Thus far many researchers have reported enzymatic properties, gene cloning, and heterologous expression of individual xylanases from A. niger. However, less attention has been paid to the relationship between xylanase genes carried on A. niger genome and xylanases produced. Therefore, we examined the xylanase genes on A. niger E-1 genome that contribute to xylan degradation though the biosynthesis of functionally active xylanases.
The A. niger E-1 genome encoded seven putative xylanase genes xynI–VII, which are closely similar to those of xylanase genes from A. niger CBS 513.88. On the other hand, strain E-1 produced three xylanases, XynII, XynIII, and XynVII, when this strain was cultured in 0.5% xylan medium supplemented with 50 mM sodium succinate (Figure 2a and Figure 3). There are no reports, to our knowledge, on the expression of xynIV and xynV belonging to cluster II in a phylogenetic tree (Figure 1) or xynI and xynVI in other A. niger strains. These results suggest that these xylanases may play physiologically distinct roles from XynII, XynIII, and XynVII to adapt to quite different environments and assimilate xylan.
XynIII activity represented 51% of total activity in the culture supernatant; XynII and XynVII activities were 15% and 11%, respectively, of the total activity, suggesting that XynIII plays a primary role in the degradation of xylan backbones in this culture condition. This speculation is also supported by the fact that many XynIII and closely related enzymes have been reported from A. niger strains (Krisana et al. 2005; Fu et al. 2012). In addition to XynIII, some enzymes highly homologous to XynII have also been reported (Hmida-Sayari et al. 2012; Yang et al. 2010), but information on XynVII, which belongs to GH family 10 in A. niger, was limited. Therefore, we characterized XynVII in further experiments.
Aspergilli endoxylanases show the maximal activities at a range of 42°C–60°C, and a pH range of 4.0–7.0 (Teixeira et al. 2010). The highest activity of purified XynVII was also observed within these ranges. However, XynVII maintained more than 85% activity after 30 min of incubation at 60°C (Figure 4a), and was more stable than other xylanases from A. niger strains US368 (Hmida-Sayari et al. 2012) and A-25 (Chen et al. 2006), which retained 50% and 10% of activities, respectively. Moreover, the pH stability profile showed that XynVII is highly stable over a wide pH range from 3.0 to 10.0 (Figure 4b), compared with other reported xylanases from A. niger strains (Fu et al. 2012; Hmida-Sayari et al. 2012; Krisana et al. 2005; Yang et al. 2010). In addition, Km and Vmax values of XynVII (2.8 mg mL–1 and 127 μmol min–1mg–1, respectively) were similar to those of GH family 10 xylanases from Penicillium pinophilum strain C1 [4.3 mg mL–1 and 195.4 μmol min–1mg–1, respectively (Cai et al. 2011)] and of GH family 11 xylanase from A. niger strain US368 [1.03 mg mL–1 and 811 μmol min–1mg–1, respectively (Hmida-Sayari et al. 2012)], indicating that XynVII from strain E-1 possesses catalytic properties that are sufficient to contribute to xylan degradation although the total activity of XynVII was smaller than those of other xylanases in the culture supernatant.
As reported for various xylanases from A. niger (Chen et al. 2006; Hmida-Sayari et al. 2012; Yang et al. 2010) and GH family 10 xylanase from Flavobacterium johnsoniae (Chen et al. 2013), XynVII activity was strongly inhibited by Hg2+, which potentially caused inhibition by its interaction with an aromatic ring present in a Trp residue. This finding was consistent with the result that XynVII was strongly inhibited by NBS, which modifies a Trp residue. It is interesting that XynVII was stable in the presence of other metal ions and modifying reagents, particularly Cu2+ and Mn2+, which are well-known inhibitors of xylanases (Chen et al. 2006; Hmida-Sayari et al. 2012).
The N-terminal amino acid sequence determination of purified XynVII was unsuccessful by a gas-phase protein sequencing. Liu et al. (2012) have studied a signal peptide cleavage site in the N-terminal region of A. niger XynB, which is identical to that of XynVII, and proposed that the cleavage of a peptide bond occurs between Arg-25 and Gln-26, releasing the signal peptide. It is likely that the N-terminal Gln residue (Gln-26) of mature E-1 XynVII is changed to pyroglutamate by cyclization after hydrolysis of the peptide bond, yielding a blocked N-terminus (Ito et al. 1992).
GH family 10 and GH family 11 xylanases differ in produced xylooligosaccharides. When a glucuronoxylan, such as beechwood xylan, is digested with GH family 11 xylanases, xylotetraose substituted with a 4-o-methylglucuronic acid residue (X4MeGlcA) accumulates as the final reaction product (Biely et al. 1997; Kolenová et al. 2006). X4MeGlcA generally show resistance against hydrolysis with xylosidase, when substitution by glucuronic acid occurs at X1 of the non-reducing end or the second X1 next to the non-reducing end (Tenkanen et al. 1996; Rasmussen et al. 2012). Although glucuronic acid is released from short xylooligosaccharides by the actions of α-glucuronidase, X4MeGlcA is too large to be hydrolyzed with the enzyme (Kolenová et al. 2006). As a result, it is assumed that X4MeGlcA remains and complete glucuronoxylan degradation is unsuccessful when glucuronoxylan is digested with GH family 11 xylanases. On the other hand, X3MeGlcA, which is produced by GH family 10 xylanase reaction, is hydrolyzed with α-glucuronidase, and the resulting X3 is acceptable for hydrolysis of xylosidase. Thus, the GH family 10 xylanase XynVII appears to contribute to efficient glucuronoxylan assimilation by strain E-1. Zheng et al. (2013) recently cloned a cDNA encoding GH family 10 xylanase from A. niger and characterized the purified recombinant enzyme. Characteristics of the recombinant enzyme were similar to those of E-1 XynVII. In addition to these characteristics, we identified the reaction products of E-1 enzyme and suggested the physiological significance of XynVII in glucuronoxylan assimilation based on the reaction products as described above.
In this study, we found only one gene encoding GH family 10 xylanase and four genes encoding GH family 11 xylanases from A. niger E-1. Wakiyama et al. (2008) showed that GH family 10 xylanases from Penicillium spp. and Aspergillus spp. could be classified into two groups by phylogenetic analysis: one is a xylanase group from Penicillium spp. and black aspergilli such as A. kawachii and A. niger, and another is from other aspergilli, such as A. fumigatus, A. nidulans, and A. oryzae. In the aspergilli described above, it has been reported that A. fumigatus, A. nidulans, and A. oryzae, of which the genomes have been sequenced, possess more than two GH family 10 genes on their genome. By contrast, the genome of the black aspergillus A. niger carried only one GH family 10 xylanase gene in both strains E-1 and CBS 513.88. Genome information about other black aspergilli is not available for database search, but numbers of GH family 10 xylanase genes on each aspergillus genome may be related to the diversity of GH family 10 xylanases in the evolution processes of aspergilli genomes.
A. niger E-1 possesses seven putative xylanase genes (xynI–VII), but only three xylanases (XynII, XynIII, and XynVII) were produced, strongly suggesting that potential regulatory mechanisms control the expression of the other four xylanase genes, xynI, xynIV, xynV, and xynVI, for which no translated products were detected. Further research is needed to comprehensively understand xylan degradation by A. niger E-1.