Schematic presentation of the mode of TlXyn30A action on glucuronoxylan. Initially, the polysaccharide is converted to a mixture of aldouronic acids of a general structure MeGlcA 2 Xyl n . Later, the unsubstituted regions are also attacked by the enzyme, yielding aldotriouronic acid MeGlcA 2 Xyl 2 and Xyl 2 as major products (in solid boxes) that are accompanied with small amounts of aldotetraouronic acid MeGlcA 2 Xyl 3 , Xyl, and Xyl 3 (dashed boxes) as limit hydrolysis products.
The recognition of MeGlcA-substituted Xylp residue in the −2 subsite of TlXyn30A is not the only substitution that the enzyme can tolerate. Hydrolysis of both 2-O-and 3-Osingly as well as 2,3-di-O-arabinosylated xylooligosaccharides showed that the arabinosylation at these positions does not abolish the enzyme action, and all types of arabinosylated xylose can also be accommodated in the −2 subsite ( Figure 4B). Based on the relatively fast chromatographic mobility of the shortest arabinoxylan fragments generated by TlXyn30A and their resistance to the GH3 β-xylosidases, which is unable to liberate the non-reducing end terminal Xylp residue linked to 3-O-arabinoxylated Xylp residue [13], we suggest that the arabinoxylooligosaccharides are of two types with regard of the non-reducing end. Those singly substituted at position 2 are terminated by the 2-O-arabinosylated Xylp residue, while those comprising 3-O-singly or 2,3-doubly arabinosylated Xylp residue (the vast majority of the fragments) may be terminated at the non-reducing end with an additional non-substituted xylose. Despite the uncertainty about the structure of arabinoxylooligosaccharides, efficient AX degradation to the short fragments is a unique and important capability of the enzyme in view of biotechnological applications.
The mode of cleavage of arabinosylated and glucuronylated xylooligosaccharides showed that a prerequisite for their hydrolysis is the presence of at least two unsubstituted Xylp residues from the branch towards the reducing end. In all cases, the decorated Xylp residue is accommodated in the −2 subsite and the pair of unsubstituted Xylp residues are bound to −1 and +1 subsites. The occupation of the +1 subsite by a short aliphatic aglycone, such as the methyl group, is not sufficient for the formation of a productive enzyme-substrate complex. This fact was documented by the resistance of methyl glycoside of 3 -arabinosylxylobiose (compound 24) to TlXyn30A ( Figure 4A) and extremely slow hydrolysis of methyl β-1,4-xylobioside (compound 25). This was in contrast to a single productive binding of methyl β-1,4-trioside (compound 11), which was more efficiently cleaved to methyl xyloside and Xyl 2 . It is worth mentioning the strong affinity of the +1 subsite for aromatic aglycones, specifically for the 4-nitrophenyl moiety, which results in liberation of the chromophore from 4-nitrophenyl xylobioside and trioside (compounds 3 and 2) ( Figure 4A). In addition, the binding of the aromatic aglycone in the +1 subsite enhances the hydrolysis to such an extent that the aromatic glycosides derived from both linear β-1,4-xylooligosaccharides are hydrolyzed as efficiently as the short aldouronic acids MeGlcA 3 Xyl 3 and MeGlcA 3 Xyl 4 .

Structure-Function Relationship
TlXyn30A is composed of an N-terminal catalytic GH30 domain that is linked to a C-terminal CBM1 domain. The presence of the cellulose-binding domain indicates that the enzyme plays a role in the degradation of complex lignocellulose material, wherein hemicellulose xylan is present in close proximity to cellulose. Of the characterized GH30_7 enzymes, TcXyn30C has a similar architecture [14], although both full-length proteins and catalytic domains are about 50% identical. A similar identity of the catalytic domain was found for other characterized GH30_7 single-domain enzymes, namely Hypocrea jecorina glucuronoxylanase XynVI (54.8%) and Thermothelomyces thermophila glucuronoxylanase/xylobiohydrolase TtXyn30A (53.0%). The catalytic domain of TlXyn30A has the highest homology (up to 75% identity and 85% similarity) to the sequences found in the genomes of several Aspergillus species, such as A. terreus (Uniprot ID: Q0CUH3), A. thermomutatus (A0A397HZ03), A. turcosus (A0A421D0F6), A. udagawae (A0A0K8KZ21), A. lentulus (A0A0S7DIT3), A. clavatus (A1C6U9), and A. novofumigatus (A0A2I1BUY5), of which the A. thermomutatus and A. clavatus proteins comprise the CBM1 domain at the C-terminus. Such a high homology suggests that the aforementioned proteins are most probably uncharacterized GH30_7 xylanases that appear to be widespread among Aspergillus species.
All the above mentioned Aspergillus proteins as well as TlXyn30A contain an arginine residue (eukaryotic arginine) that is conserved in the GH30_7 sequences, including TcXyn30B, for which it has been shown to interact with the carboxyl group of the MeGlcA moiety of the glucuronoxylan-type substrate [15]. The MeGlcA carboxyl group is also hydrogen bonded to TcXyn30B Glu345 and Ser351 [15]. In the corresponding positions, the same amino acid residues are also found in the sequence of TlXyn30A ( Figure 6B), thus explaining its preference for GX and the mode of its initial cleavage to aldouronic acids MeGlcA 2 Xyl n . However, other factors finely tuning the substrate specificity, in particular the xylobiohydrolase activity of the GH30_7 enzymes, are the length and primary structure of the β2-α2 loop in the GH30_7 members. If the loop is long enough, it may interfere with the accommodation of the substituted Xylp residue in the −3 subsite and also obstruct the occupation of more distant negative binding subsites by larger substrates, thus endowing the enzyme with xylobiohydrolase activity [5]. The loop of all the GH30_7 enzymes exhibiting xylobiohydrolase activity, namely TcXyn30B, TtXyn30A, and AaXyn30A, is five amino acids longer than that of TlXyn30A ( Figure 6A) and also the Aspergillus proteins of high sequence identity. The shorter β2-α2 loop of TlXyn30A could be the reason that the loop does not represent any obstacle to bind larger substrates to distant negative subsites, which is a prerequisite for the endoxylanase activity of the enzyme.
(A) (B) Figure 6. Multiple sequence alignment of TlXyn30A and selected GH30 xylanases. The first two sequences, TcXyn30A (Uniprot ID: A0A0B8MZ29) and TrXynIV (A0A024RWW9), represent reducing-end xylose-releasing enzymes. For the next three sequences, AaXyn30A [5], TcXyn30B (A0A4V8H018), and TtXyn30A (G2Q1N4), non-reducing end xylobiohydrolase activity has been demonstrated. The latter two enzymes are specific glucuronoxylanases, similar to TrXynVI (G0RV92). In contrast, TcXyn30C (A0A6N4SL16) is a non-specific endoxylanase. The next sequence, At_QUCUH3, corresponding to an uncharacterized Aspergillus terreus protein, shows the highest similarity to TlXyn30A. The last 5 sequences are of bacterial origin and belong to the GH30_8 subfamily. HcXyn30A (G8LU16) is a unique prokaryotic non-reducing end xylobiohydrolase. EcXyn30A (Q46961) and BsXynC (Q45070) are canonical GH30_8 glucuronoxylanases. CaXyn30A (Q97TI2) and RpXyn30A (F1TBY8) are exceptional GH30_8 non-specific endoxylanases. Only the regions that are believed to have the highest impact on the unique catalytic properties of TlXyn30A (typed in violet color) are shown. The corresponding secondary structure elements of TcXyn30B (green) and EcXyn30A (red) are shown on the top and bottom. Part (A) displays the sequences in the β2-α2 loop, which is considerably larger in eukaryotic GH30_7 enzymes, compared to prokaryotic GH30_8 members. However, even in the eukaryotic sequences, there are differences in the length of the loop that are presumably related to their substrate specificity. Part (B) shows the sequences in the β8-α8 region, which contains amino acids interacting with the MeGlcA moiety of the acidic substrates like GX. Among the GX-interacting amino acids is a prokaryotic arginine (typed in blue and marked by an up triangle), which endows the canonical GH30_8 members with glucuronoxylanase specificity. For multiple sequence alignment of entire amino acid sequences, the reader is referred to Supplementary Materials ( Figure S2).

Enzymes
Gene cloning and expression as well as encoded protein purification and verification of the purity of Talaromyces leycettanus CBS 398.68 GH30 xylanase (55 kDa by SDS PAGE) were described previously [9]. GH3 β-xylosidase is a recombinant Aspergillus niger enzyme expressed in Saccharomyces cerevisiae [16]. . Multiple sequence alignment of TlXyn30A and selected GH30 xylanases. The first two sequences, TcXyn30A (Uniprot ID: A0A0B8MZ29) and TrXynIV (A0A024RWW9), represent reducing-end xylose-releasing enzymes. For the next three sequences, AaXyn30A [5], TcXyn30B (A0A4V8H018), and TtXyn30A (G2Q1N4), non-reducing end xylobiohydrolase activity has been demonstrated. The latter two enzymes are specific glucuronoxylanases, similar to TrXynVI (G0RV92). In contrast, TcXyn30C (A0A6N4SL16) is a non-specific endoxylanase. The next sequence, At_QUCUH3, corresponding to an uncharacterized Aspergillus terreus protein, shows the highest similarity to TlXyn30A. The last 5 sequences are of bacterial origin and belong to the GH30_8 subfamily. HcXyn30A (G8LU16) is a unique prokaryotic non-reducing end xylobiohydrolase. EcXyn30A (Q46961) and BsXynC (Q45070) are canonical GH30_8 glucuronoxylanases. CaXyn30A (Q97TI2) and RpXyn30A (F1TBY8) are exceptional GH30_8 non-specific endoxylanases. Only the regions that are believed to have the highest impact on the unique catalytic properties of TlXyn30A (typed in violet color) are shown. The corresponding secondary structure elements of TcXyn30B (green) and EcXyn30A (red) are shown on the top and bottom. Part (A) displays the sequences in the β2-α2 loop, which is considerably larger in eukaryotic GH30_7 enzymes, compared to prokaryotic GH30_8 members. However, even in the eukaryotic sequences, there are differences in the length of the loop that are presumably related to their substrate specificity. Part (B) shows the sequences in the β8-α8 region, which contains amino acids interacting with the MeGlcA moiety of the acidic substrates like GX. Among the GX-interacting amino acids is a prokaryotic arginine (typed in blue and marked by an up triangle), which endows the canonical GH30_8 members with glucuronoxylanase specificity. For multiple sequence alignment of entire amino acid sequences, the reader is referred to Supplementary Materials ( Figure S2).

Enzymes
Gene cloning and expression as well as encoded protein purification and verification of the purity of Talaromyces leycettanus CBS 398.68 GH30 xylanase (55 kDa by SDS PAGE) was added to a final concentration of 2.11 µM. During the reaction course, aliquots of 5 µL were spotted on silica gel-coated aluminum sheets (Merck, Darmstadt, Germany). After 24 h, the reaction was terminated by heating at 100 • C for 5 min. Subsequent treatment with β-xylosidase (1 U/mL) was done overnight at 40 • C. Low-molecular-weight substrates, i.e. linear xylooligosaccharides (Xyl 2 -Xyl 6 ), aldouronic acids MeGlcA 3 Xyl 3 and MeGlcA 3 Xyl 4 , arabinoxylooligosaccharides, methyl and 4-nitrophenyl glycosides, were used at 2.5 mM. Their hydrolysis with 6.25 µM TlXyn30A was conducted in 50 mM sodium acetate buffer, pH 3.5, at 35 • C, and 2-µL aliquots were spotted on the TLC plate. The TLC plate was developed either twice in a solvent system of ethyl acetate/acetic acid/2propanol/formic acid/water 25:10:5:1:15 (v/v) (polysaccharides, aldouronic acids, and linear xylooligosaccharides) or once in a solvent system of n-butanol/ethanol/water 10:8:5 (v/v), and the sugars were visualized with the orcinol reagent.

MALDI-ToF MS
The hydrolysates were decationized by Dowex 50 (H + form) and 1 µL was mixed with 1 µL of the matrix (1% solution of 2,5-dihydroxybenzoic acid in 30% acetonitrile) directly on the MS target plate. After air-drying, the samples were analyzed by a UltrafleXtreme MALDI ToF/ToF mass spectrometer (Bruker Daltonics, Bremen, Germany) operating in reflectron positive mode.

Conclusions
Although T. leycettanus GH30_7 xylanase behaves as a typical endo-β-1,4-xylanase, its catalytic properties are unique in comparison with other types of non-specific endoxylanases. The enzyme hydrolyzes all types of xylan to a higher degree than GH10, GH11, and other GH30 xylanases. The shortest and most abundant fragment yielded by TlXyn30A from glucuronoxylan (MeGlcA 2 Xyl 2 ) is smaller by one and two xylosyl residues than the shortest aldouronic acids generated by GH10 and GH11 xylanases, respectively. Unbranched regions of the polysaccharides and larger oligosaccharides as well as linear β-1,4-xylooligosaccharides are degraded mainly to Xyl 2 . This is important in view of possible biotechnological applications of the enzyme.
Supplementary Materials: The following are available online. Figure S1: pH and temperature optimum and temperature stability of TlXyn30A. Figure S2: Multiple sequence alignment of GH30 catalytic domain of TlXyn30A and selected GH30 xylanases.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.