INTRODUCTION

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-Galactosidases (EC 3.2.1.22) and -galactosidases (EC 3.2.1.23) are enzymes which are commonly found in nature and which are able to release - or -linked D-galactose from a wide range of compounds. Several fungal -galactosidases have been purified, in particular from Aspergillus sp., and these enzymes have different physicochemical and kinetic properties (2, 9, 23, 30, 34, 40). These enzymes can be divided into different classes based on their characteristics. Some of these -galactosidases have molecular masses of 70 to 95 kDa (9, 30, 40), whereas others are in the range of 45 to 56 kDa (2, 6, 23). The pI of Aspergillus -galactosidases is between 4.5 and 5.0, and all of the enzymes have activity against galactose-containing oligosaccharides such as raffinose. Induction of -galactosidases has been observed on arabinoxylan (23), galactose (34), galactomannan (9), and wheat and rice bran (40). Up to now, two -galactosidase-encoding genes have been cloned from Aspergillus niger. Den Herder et al. (9) purified an -galactosidase which was expressed on galactomannan and cloned the corresponding gene (aglA). A second gene encoding an -galactosidase from A. niger with activity against galactose-containing oligosaccharides has been described more recently (18). We have designated this gene aglC. An extracellular -galactosidase has also been purified from A. niger, and the corresponding gene (lacA) has been cloned (20). There are no indications of other genes encoding additional extracellular -galactosidases in A. niger. One paper reported the purification of three forms of A. niger -galactosidase (47), but these are most likely different glycoforms of the same enzyme. A. niger -galactosidase is induced during growth on polygalacturonic acid (27) and arabinoxylan (23).

Galactose is present in different oligosaccharides (e.g., raffinose, stachyose, and melibiose) and polysaccharides (galactomannan, pectin, and xylan) from plants. In pectin, galactose is mainly present as branched side chains (31), whereas in arabinoxylan, single galactose residues are attached to xylose or arabinose (13, 49).

A. niger has a very efficient extracellular enzyme spectrum specialized in degrading plant-derived oligo- and polysaccharides, including those hydrolyzing - and -linked galactosides. Some of these enzymes have a high substrate specificity, resulting in the production of a number of enzymes with similar functions. -Galactosidases from Aspergillus have been shown to catalyze the hydrolysis of -1,6-linked galactose residues from oligomeric (e.g., melibiose, raffinose, and stachyose) and polymeric (e.g., galactomannan) compounds (23, 34). The potential of A. niger to produce several -galactosidases could indicate that these enzymes are active on different substrates and might therefore have different expression patterns.

-Galactosidase (lactase) is able to cleave -linked galactose residues from various compounds and is commonly used to cleave lactose into galactose and glucose (48). The role of this enzyme of A. niger in nature is more likely in removing -linked galactose residues from plant-derived oligo- and polysaccharides than in the hydrolysis of lactose.

Here we describe the cloning and characterization of the aglB gene encoding a third -galactosidase from A. niger which was previously purified in our laboratory (23). Also, we studied the expression patterns of the three - and the -galactosidase genes from A. niger in detail.

	MATERIALS AND METHODS

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Strains, libraries, and plasmids. All strains were derived from wild-type A. niger N400 (CBS 120.49). N402 has short conidiophores (cspA1). CreA mutant strain NW200 (bioA1 cspA1 creAd4 pyrA13::pGW635 areA1::pAREG1) was selected in an areA1 background (35) and subsequently cotransformed with pAREG1 (containing the A. niger areA gene; 26) and pGW635 (containing the pyrA selection marker) to restore the areA wild type. The prtF mutation present in strain NW156 (leuA1 pyrA6 prtF28) was previously described (43), as was A. niger NXA1-4 [argB13 cspA1 nicA1 pyrA6 UAS(xlnA)-pyrA xlnR1], which has a defect in the xylanolytic transcriptional activator gene xlnR (44). Escherichia coli DH5F' was used for routine plasmid propagation. E. coli LE392 was used as a host for phage EMBL3. pBluescript (39) and pGEM-T (Promega) were used for subcloning. The genomic and cDNA libraries of A. niger have previously been described (14, 16).

Media and culture conditions. Minimal medium (MM) contained (per liter) 6.0 g of NaNO₃, 1.5 g of KH₂PO₄, 0.5 g of KCl, 0.5 g of MgSO₄, trace elements (46), and 1% (mass/vol) glucose as a carbon source unless otherwise indicated. For complete medium (CM), MM was supplemented with 0.2% (mass/vol) tryptone, 0.1% (mass/vol) yeast extract, 0.1% (mass/vol) Casamino Acids, and 0.05% (mass/vol) yeast RNAs. Liquid cultures were inoculated with 10⁶ spores/ml and incubated at 30°C in an orbital shaker at 250 rpm. Agar was added at 1.5% (mass/vol) for solid medium. For the growth of strains with auxotrophic mutations, the necessary supplements were added to the medium.

Chemicals. D-Xylose, D-glucose, D-fructose, D-galactose, D-mannose, and lactose were obtained from Merck (Darmstadt, Germany). D-Glucuronic and D-galacturonic acid were from Fluka (Buchs, Switzerland). Melibiose, raffinose, stachyose, L-arabinose, gum arabic, gum karaya, locust bean gum, methylumbelliferyl--D-galactoside, and beechwood xylan were from Sigma (St. Louis, Mo.). Potato pectic galactan was from Megazyme International (Bray, Ireland). Taq polymerase was from Gibco BRL (Breda, The Netherlands).

PCR cloning of specific fragments of lacA, aglA, aglB, and aglC. Based on the nucleotide sequences of lacA, aglA, and aglC, specific oligonucleotides were designed (5'-GGTCTCTCTGAGGCAGGC-3' and 5'-TAGTATGCACCCTTCCGC-3' for lacA, 5'-ACGGCTCTATCGAGCAGCCC-3' and 5'-CTCCCCGTATATCGGGACCC-3' for aglA, and 5'-ATGATCGGTCTTCCCATGCTG-3' and 5'-TCGTCCATGACAAAGAGGTGG-3' for aglC) and used in PCRs under the following conditions: melting at 95°C, annealing at 50°C, and elongation at 72°C. Chromosomal DNA of A. niger N402 was used as the template. This resulted in specific DNA fragments of 373, 593, and 1,276 bp for lacA, aglA, and aglC, respectively. For the isolation of a specific fragment of aglB, one oligonucleotide was designed based on the N-terminal amino acid sequence of AglB (23) and one was based on a highly conserved region in a number of -galactosidases (5'-GGNTGGAAYTCNTGGAAYGC-3' and 5'-CATNCCNCCRTTNCCNACYTC-3', with Y and R representing C/T and A/G, respectively). PCRs at 42°C using these two oligonucleotides and total cDNA from the A. niger cDNA library resulted in a fragment of 762 nucleotides. All three fragments were cloned in the PGEM-T vector system (Promega). Sequence analysis was performed as described below.

Isolation, cloning, and characterization of the aglB gene. Plaque hybridization using nylon replicas was performed as described by Benton and Davis (5). Hybridizations were performed overnight at 65°C by using the aglB PCR fragment as a probe. Filters were washed in 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M Na₃-citrate, pH 7.6)-0.5% (mass/vol) sodium dodecyl sulfate (SDS). Positive plaques, identified on duplicate replicas after autoradiography, were recovered from the original plates and purified by rescreening at low plaque density. Standard methods were used for other DNA manipulations, such as Southern and Northern analyses, subcloning, DNA digestions, and lambda phage and plasmid DNA isolations (36). Chromosomal DNA was isolated as previously described (8). Sequence analysis was performed on both strands of DNA by using either the Cy5 AutoCycle Sequencing kit or the Cy5 Thermo Sequenase Dye Terminator Kit (Pharmacia Biotech, Uppsala, Sweden). The reactions were analyzed with an ALFred DNA Sequencer (Pharmacia Biotech). Nucleotide sequences were analyzed with computer programs based on those of Devereux et al. (10) (PCGene; Intelligenetics, Geneva, Switzerland). Aspergillus cotransformations were performed as described by Kusters-van Someren et al. (21) by using the pyrA gene as a selection marker.

Northern analysis. Total RNA was isolated from powdered mycelium using TRIzol Reagent (Life Technologies) in accordance with the supplier's instructions. For Northern analysis, 5 µg of total RNA was incubated with 3.3 µl of 6 M glyoxal-10 µl of dimethyl sulfoxide-2 µl of 0.1 M phosphate buffer (pH 7) in a total volume of 20 µl for 1 h at 50°C to denature the RNA. The RNA samples were separated on a 1.5% agarose gel using 0.01 M phosphate buffer (pH 5) and transferred to Hybond-N filters (Amersham) by capillary blotting. Filters were hybridized at 42°C in a solution of 50% (vol/vol) formamide, 10% (mass/vol) dextran sulfate, 0.9 M NaCl, 90 mM Na₃-citrate, 0.2% (mass/vol) Ficoll, 0.2% (mass/vol) polyvinylpyrrolidone, 0.2% (mass/vol) bovine serum albumin, 0.1% (mass/vol) SDS, and 100 µg of single-stranded herring sperm DNA per ml. Washing was performed under homologous conditions in 30 mM NaCl-3 mM Na₃-citrate-0.5% (mass/vol) SDS at 68°C. A 0.7-kb EcoRI fragment of the 18S rRNA subunit (28) was used as a probe for RNA loading control.

Sequence alignments. Amino acid sequence alignments were performed by using the Blast programs (3) at the server of the National Center for Biotechnology Information (Bethesda, Md.).

Nucleotide sequence accession number. The EMBL accession number for aglB from A. niger is Y18586.

	RESULTS

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Cloning and sequence analysis of aglB from A. niger. Based on the N-terminal amino acid sequence (LVRPDGVGLTPALGWNSWNAY) of AglB (23) and a highly conserved region present in a number of -galactosidases, two degenerate oligonucleotides were designed and a specific fragment of aglB cDNA was isolated as described in Materials and Methods.

View larger version (57K): [in this window] [in a new window]   FIG. 1.   Nucleotide and derived amino acid sequences of aglB. The introns (lowercase letters), putative regulatory sequences (boldface and underlined), signal peptide (lowercase letters), and putative N-glycosylation sites (boldface capital letters) are indicated.

Overexpression of aglB. A. niger NW156 was transformed with plasmid pIM3214 to generate multicopy transformants. A total of 20 transformants were selected and inoculated on MM plates containing 1% (mass/vol) xylose and methylumbelliferyl--D-galactoside. Five transformants were identified which showed increased -galactosidase activity, and these were purified. These transformants and wild-type N402 were grown overnight on CM containing 1% (mass/vol) fructose and transferred to MM containing 1% (mass/vol) xylose. After 4 h, mycelium was harvested and RNA and chromosomal DNA were isolated. Southern and Northern analyses were performed, and autoradiographs were scanned by using an LKB Ultroscan XL laser densitometer and subsequently normalized for the loading control (18S expression) to determine copy numbers and expression levels. The estimated copy numbers of the transformants ranged from 3 to 40 (Table 1), and expression levels were between 2.5 and 40 times wild-type expression.

Comparison of the amino acid sequence of AglB with those of other -galactosidases. The deduced amino acid sequence of AglB was compared to the amino acid sequences of other -galactosidases. AglB showed the highest overall similarity to an -galactosidase from Penicillium purpurogenum and Agl1 from Trichoderma reesei (62 and 54% amino acid sequence identity, respectively). Two regions, in particular, were highly similar to those of a number of other -galactosidases from various organisms, including A. niger AglA, but not to A. niger AglC (Fig. 2). The highest similarity is found in a region of approximately 120 amino acids starting just C terminal of the signal peptide of AglB. High sequence similarity between the enzymes is also observed in a region of approximately 70 amino acids which starts at residue 233. As already stated, AglC had no significant similarity to A. niger AglA and AglB but was found to be related to T. reesei Agl2 (64% amino acid sequence identity) and to a number of bacterial -galactosidases (Fig. 3).

View larger version (72K): [in this window] [in a new window]   FIG. 2.   Alignment of the amino acid sequences of -galactosidases from A. niger (AglB and AglA; 9), P. purpurogenum (38), T. reesei (AglI; 24), Coffea arabica (51), Cyamopsis tetragonoloba (32), Phaseolus vulgaris (7), Mortierella vinacea (37), Saccharomyces cerevisiae (41), S. paradoxus (29), and Zygosaccharomyces cidri (42). In the consensus sequence, amino acids are depicted which are conserved in at least 7 of the 11 -galactosidases. Amino acids which are identical in all 11 -galactosidases are in boldface type.

View larger version (68K): [in this window] [in a new window]   FIG. 3.   Alignment of the amino acid sequences of -galactosidases from A. niger (AglC; 18), T. reesei (AglII; 24), Thermoanaerobacter ethanolicus (50), Pediococcus pentosaceus (22), Streptococcus mutans (1), and E. coli (4). In the consensus sequence, amino acids are depicted which are conserved in at least four of the six -galactosidases. Amino acids which are identical in all six -galactosidases are in boldface type.

Differential expression of A. niger galactosidases. The expression of aglA, aglB, aglC, and lacA was studied by carrying out transfer experiments. A. niger N402 was grown for 16 h in CM containing 2% (mass/vol) fructose. The mycelium was harvested and washed with MM without a carbon source, and aliquots were transferred to MM with different carbon sources and incubated for 4 h as described in Materials and Methods. A Northern analysis was performed by using RNA isolated from the mycelium samples and using the PCR fragments of aglA, aglB, aglC, and lacA and a fragment of the 18S rRNA gene (28) as probes (Fig. 4).

View larger version (55K): [in this window] [in a new window]   FIG. 4.   Expression patterns of galactosidase genes from A. niger on different compounds after 4 h of transfer. The 18S rRNA served as an RNA loading control. Percentages are in mass per volume. Abbreviations: glc, glucose; xyl, xylose; gal, galactose.

Influence of XlnR on the expression of aglB and lacA. Based on the high expression of lacA on xylose, arabinose, xylan, and pectin and of aglB on xylan, a second transfer experiment was performed to study the influence of the xylanolytic transcriptional activator (XlnR; 44) on the expression of aglB and lacA. A. niger N402 and an XlnR mutant (NXA1-4) were grown for 16 h in CM containing 2% (mass/vol) fructose as a carbon source at 30°C. The mycelium was harvested and washed with MM without a carbon source, and aliquots were transferred to MM containing different levels of xylose (1 and 0.03%, mass/vol) or arabinose (1%, mass/vol) as a carbon source. After a 2-h incubation period, mycelium was harvested and a Northern analysis was performed by using RNA isolated from the samples. Both the -galactosidase- and -galactosidase B-encoding genes were expressed on all three carbon sources in the wild-type strain (Fig. 5). The expression on 1% xylose was lower than the expression on 0.03% xylose for both genes. In the XlnR mutant, no expression of lacA was detected, but low levels of aglB expression were still observed on xylose.

View larger version (25K): [in this window] [in a new window]   FIG. 5.   Influence of XlnR on the expression of lacA and aglB. Northern blot analysis was performed after 2 h of transfer. The 18S rRNA served as an RNA loading control. Percentages are in mass per volume.

	DISCUSSION

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Analysis of the derived amino acid sequence of AglB resulted in a molecular mass of 48,835 Da, whereas the experimentally determined molecular mass is 54 kDa (23). The difference in molecular mass suggests that AglB is a glycoprotein and that several of the putative N-glycosylation sites identified in the amino acid sequence are, in fact, glycosylated. The predicted pI of 4.6 is in good agreement with the experimentally determined pI value of 4.2 to 4.6 (23). The variation observed for the purified enzyme is most likely caused by different glycoforms of the enzyme. The amino acid sequence of AglB is similar to the amino acid sequences of a number of other -galactosidases of eukaryotic origin, including another -galactosidase (AglA) from A. niger (9) and an -galactosidase (Agl1) from T. reesei (24). All of these enzymes belong to family 27 of the glycosyl hydrolases (17). Two regions with a high level of similarity can be identified by comparing the amino acid sequences of these enzymes, suggesting that they belong to a subfamily of -galactosidases. AglC belongs to a different subfamily of -galactosidases, together with T. reesei Agl2 (24) and a number of bacterial -galactosidases, which have been assigned to family 36 of the glycosyl hydrolases (17). A third -galactosidase isolated from T. reesei (Agl3; 24) does not contain the conserved regions of either of these subfamilies and might therefore belong to yet another subfamily. Den Herder et al. (9) suggested the presence of four different -galactosidases in A. niger based on the analysis of -galactosidase activities, which could be an indication of the presence of an A. niger homologue of T. reesei Agl3. However, several glycoforms of AglB were previously isolated (23), indicating that the total number of -galactosidases could be lower than four.

The expression of the four galactosidases studied here is specific for each gene. The carbon sources resulting in the highest expression of -galactosidase-encoding lacA are xylose, arabinose, xylan, and pectin. The expression on arabinose is probably caused by the presence of a small amount of xylose in the arabinose that is commercially available (11). Expression levels of a number of xylanolytic genes on xylose have been shown to be the result of a balance between XlnR-mediated induction and CreA-mediated repression of expression (12). This appears also to be the case for lacA and would explain the different expression levels observed for the different xylose concentrations. The absence of lacA expression in the XlnR-deficient mutant indicates that the expression of this gene on xylose and xylan is regulated by XlnR, as has been shown for a number of other genes (45). Production of -galactosidase has been performed by using wheat bran (33) that is rich in arabinoxylan, confirming the expression data obtained in this study. The function of LacA as a member of the xylanolytic spectrum may therefore be in removing -linked galactose residues from xylan. The expression of lacA on galactose is much lower than the expression on arabinose or on xylose. Although -galactosidase is commonly used for the hydrolysis of lactose (48), plant -galactosidases have been suggested to play a role in pectin degradation (15) and production of Aspergillus -galactosidase on polygalacturonic acid has been reported (27). The expression of lacA on pectin observed in this study confirms that a pectin-related compound is also able to induce lacA gene expression. As for xylan, this could indicate a role for LacA in the degradation of pectin by A. niger.

The expression of aglA was high on galactose and galactose-containing oligosaccharides but was fully repressed in the presence of glucose. No expression was observed on other carbon sources, except arabinose and glucuronic acid, whereas moderate expression was also observed on galactose-containing gums. In contrast, aglB was expressed on all of the carbon sources tested. This suggests a basic level of expression of the gene, which is confirmed by the fact that the increase in expression in multicopy transformants is similar to the increase in copy number. High levels of expression of aglB were observed on galactose, xylose, and beechwood xylan but not on galactose-containing oligo- and polysaccharides (other than xylan). The expression on glucose and fructose suggests that although the promoter of aglB contains two putative CreA binding sites, aglB is not, or is only to a small extent, subject to CreA-mediated repression of gene expression. Previous studies demonstrated induction of -galactosidases on galactomannan (6, 9), lactose (23, 34), locust bean gum (24), wheat and rice bran (40), and galactose (34). The aglA gene seems to represent an -galactosidase which is specifically induced on galactose. The high expression levels on galactose-containing oligosaccharides could indicate a preference for these structures (stachyose, melibiose, and raffinose) as the natural substrates. The aglB gene is expressed on all carbon sources at a high basal level. The product of this gene might therefore be important for the induction of other -galactosidases by releasing small amounts of galactose from polymeric compounds. The high level of expression on xylan suggests a role for the xylanolytic activator XlnR in the expression of aglB. The results from the experiment with the XlnR mutant confirm that XlnR has a function in the expression of aglB, although it is different from the effects observed for other xylanolytic genes (45). These genes are not induced on other sugars than xylose, and expression on xylose in the XlnR mutant is abolished. The expression of aglB on xylose is decreased in the XlnR mutant but not abolished. Thus, the expression of this gene does not exclusively depend on XlnR. The effect of XlnR does suggest a role for AglB in the xylanolytic spectrum, indicating that AglB might be involved in releasing -linked galactose from the xylan backbone. The different expression levels at different xylose concentrations cannot be explained as described above for lacA, since no indications for CreA-mediated repression were observed for aglB. The difference might be caused by a more indirect effect, possibly mediated by CreA, in xylanolytic induction of gene expression. The xylanolytic genes tested for modulation of expression on xylose (12) also did not have identical expression patterns at different xylose concentrations, but for all of the genes, expression decreased with increasing xylose concentrations. The results in this paper demonstrate that the xylanolytic activator XlnR is also involved in the regulation of an - and a -galactosidase gene of A. niger, emphasizing its key role in hemicellulose degradation.

The expression pattern of aglC is remarkable for a gene encoding an -galactosidase. Expression was only observed on glucose, fructose, or combinations containing glucose. Similar results have been obtained for T. reesei agl3, when expression of the latter and of a number of other hemicellulase genes was studied on a set of different carbon sources (25). Expression was only observed on cellulose, sorbitol, and glucose but not on galactose, xylose, or other monomeric or polymeric compounds. The aglC gene of A. niger has been clearly demonstrated to encode -galactosidase activity by using p-nitrophenyl--D-galactoside, raffinose, and stachyose as substrates, although no activity was found by using guar gum (18). This could be an indication that AglC activity is specific for galactose residues linked to glucose or fructose in galactose-containing oligosaccharides such as raffinose and stachyose and that the gene is therefore only expressed in the presence of glucose.

Whether the expression of the four genes tested indeed mirrors the substrate preferences of the encoded enzymes requires further study, involving activity measurements of the enzymes against oligo- and polysaccharides. It is clear that the differences in expression of the genes will result in specific enzyme spectra on different polymeric substrates. The polymeric substrate used to produce A. niger enzyme preparations will therefore reflect its composition.