Tài liệu Báo cáo Y học: The b-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions pdf

The b-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions Ronald P. de Vries 1† , Lucie Par ˇ enicova ´ 1‡ , Sandra W. A. Hinz 2 , Harry C. M. Kester 1 , Gerrit Beldman 2 , Jacques A. E. Benen 1 and Jaap Visser 1§ 1 Molecular Genetics of Industrial Microorganisms and 2 Food Chemistry, Wageningen University, Wageningen, The Netherlands The Aspergillus niger b-1,4-endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, D -galacturonic acid and L -arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site resi- dues identified previously for the Pseudomonas fluorescens b-1,4-endogalactanase. The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of ara- binogalactans of potato, onion and soy with GALA resulted initially in the release of D -galactotriose and D -galactotetra- ose, whereas prolonged incubation resulted in D -galactose and D -galactobiose, predominantly. MALDI-TOF analysis revealed the release of L -arabinose substituted D -galacto- oligosaccharides from soy arabinogalactan. This is the first report of the ability of a b-1,4-endogalactanase to release substituted D -galacto-oligosaccharides. GALA was not active towards D -galacto-oligosaccharides that were substi- tuted with D -glucose at the reducing end. Keywords: Aspergillus niger; b-1,4-endogalactanase; galac- turonic acid; expression; galactan degradation. Endogalactanases are involved in the degradation of plant cell wall polysaccharides, in particular pectin. Two types of arabinogalactan side chains are present in pectin. Type I consists of a chain of b-1,4 linked D -galactopyranose linkages, while type II contains a backbone of b-1,3-linked D -galactopyranose residues that can be substituted with b-1,6-linked D -galactopyranose residues [1]. Both types can be substituted with a)1-,3-linked L -arabinofuranose chains. Type I arabinogalactan is degraded by b-1,4-endogalacta- nase and b-galactosidase. b-1,4-Endogalactanases cleave within the galactan moiety of type I arabinogalactan, releasing D -galacto-oligosaccharides. Bacterial b-1,4-endo- galactanases release mainly galactotriose and galactotetra- ose [2–6], while some also release galactobiose [3,5,7]. Eukaryotic b-1,4-endogalactanases release predominantly galactobiose and galactose from galactan [8–10]. Genes encoding b-1,4-endogalactanase have been cloned from both bacteria and fungi [11–15]. Based on their derived amino acid sequence, the corresponding enzymes have been assigned to family 53 of the glycosyl hydrolases [16]. These enzymes have a retaining mechanism and for the Pseudo- monas fluorescens b-1,4-endogalactanase the catalytic resi- dues have been determined [11]. Recently, the Aspergillus aculeatus b-1,4-endogalactanase has been expressed in vivo in potato resulting in a 30% reduction of the galactosyl content of the pectin fraction of the cell walls [17]. In this paper we describe the cloning, characterization and expression analysis of the A. niger galA gene, encoding b-1,4-endogalactanase. This is the first paper that describes the expression of this gene in detail and that compares the activity of the gene product, GALA, against arabinogalac- tans with a different degree of L -arabinose substitution. MATERIALS AND METHODS Strains and growth conditions All A. niger strains were derived from A. niger N400 (CBS 120.49) and are described in Table 1. Escherichia coli DH5aF¢ was used for routine plasmid propagation. E. coli LE392 was used as a host for phage kEMBL3. Subcloning was performed using pBluescript SK + [18] and pGEM-T (Promega, Madison, WI, USA). The genomic library of A. niger has been described previously [19]. Minimal medium and complete medium were descri- bed before [20]. Liquid cultures were inoculated with Correspondence to R. P. de Vries, Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. Fax: +31 302513655, Tel.: +31 302533016, E-mail: r.p.devries@bio.uu.nl Abbreviations: CREA, carbon catabolite repressor protein; HPAEC, high performance anion exchange chromatography; GALA, the A. niger b-1,4-endogalactanase; galA,geneencodingtheA. niger b-1,4-endogalactanase; Galp, galactopyranose; Glcp, glucopyranose; LACA, A. niger a-galactosidase; PACC, pH regulatory protein; TOS, transgalactooligosaccharides. Present address: Microbiology, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands. àPresent address:DipartimentodiBiologia,Universitadeglistudidi Milano, Via Celoria 26, 20133 Milano, Italy. §Present address: FGT Consultancy, PO Box 396, 6700 AJ Wagenin- gen, The Netherlands. (Received 27 March 2002, revised 1 July 2002, accepted 21 August 2002) Eur. J. Biochem. 269, 4985–4993 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03199.x 10 6 sporesÆmL )1 , and incubated at 30 °Cinanorbital shaker at 250 r.p.m. For the growth of strains with auxotrophic mutations, the necessary supplements were added to the medium. Transfer experiments were performed by pregrowing the strains for 16 h in complete medium containing 2% (w/v) fructose as carbon source, after which the mycelium was harvested and washed with minimal medium without carbon source. Aliquots (1.5 g) of wet mycelium were then transferred to 50 mL minimal medium containing carbon sources as indicated in the text. After 4 h of incubation in a rotary shaker at 250 r.p.m and 30 °C mycelium was harvested, dried between tissue paper, frozen in liquid nitrogen and stored at )70 °C. For purification of the b-1,4-endogalactanase, A. niger transformant NW290:: pIM3982.77 was grown in minimal medium supplemented with 0.003% (w/v) yeast extract and 3% (w/v) fructose. The pH was adjusted to 3.8. Cultivation was performed at 30 °C in a 5-litre jacketed stirred tank reactor (Applikon). Air-saturated culture medium was inoculated with 10 6 spores per litre. The spores were allowed to germinate for 6 h at low stirring speed (400 r.p.m) after which the cultivation was continued for 18 h at 750 r.p.m. Culture pH was maintained at 3.8 by the addition of a 5- M sodium hydroxide solution. The cultures were sparged with air (2 v.v.m) and 0.5 mL 30% (v/v) polypropylene glycol in ethanol was added per litre of medium as antifoam agent. Materials 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). Mellibiose, raffinose, stachyose, L -arabinose, gum arabic, gum karaya, locust bean gum, and beechwood xylan were from Sigma (St. Louis, Mo.). Potato pectic galactan was from Megazyme Interna- tional (Bray, Ireland). Taq polymerase was from Gibco BRL (Breda, The Netherlands). All other standard chemicals were either obtained from Sigma or Merck. Potato arabi- nogalactan and onion arabinogalactan were obtained as described previously (Fractions F44) [21]. Soy arabinoga- lactan was kindly provided by NOVO Nordisk (Dittingen, Switzerland). Sugar compositions were determined as des- cribed [21]. Borculo Whey Products (Borculo, The Nether- lands) kindly provided transgalactooligosaccharides (TOS). PCR cloning of a specific fragment of galA Two oligonucleotides were designed based on the sequence of the A. aculeatus b-1,4-endogalactanase encoding gene (5¢-CTCTTCTCTCTTGCTCTTG-3¢ and 5¢-GTTCGTCT CCACAACCAC-3¢, respectively) and used in PCRs under the following conditions: 1 min denaturing at 95 °C, 1 min annealing at 50 °C and 2 min amplification at 72 °C, 30 cycles. Chromosomal DNA of A. niger N402 was used as a template. This resulted in a fragment of approximately 700 bp which was cloned in pGEM-T easy (Promega). Sequence analysis was performed as described below. Cloning and characterization of galA Plaque hybridizations were performed as previously des- cribed [22]. Hybridizations were performed overnight at 65 °CbyusingthegalA PCR fragment as a probe. Filters were washed to 0.2 · NaCl/Cit (1 · NaCl/Cit is 0.15 M NaCl plus 0.015 M Na 3 -citrate, pH 7.6)-0.5% (mass/vol) 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 analysis, subcloning, DNA digestions, and lambda phage and plasmid DNA isolations [23]. Chromosomal DNA was isolated as previously described [24]. Sequence analysis was performed on both strands of DNA by using the Cy5 AutoCycle Sequencing 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. [25]. RT-PCRs were performed using the Enhanced Avian RT-PCR kit (Sigma) according to the suppliers instructions using galA-specific oligonucleo- tides (5¢-GATGATCTACCCTCTGCTTC-3¢ and 5¢-GTC ACGGACGGACTGGGT-3¢). Northern analysis was per- formed as described previously [20]. Per sample, 5 lgof total RNA was loaded on the gel. The A. niger galA accession number is AJ305303. Sequence alignments Nucleic acid and amino acid sequence alignments were performed by using the Blast programs [26] at the server of the National Center for Biotechnology Information (Bethesda, Md., USA). Purification of b-1,4-endogalactanase Culture fluid was collected by filtration through cheesecloth and diluted twofold with distilled water, after which the pH was adjusted to 6.5 by the addition of sodium hydroxide. Proteins were collected by batchwise adsorption to Stream- line Q XL (Amersham Pharmacia Biotech, Sweden). For this 40 mL of the matrix was added to the culture fluid and stirred for 2 h. Bound protein was eluted by a pulse of 10 m M piperazine/HCl pH 6.0, 1 M NaCl. After extensive dialysis against 10 m M piperazine/HCl pH 6.0, the protein was loaded onto a Source 30 Q column (Amersham Pharmacia Biotech, Sweden, 16 mL) followed by elution with a 300-mL linear NaCl gradient (0–1 M ). Fractions (10 mL) were collected and assayed for b-1,4-endogalac- tanase activity. b-1,4-Endogalactanase containing fractions were pooled, diluted fivefold with buffer and reapplied to the same column. Elution was performed with a 300-mL linear NaCl gradient (0–0.5 M ). The purity of b-1,4-endo- galactanase was determined by SDS/PAGE. Table 1. Strains used in this study. Strain Genotype Reference N402 cspA1 [42] NW200 cspA1, bioA1, creA d 4, pyrA13/pyrA + , areA1/areA + [43] NW290 cspA1, fwnA12, DargB/argB + , pyrA6, prtF28, DpgaA, DpgaB, goxC17 [44] 4986 R. P. de Vries et al.(Eur. J. Biochem. 269) Ó FEBS 2002 Enzyme assays Transformants producing elevated levels of b-1,4-endoga- lactanase were selected using AZCL-galactan (Megazyme, Bray, Ireland) according to the supplier’s instructions. During the purification endogalactanase activity was moni- tored using azo-galactan (Megazyme) as the substrate. Two hundred microlitres of a solution of 1% (w/v) azo-galactan in water was mixed with 50 lL of a 50-m M sodium acetate buffer (pH 4.2) followed by the addition of 50 lL enzyme solution. Incubations were performed for 20 min at 40 °C. Reactions were stopped by the addition of 650 lL ethanol. The precipitated substrate was removed by centrifugation and the supernatant was used to measure absorbance at 590 nm. The pH optimum of GALA was determined using azo-galactan as described above with a pH range of 3.5–7.5. The specific activity of GALA was determined using GALACTAZYME tablets (Megazyme) with conditions as indicated by the supplier and using 0, 10, 25, 50, 100, 200 and 500 lL of the GALA preparation. Incubations were performed in duplicate. The hydrolysis activity of GALA with different arabino- galactans was measured in duplicate by HPAEC and MALDI-TOF MS after incubation of 1 mgÆmL )1 of potato, onion and soy arabinogalactan with 1.33 lgÆmL )1 GALA at 30 °C, pH 4.5 (McIlvain buffer) for 15 min, 30 min, 2 h and 20 h, respectively. The incubation was stopped by heating the samples for 5 min at 100 °C. Transgalactooligosaccharides (TOS) were incubated in duplicate with GALA to analyse the activity of the enzyme towards these oligosaccharides. The oligosaccharides used all contained a glucose residue at the reducing end and were: b-D-Galp-1,4-D-Glcp (b-D-Galp-1,4-) 2 D-Glcp (b-D-Galp- 1,4-) 3 D-Glcp (b-D-Galp-1,4-) 4 D-Glcp (b-D-Galp-1,4-) 5 D- Glcp. Incubations were performed in duplicate. For this, 0.5 mgÆmL )1 of TOS was incubated with 1.33 lgÆmL )1 GALA at 30 °C, pH 4.5 (McIlvain buffer) for 15 min, 30 min, 2 h and 20 h, respectively. The incubation was stopped by heating the samples for 5 min at 100 °C. a-L-1,3-Arabinofuranosidase activity in the GALA pre- paration was measured as described before [27], using 10 and 100 lL of the enzyme preparation (in duplicate) and incubation times of 1 and 4 h. Analytical methods SDS/PAGE. Electrophoresis of proteins was performed under denaturing conditions on 10% (w/v) gels using the method of Laemmli [28] in a Mini-V system (Life Technologies B.V., Breda, The Netherlands). HPAEC. High performance anion exchange chromato- graphy (HPAEC) was performed using a SpectraSystem P4000 (Thermo Separation Products) equipped with a Dio- nex CarboPac PA-1 (4 · 250 mm) column and a Dionex ED40 Electrochemical Detector in de Pulsed Amperometric Detection mode. The samples were analyzed using a linear gradient of 0–400 m M sodium acetate in 100 m M sodium hydroxide for 40 min D -galactose, D -galactobiose and D -galactotetraose were used as standards to identify the D -galactose and D -galacto-oligosaccharides. The calculated areas for D -galactose and D -galacto-oligosaccharides were expressed as percentages of the area of 1 m MD -galactose. MALDI-TOF MS. MALDI-TOF MS was performed with a Voyager-DE RP Biospectrometry Workstation (PerSeptive Biosystems, Framingham, VS) in the positive mode. The laser intensity was 2300, the pulsed delay time was 200 nsec, the accelerating voltage was 12 000 V, the grid voltage was 7200 V and the guide wire voltage was 9.6 V. The instrument was used in the reflector mode. The mass spectrometer was calibrated with maltodextrins. RESULTS Cloning and characterization of galA Based on the sequence of the Aspergillus aculeatus b-1,4- endogalactanase encoding gene, two oligonucleotides were designed and used in PCRs, resulting in a specific fragment of A. niger galA, as described in Material and methods. Screening of a genomic library of A. niger using this fragment as a probe resulted in the isolation of two galA-containing phage k clones. From one of these clones a4.5-kbEcoRI fragment was cloned into pBluescript SK + , resulting in plasmid pIM3980. Double stranded sequence was determined for a region of this construct containing galA and some of the flanking regions, resulting in the genomic sequence of A. niger galA.The presence of one putative intron was confirmed by RT- PCR using total RNA of A. niger mycelium (transferred to minimal medium containing 15 m MD -galacturonic acid) as a template. The galA gene has a length of 1122 bp, is interrupted by one intron of 72 bp, and encodes a protein of 350 amino acids. Computer analysis predicted a eukaryotic signal sequence of 16 amino acids. The putative mature enzyme has a calculated pI of 3.67, a calculated molecular mass of 37 053.9 Da and contains one potential N-glycosylation site. BLAST analysis of the deduced amino acid sequence of GALA revealed very high similarity to the endogalactanases of A. tubingensis (95.7% amino acid sequence identity) and A. aculeatus (78.9% amino acid sequence identity) and lower similarity to bacterial endogalactanases (between 21% and 28% amino acid sequence identity). This is illustrated by a CLUSTALW analysis [29] of the deduced amino acid sequences of GALA and b-1,4-endogalactanases of Aspergillus tubingensis [15], A. aculeatus [12], Bacillus circulans (Acc. No. P48843), Yersinia pestis [14], Bacillus subtilis (Acc. No. O07013), Clostridium acetobutylicum [13], and Pseudomonas fluorescens [11] (Fig. 1). Both catalytic residues identified in P. fluorescens (E161, E270) [11] are present in the eukaryotic b-1,4-endogalactanases. Sequence analysis of the promoter of galA revealed the presence of several sequences possibly involved in the regulation of galA expression. Putative CREA binding sites [30] were detected at position )204, )218, 299, )482, and )580 from the start codon. In addition, one putative PACC site ()549) [31] and two CCAAT sites ()280, )300) were detected. Expression analysis of galA The expression pattern of galA on a selection of mono- saccharides, oligosaccharides and polysaccharides was com- pared to the previously reported expression pattern of lacA (encoding b-galactosidase) (Fig. 2) [32]. For this, mycelium Ó FEBS 2002 A. niger b-1,4-endogalactanase (Eur. J. Biochem. 269) 4987 was pregrown as described in Material and methods, and transferred for 4 h to minimal medium containing carbon sources as indicated in Fig. 2. Expression of galA was only detected in the presence of sugar beet pectin, whereas expression of lacA was detected in the presence of L -arabi- nose, D -xylose, sugar beet pectin and xylan (Fig. 2). To determine which of the monosaccharides present in sugar beet pectin is the actual inducer of galA expression a Fig. 1. Alignment of the derived amino acid sequences of fungal and bacterial b-(1,4)-endogalactanase encoding genes. In the consensus sequence the amino acids indicated are those present in at least four sequences. Amino acids present in all sequences are in bold. The eukaryotic signal sequence of A. niger GALA is depicted in lower case letters. The putative N-glycosylation site of A. niger GALA is in bold, italics, underlined and indicated above the sequence ("). The two catalytic residues identified in the P. fluorescens endogalactanase [11] are indicated above the alignment (^)andare in white against a grey background. 4988 R. P. de Vries et al.(Eur. J. Biochem. 269) Ó FEBS 2002 second experiment with identical experimental setup was performed in which the expression of galA was studied in two strains, a wild type (N402) and a mutant with a derepressed phenotype for CREA repression (NW200). Five of the carbon sources used in this experiment ( D -xylose, L -arabinose, D -galactose, L -rhamnose, D -galacturonic acid, Fig. 3) have been identified as components of sugar beet pectin [1], whereas the others served as controls. Expression of galA in the wild type strain was only detected in the presence of D -galacturonic acid (Fig. 3). However, in the CREA mutant expression of galA was detected in the presence of D -galacturonic acid and L -arabinose. The expression of galA is very low on the inducing compounds. Using approximately 50 ng of the PCR product for labelling, the radiation level of the probe after purification was 42.6 kBq. It was necessary to expose the autoradiogram for two weeks in order to visualize the hybridizing bands. Purification and characterization of GALA To obtain an A. niger transformant that produces increased levels of b-1,4-endogalactanase, a construct was made in which galA was fused to the promoter of the A. niger pkiA gene, encoding pyruvate kinase [24]. This gene has a high constitutive expression level and has been used previously for the production of high levels of proteins in A. niger [33]. The fusion construct (pIM3982) was made by introducing an NsiI site at the translation start point of galA and by then cloning a fragment, starting at this NsiI site and containing the galA gene and approximately 700 bp 3¢-flanking region, in pIM4700 (containing the pkiA promoter). A. niger NW290 was transformed with pIM3982 and transformants were analyzed for b-1,4-endogalactanase production using azo-galactan. Transformant NW290::pIM3982.77 was selected as the highest b-1,4-endogalactanase producing transformant and was used for the purification of GALA. Growth of this transformant and subsequent purification of the enzyme were performed as described in Materials and methods. Purity of the preparation was checked by SDS/ PAGE (Fig. 4). The purification resulted in 10 mL of the GALA preparation with a concentration of 31.6 lgÆmL )1 . Using galactazyme tablets a specific activity of 12.3 UÆmg )1 was determined for GALA. The purified protein had a molecular mass of 48.5 kDa as determined by SDS/PAGE (Fig. 4). The pH optimum was between4and4.5. No activity of GALA could be detected against any of the transgalactooligosaccharides listed in Materials and methods. Hydrolysis of arabinogalactans The sugar composition of the arabinogalactans from potato, onion and soy was determined as described [21]. Onion arabinogalactan consists of 99% D -galactose and 0.3% L -arabinose and is predominantly linear. Potato arabinogalactan consists of 86% D -galactose and 6.6% L -arabinose, while soy arabinogalactan consists of 57% D -galactose and 38% L -arabinose. Methylation analysis demonstrated that a substantial amount of the L -arabinose residues (14%) in soy arabinogalactan is present as terminal residues [21], suggesting that of these arabinogalactans, soy arabinogalactan is the most highly branched substrate. Fig. 2. Comparison of the expression of the A. niger b-(1,4)-endogalactanase (galA)and b-galactosidase (lacA) encoding gene. A frag- ment of the 18S rRNA gene [45] was used as an RNA loading control. Fig. 3. Expression of galA in the presence of different monomeric carbon sources in the wild type and CREA derepressed A. niger strains. A fragment of the 18S rRNA gene [45] was used as an RNA loading control. Ó FEBS 2002 A. niger b-1,4-endogalactanase (Eur. J. Biochem. 269) 4989 The three arabinogalactans were incubated with GALA for 15 min, 30 min, 2 h and 20 h to determine the substrate specificity of the enzyme. The degradation of the polymer and subsequent oligosaccharide formation was analyzed by HPAEC and MALDI-TOF MS. HPAEC analysis demon- strated that already after 15 min the formation of tetramers, trimers, dimers and monomers of D -galactose was visible for all three substrates (Fig. 5). Using potato and onion arabinogalactan as substrate, a small increase in all four products is observed during the incubation. The product formation using soy arabinogalactan as a substrate is clearly different (Fig. 5). After initial release of trimers and tetramers, these products disappear during prolonged incubation. A relatively higher amount of monomers and dimers is released from soy arabinogalactan, which increased upon prolonged incubation. MALDI-TOF MS analysis allowed for a more detailed analysis of the products formed during the incubations, although the method used cannot detect monomers and dimers. GALA initially released D -galactotriose, D -galacto- tetraose and D -galactopentaose from potato and onion arabinogalactan, while after prolonged incubations pre- dominantly D -galactotriose and D -galactotetraose could be detected (Fig. 6). Using soy arabinogalactan as a substrate, not only D -galactotriose, D -galactotetraose and D -galacto- pentaose were detected but also L -arabinose substituted forms of these three oligosaccharides (Fig. 6). After pro- longed incubations (2 h), only D -galactotriose could be detected. However, HPAEC demonstrated that after even longer incubations (20 h) D -galactotriose also disappears. To determine whether a small amount of a- L -1,3-arabino- furanosidase was responsible for the disappearance of Fig. 4. SDS/PAGE-of the purified b-(1,4)-endogalactanase prepar- ation. 1, 2: purified GALA preparation; 3: molecular mass marker proteins. Fig. 5. HPAEC analysis of the hydrolysis of arabinogalactans by GALA. Three different arabinogalactans were used: potato (r), onion (j)andsoy (m). The release of monomers (A), dimers (B), trimers (C) and tetramers (D) was studied during the incubations. The areas calculated for D -galactose and D -galacto-oligosaccharides were expressed as percentages of the area of 1 m MD -galactose. The values used for the figure are the average of duplicate incubations. 4990 R. P. de Vries et al.(Eur. J. Biochem. 269) Ó FEBS 2002 the L -arabinose-substituted oligosaccharides the GALA preparation was assayed for this activity. However, no a- L -1,3-arabinofuranosidase activity could be detected in the preparation (data not shown). This paper demonstrates the important role of GALA in the degradation of both linear and L -arabinose-substituted galactan side chains of pectin. This is in agreement with a previous study in which the synergy of enzymes degrading the pectin side chains was studied [34]. GALA had a positive effect on the activity of the other enzymes involved in the degradation of these side chains. DISCUSSION A. niger galA is highly similar to the b 1,4-endogalactanase encoding genes from A. tubingensis and A. aculeatus.The similarity of the galA genesishigherbetweenthetwo biseriate species, A. niger and A. tubingensis,thanbetween the monoseriate A. aculeatus galA gene and either of the other two Aspergillus genes. A similar observation has previously been reported for pectin lyase encoding genes of these species [35]. Significant similarity was also detected to a number of bacterial b-1,4-endogalactanases. The highest similarity was detected in the region around the first catalytic residue identified for the P. fluorescens b-1,4- endogalactanase [11]. The similarity around the second catalytic residue is lower. The determined molecular mass for A. niger GALA (48.5 kDa) is higher than the molecular mass based on the amino acid sequence (37 kDa). This suggests that the puta- tive N-glycosylation site identified in the sequence is indeed functional. The determined molecular mass and pH opti- mum (pH 4–4.5) of GALA are similar to those reported pre- viously for Aspergillus b-1,4-endogalactanases [10,12,36–40]. Expression of galA was observed in the presence of sugar beet pectin, D -galacturonic acid and L -arabinose. As A. niger is not able to import pectin, it is likely that the latter two compounds, or metabolites derived from them, are the true inducers of galA expression. This indicates that galA is coexpressed with both the pectinolytic genes encoding main chain cleaving enzymes as well as with the arabinanolytic genes [1]. The absence of expression of galA in the presence of pectic galactan indicates that galactose is not an inducer of galA expression. This is in agreement with a role for L -arabinose and D -galacturonic acid as inducers as men- tioned above. Expression of lacA is also detected in the presence of these compounds, but in the presence of D -xylose and xylan as well. This can be explained by the structure of the polysaccharides. Xylan contains single b-1,4-linked D -galactose residues but no b-1,4-linked galactan chains. Therefore it only requires an exo-acting galactose releasing enzyme (LACA). Previously it was shown that lacA is under the control of the xylanolytic activator protein [32,41]. The increase in expression levels of galA in the CREA mutant, indicates that at least one of the detected putative CREA binding sites in the promoter of galA is functional. Hydrolysis of arabinogalactans with a different degree of L -arabinose substitution by GALA results in different products. Arabinogalactans with a low degree of L -arabi- nose substitution (potato, onion) as substrates result in the liberation of monomers, dimers, trimers and tetramers of D -galactose. Using arabinogalactans with a high degree of L -arabinose substitution (soy), higher oligomers appear initially, but these disappear if the incubation is continued. Another difference is that only with the latter substrate arabinose-substituted galacto-oligosaccharides are detected. These disappear in time, suggesting the presence of an arabinose-releasing activity in the GALA preparation. However, no a-1-,3- L -arabinofuranosidase activity could be detected using PNP-a- L -arabinofuranoside as a sub- strate. This most likely means that the removal of the L -arabinose residues from the galactomannan oligosaccha- rides is caused by traces of an enzyme in the b-1,4- endogalactanase preparation that is not active against the PNP-substrate. Galacto-oligosaccharides that were sub- stituted with D -glucose at the reducing end were not Fig. 6. Maldi-TOF MS analysis of the hydrolysis of arabinogalactans by GALA. Three different arabinogalactans were used: potato (A), onion (B), and soy (C). Spectra were taken after 15 min (1) and 2 h (2) of incubation with GALA. Ó FEBS 2002 A. niger b-1,4-endogalactanase (Eur. J. Biochem. 269) 4991 hydrolyzed by GALA. So, heterosugar oligosaccharides with the structures Gal–Gal–Glc and Gal–Gal–Gal–Glc can not be hydrolyzed, while their corresponding homosugar oligosaccharides Gal3 and Gal4 are degradable (Fig. 5, panels C and D). This indicates that the substrate binding site in GALA that binds the reducing-end sugar is very important in enzyme substrate interaction. Previous studies using an Aspergillus b-1,4-endogalactanases reported release of D -galactose and D -galactobiose from arabinogalactan, after initial release of D -galactotriose and D -galactotetraose [10,12,36,39]. However, none of these papers reported the release of L -arabinose substituted galacto-oligosaccharides. ACKNOWLEDGEMENTS The authors thank Simon Flitter for the identification and isolation of the galA containing phage clones and Matthew Illsley for the analysis of a- L -1,5-arabinofuranosidase activity in the endogalactanase preparation. REFERENCES 1. de Vries, R.P. & Visser, J. (2001) Aspergillus enzymes involved in degradation of plant cell wall polysaccharides. Microb.Mol.Biol. Rev. 65, 497–522. 2. Labavitch, J.M., Freeman, L.E. & Albersheim, P. 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The b-1,4-endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation. isolation of the galA containing phage clones and Matthew Illsley for the analysis of a- L -1,5-arabinofuranosidase activity in the endogalactanase preparation. REFERENCES 1.