from Penicillium purpurogenum - Applied and Environmental

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2010, p. 5247–5253 0099-2240/10/$12.00 doi:10.1128/AEM.00214-10 Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Vol. 76, No. 15

Novel Bifunctional ␣-L-Arabinofuranosidase/Xylobiohydrolase (ABF3) from Penicillium purpurogenum䌤† María Cristina Ravanal,1 Eduardo Callegari,2 and Jaime Eyzaguirre1* Departamento de Ciencias Biolo ´gicas, Universidad Andre´s Bello, Santiago, Chile,1 and BRIN-USDSSOM Proteomics Facility, University of South Dakota, Vermillion, South Dakota2 Received 27 January 2010/Accepted 9 June 2010

The soft rot fungus Penicillium purpurogenum grows on a variety of natural substrates and secretes various isoforms of xylanolytic enzymes, including three arabinofuranosidases. This work describes the biochemical properties as well as the nucleotide and amino acid sequences of arabinofuranosidase 3 (ABF3). This enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700 and can bind cellulose. The enzyme is active with p-nitrophenyl ␣-L-arabinofuranoside and p-nitrophenyl ␤-D-xylopyranoside with a Km of 0.65 mM and 12 mM, respectively. The enzyme is active on xylooligosaccharides, yielding products of shorter length, including xylose. However, it does not hydrolyze arabinooligosaccharides. When assayed with polymeric substrates, little arabinose is liberated from arabinan and debranched arabinan; however, it hydrolyzes arabinose and releases xylooligosaccharides from arabinoxylan. Sequencing both ABF3 cDNA and genomic DNA reveals that this gene does not contain introns and that the open reading frame is 1,380 nucleotides in length. The deduced mature protein is composed of 433 amino acids residues and has a calculated molecular weight of 47,305. The deduced amino acid sequence has been validated by mass spectrometry analysis of peptides from purified ABF3. A total of 482 bp of the promoter were sequenced; putative binding sites for transcription factors such as CreA (four), XlnR (one), and AreA (three) and two CCAAT boxes were found. The enzyme has two domains, one similar to proteins of glycosyl hydrolase family 43 at the amino-terminal end and a family 6 carbohydrate binding module at the carboxyl end. ABF3 is the first described modular family 43 enzyme from a fungal source, having both ␣-L-arabinofuranosidase and xylobiohydrolase functionalities. have been isolated from a number of bacteria, fungi, and plants (28). Beldman et al. (3) distinguished the following classes of exo ␣-L-arabinosyl hydrolases based on their mode of action and substrate specificity: 1, not active toward polymers; 2, active toward polymers; 3, specific for arabinoxylans; 4, not active on the synthetic substrate p-nitrophenyl-␣-L-arabinofuranoside (pNPAra). ABFs can also be distinguished by their amino acid sequence. They belong to families 3, 43, 51, 54, and 62 of the glycosyl hydrolases as defined by Henrissat and Davies (18) based on amino acid sequence similarities. As mentioned earlier, ABFs hydrolyze L-arabinofuranose side chains from xylan and pectin. In the case of xylan, the L-arabinofuranose residues may be linked to carbon-2 or -3 of the xylose residues in the backbone (21). In pectins, arabinofuranose residues are bound to arabinans in position 2 or 3, and in rhamnogalacturonan II they are bound to galactose (position 2, 3, 4, or 6) or rhamnose (carbon 2, 3, or 4) (32). These poorly understood regioselectivities may explain at least in part the production of multiple ABFs by some fungi. Both Aspergillus terreus (24, 25) and Penicillium purpurogenum (10) secrete three ABFs, and two are produced by Aspergillus awamori (22), Aspergillus niger (27), Penicillium capsulatum (12), Penicillium canescens (34), and Penicillium chrysogenum (29). We have previously reported the purification, characterization, and sequences of two ABFs (ABF1 and -2) from P. purpurogenum. ABF1 belongs to family 54 of the glycosyl hydrolases, while ABF2 belongs to family 51 (8, 10, 15). In this work the properties of ABF3 are studied and compared to those of the other two ABFs. It shows significant differences in

The simple sugar L-arabinose is the most common pentose with the L-configuration present in nature. It is found principally in plant cell wall components such as pectin and xylan. In pectin it is present in the arabinan backbone and side chains and in the arabinogalactan side chains. In xylan it is found as side chains of arabinoxylan (3). The biodegradation of plant cell wall polysaccharides is an important biotechnological process for obtaining monosaccharides useful in different industrial applications, such as fermentation for the generation of bioethanol (28). Among the enzymes participating in this process are those releasing arabinose residues. These include endo-acting arabinanases (hydrolyzing arabinan backbone) and exo-acting arabinofuranosidases (ABFs), which hydrolyze arabinofuranose side chains (3). Considering the variety of substrates and linkages associated with arabinofuranose-derived complex polysaccharides, the existence of a number of ABFs with different specificities can be envisioned. ␣-L-Arabinofuranosidases (␣-L-arabinofuranoside arabinofuranohydrolases; EC 3.2.1.55) are exo-acting enzymes which hydrolyze nonreducing arabinofuranose residues from arabinoxylan, pectins, and shorter oligosaccharides. These enzymes

* Corresponding author. Mailing address: Departamento de Ciencias Biolo ´gicas, Universidad Andre´s Bello, Repu ´blica 217, Santiago, Chile. Phone: (562) 6618070. Fax: (562) 6980414. E-mail: jeyzaguirre @unab.cl. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 18 June 2010. 5247

5248

RAVANAL ET AL.

amino acid sequence and biochemical properties: ABF3 is a bifunctional ␣-L-arabinofuranosidase/xylobiohydrolase and it belongs to family 43 of the glycosyl hydrolases. MATERIALS AND METHODS Fungal strain and culture conditions. Penicillium purpurogenum ATCC strain MYA-38 was grown in Mandel’s medium as described previously (19). Liquid cultures were grown (1 liter of medium in a 2-liter Erlenmeyer flask) for 8 days at 28°C in an orbital shaker (200 rpm) using 1% oat spelt xylan (Sigma-Aldrich, St. Louis, MO) as carbon source. Nitrogen sources were 0.75 g/liter Neopeptone (Difco, Franklin Lakes, NJ), 0.3 g/liter urea, and 1.4 g/liter (NH4)2SO4. Enzyme purification. The culture supernatant was used for purification. The initial purification steps were performed as described in reference 15. Two enzyme active fractions were obtained from the SP Sepharose column, one containing ABF2 and the other ABF3. These fractions were separately loaded on a 150-ml BioGel P-300 gel filtration column (Bio-Rad, Hercules, CA) equilibrated with 50 mM sodium acetate buffer (pH 4.0) and eluted with the same buffer. Active fractions were pooled, and the purities of ABF2 and -3 were analyzed by SDS-PAGE. ABF1 was purified as described previously (10). Xylanase A was purified as described in reference 2. Enzyme activity assays and protein concentration determinations. Semiquantitative activity analysis of ABFs in the chromatography eluates was performed as described previously (15) with pNPAra (Sigma) as substrate. For the determination of kinetic constants, 330 ␮l of 50 mM sodium citrate buffer (pH 4.0), 200 ␮l of substrate (pNPAra, 0.2 to 4 mM; p-nitrophenyl ␤-D-xylopyranoside [pNPXyl; Sigma], 2.5 to 20 mM) dissolved in the same buffer, and 20 ␮l of enzyme were mixed. After 10 min of incubation at 30°C the reaction was stopped by adding 450 ␮l of 0.2 M Na2CO3, and the absorbance was measured in a spectrophotometer at 405 nm. One unit of activity was defined as the amount of enzyme required to hydrolyze 1 ␮mol of substrate per min. Substrate specificity assays with p-nitrophenyl derivatives (Sigma) were performed by the same method (10 mM substrate concentration). The pH optimum was determined with the above assay using McIlvaine buffer in the pH range 2.6 to 6.6. For optimal temperature determination, the same assay was performed at 4°C, 20°C, 30°C, 42°C, 50°C, 60°C, and 72°C. The substrate concentration for the pH and temperature optimum assays was 10 mM pNPAra or 50 mM pNPXyl. Chromatography eluates were analyzed for protein content by measuring absorbance at 280 nm. Otherwise, the DC protein assay (Bio-Rad) was used with bovine serum albumin (Sigma) as standard. Activity of ABF3 on oligosaccharides. Enzyme activity on xylooligosaccharides was assayed using xylobiose, xylotriose, xylotetraose, and xylopentaose (Megazyme; Bray, Wicklow County, Ireland) as substrates. Enzyme was incubated with 1% substrate in pH 4.0 sodium citrate buffer for 21 h at 30°C. Products were analyzed by thin-layer chromatography (TLC). An assay under the same conditions was performed with 1% arabinobiose, arabinotriose, or arabinohexaose (Megazyme). Samples were incubated for 75 min at 50°C, and the liberation of arabinose was analyzed by TLC and quantified using the K-ARGA kit from Megazyme. Activity of ABF3 on polysaccharides. Activities on wheat arabinoxylan, arabinan, and debranched arabinan from sugar beet pulp (Megazyme) were determined for the three ABFs by incubating these substrates at 10 mg/ml in 50 mM sodium citrate buffer (pH 4.0) at 28°C for 6 or 72 h. Release of arabinose was detected as described above and expressed as the percentage of the arabinose content of the substrate (information supplied by the manufacturer). Generation of oligosaccharides from arabinoxylan by ABF1 and ABF3 was assayed by thinlayer chromatography. Synergism between ABFs and xylanase. Wheat arabinoxylan (10 mg/ml) was incubated at 28°C for 6 h in 50 mM sodium citrate buffer (pH 4.0) with 3.2 ␮M each ABF and 1.53 ␮g P. purpurogenum endoxylanase A per assay mixture. Arabinose liberation was quantified as described above. A control was performed by incubating the substrate under identical conditions but in the absence of enzymes. The amount of arabinose detected in the control was subtracted from the raw data. Chromatography and electrophoresis. Thin-layer chromatography was performed as described in reference 10. The plates were developed in ethyl acetateglacial acetic acid-water (3:2:1 [vol/vol/vol]). Sodium dodecyl sulfate electrophoresis was performed according to the methods outlined in reference 23 as described in reference 4. Gels were stained with Coomassie brilliant blue R-250. Isoelectrofocusing and zymograms were performed as described previously (15). Glycosylation assays. The glycosylation of ABF3 was probed using two methods: (i) periodic acid-Schiff reagent staining of an SDS-PAGE gel, and (ii)

APPL. ENVIRON. MICROBIOL. treatment with endo-␤-N-acetyl glucosaminidase (endo H). Both methods have been described previously (17). Quaternary structure determination. A Superose 12 HR high-performance liquid chromatography (HPLC) column (GE Healthcare, Piscataway, NJ) with a 24-ml bed volume (kindly provided by Emilio Cardemil, University of Santiago de Chile) was utilized. The column was equilibrated with 50 mM sodium acetate buffer (pH 4.0) containing 100 mM KCl at 28°C. A 100-␮l volume of the buffer containing 10 ␮g of ABF3 was injected into the column, which was previously calibrated with apoferritin (443 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and phosphoenolpyruvate carboxykinase (61 kDa). Binding of ABF3 to cellulose. A 700-␮l volume of 1 M NaCl was added to each of two Eppendorf tubes containing 50 mg microcrystalline cellulose (GE Healthcare); after vigorous stirring the tubes were centrifuged for 5 min at 10,000 ⫻ g, the supernatant was discarded, and the process was repeated. The cellulose was then resuspended in 700 ␮l 50 mM sodium citrate buffer (pH 4.0), and it was washed and centrifuged three times as described above. To one tube was added 20 ␮l of 0.034 mg/ml ABF3 plus the above buffer to a complete volume of 100 ␮l. To the other tube was added 100 ␮l of buffer. Both tubes were incubated for 1 h at 4°C with constant stirring and centrifuged. The cellulose pellets were added to 100 ␮l of 1 M NaCl, and after 1 h of incubation at 4°C they were centrifuged. ABF activity was determined (as described above) in all the supernatants. Amino acid sequencing. Amino-terminal sequencing (gas-phase Edman degradation) and the sequence analysis of internal peptides used for primer design (mass spectrometry) were kindly performed by Isabel Vandenberghe in Joseph Van Beeumen’s laboratory, University of Ghent, Belgium. Identity of the protein sequence deduced from cDNA to that of the purified ABF3 was confirmed by means of extensive mass spectrometry analysis of the latter. Peptide analysis was performed using nano-LC–electrospray ionization-tandem mass spectrometry (nanoLC-ESI-MS/MS). Peptide matches were conducted with MASCOT server v2.2 (details are provided in the supplemental material). RNA and DNA preparation. Total RNA was prepared as described in reference 15. mRNA was obtained by means of the Absolutely mRNA purification kit (Agilent, Santa Clara, CA), and cDNA was prepared using the SuperScript RT kit (Life Technologies, Carlsbad, CA). Genomic DNA was prepared according to the methods described in reference 1. PCR techniques. PCR was performed in a Palm-Cycler 9600–00 (Corbett Research, Sydney, Australia) with a final volume of 20 ␮l. The reaction mixture contained 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 0.05 mM (each) dATP, dCTP, dGTP, and dTTP (Fermentas, Burlington, Canada), 1.5 mM MgCl2, and 15 ␮M concentrations of the corresponding primers. The amount of template used varied with each experiment. The general PCR conditions used were 30 cycles of 1 min denaturation at 94°C, 1 min of hybridization at variable temperatures, 1 min of elongation at 72°C, and 10 min of extension at 72°C at the end of each cycle. Primer design. Table 1 lists the sequences and orientations of the primers used. JE-1C was designed based on the amino-terminal sequence of ABF3; JE-12C was based on a conserved amino acid sequence region found when aligning sequences from fungal proteins, which show similarity to the aminoterminal sequence of ABF3. JE-2R, JE-2R*, JE-3R, JE-4R, JE-2T, JE-3E, and JE-6E were designed from known sequences as the sequencing process advanced (see Results). The 3⬘-random amplification of cDNA ends (3⬘-RACE) adapter, 3⬘-RACE outer primer, and 3⬘-RACE inner primer are part of the RLM-RACE kit (Ambion, Austin, TX). AP1 and AP2 are part of the Genome Walker kit (Clontech, West Chester, OH). General recombinant DNA techniques. Agarose gel electrophoresis was carried out by standard procedures (31). PCR products were extracted from agarose gels by means of the QIAquick gel extraction kit (Qiagen, Valencia, CA), inserted in the pGEM-T Easy vector system (Promega, Madison, WI), and cloned in Escherichia coli DH5␣. DNA sequencing was performed in both strands of cDNA and abf3 by Macrogen Inc., Seoul, South Korea, using a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). Sequencing strategy for the abf3 gene and its cDNA. The cDNA was used as template for PCR: a first PCR was performed with primers JE-1C and JE-12C, and a product of about 1,000 bp was obtained. This product was cloned and sequenced. Next, 3⬘-RACE was performed using primers JE-3R and JE-4R (forward) and 3⬘-RACE outer primer and 3⬘-RACE inner primer (reverse) for a nested PCR. A product of about 400 bp was obtained, which was cloned and sequenced. Thus, the complete sequence of the cDNA was obtained. For the sequence of abf3 the following strategy was utilized: a PCR was performed with genomic DNA as template and primers JE-1C and JE-12C, and the product was cloned and sequenced. To obtain the 3⬘ end, a PCR was performed with primers JE-4R and JE-2T (the last primer was designed based on the known cDNA

VOL. 76, 2010

ARABINOFURANOSIDASE FROM PENICILLIUM PURPUROGENUM

5249

TABLE 1. Primers used in this work Primer

Sequence (5⬘ to 3⬘)

Orientation

JE-1C JE-12C JE-2R JE-2R* JE-3R JE-4R JE-2T JE-3E JE-6E 3⬘-RACE adapter 3⬘-RACE outer 3⬘-RACE inner AP1 AP2

5⬘-GA(T/C) AAC CC(A/T) AT(C/T) AT(C/T) CAG ACC AT(C/T) TAC-3⬘ 5⬘-(C/G)C CCT TGA CTT TGA TGT A(G/A)TC-3⬘ 5⬘-CAC GAC CGG TGC TGG ATC TGC TGT GTA-3⬘ 5⬘-TCG TGA TCG GTA AAG CAG TAA AGG CGC CCA-3⬘ 5⬘-C AGT GAA GGC GGT CTT AAT GTC TGC-3⬘ 5⬘-AT GGC GAT TAC ATC AAA GTC AAG GGC-3⬘ 5⬘-CT AGA CCA ACC TCT ACC TGA GCA GT-3⬘ 5⬘-ACC CGT CGC GTT GTG CAA ATG AAT ATT GG-3⬘ 5⬘-CGA GTT TAC ACA GGG TGA AGC ACC AAT ACC-3⬘ 5⬘-GCG AGC ACA GAA TTA ATA CGA CTC ACT ATA GGT TTT TTT TTT TT-3⬘ 5⬘-GCG AGC ACA GAA TTA ATA CGA CT-3⬘ 5⬘-CGC GGA TCC GAA TTA ATA CGA CTC ACT ATA GG-3⬘ 5⬘-GTA ATA CGA CTC ACT ATA GGG C-3⬘ 5⬘-ACT ATA GGG CAC GCG TGG T-3⬘

Sense Antisense Antisense Antisense Sense Sense Antisense Antisense Sense Antisense Antisense Antisense

sequence), and the product was cloned and sequenced. In order to obtain the upstream sequence to cover the amino-terminal end and part of the promoter, the Genome Walker kit was utilized, following the manufacturer’s instructions. A first PCR was performed with primers AP1 and JE-2R* and the four Genome Walker libraries as templates. A nested PCR was performed with primers AP2 and JE-2R, and products were obtained from two libraries: one of ⬃300 bp and the other of ⬃1,500 bp. These products were cloned and sequenced, demonstrating that the sequence of the 300-bp fragment is included in the larger fragment. The promoter sequence (about 500 bp) was confirmed using primers JE-6E and JE-3E. Sequencing was performed for both strands. Nucleotide sequence accession number. The sequence of abf3 and its promoter has been deposited in GenBank with accession number FJ906695.

RESULTS Purification and molecular properties of ABF3. Table 2 summarizes the purification of ABF3. The enzyme was purified 50-fold with a final yield of 5.8%. The purified enzyme revealed a single band when analyzed by SDS-PAGE (Fig. 1A). The apparent molecular weight deduced from the gel was 50,700, while those of ABF1 and -2 were 58,000 and 70,000, respectively (10, 15). Using chromatography in a calibrated HPLC-Superose 12 HR column (as described in Materials and Methods), a molecular weight of 28,000 was estimated for the native enzyme, indicating that ABF3 is a monomer. This low value may be due to retardation in the column by an interaction between the enzyme and the resin. The enzyme is glycosylated, as shown by staining of an SDS gel with Schiff’s reagent (Fig. 1B). Treatment of ABF3 with endo H did not change the molecular weight compared to the native enzyme in an SDS gel (data not shown). When ABF3 was incubated with cellulose as described in Materials and Methods, no activity was detected in the supernatant. On the other hand, when the samples were

treated with 1 M NaCl, enzyme was dissociated and activity was found in the supernatant, showing that ABF3 binds cellulose. Kinetic properties and substrate specificity of ABF3. The substrate specificity of ABF3 was tested with a series of nitrophenyl derivatives: pNPXyl, pNPAra, p-NP-␣-L-arabinopyranoside, o-NP-␤-D-xylopyranoside, p-NP-␤-D-glucopyranoside, p-NP-␤-Dmannopyranoside, p-NP-␣-D-glucopyranoside, p-NP-␤-D-fucopyranoside, p-NP-␣-D-galactopyranoside, and p-NP-␤-D-galctopyranoside. Significant activity was observed only with pNPAra and pNPXyl, indicating that ABF3 is bifunctional; all others showed less than 2% of the activity. The enzyme followed MichaelisMenten kinetics with both substrates. Table 3 gives the kinetic parameters for pNPAra and pNPXyl. The enzyme showed much higher affinity (lower Km) for pNPAra. However, both the kcat and kcat/Km values showed higher catalytic efficiency with pNPXyl. The pH optimum for the enzyme (for pNPAra and pNPXyl) was 5.0. The optimal temperature (determined with pNPAra) and assayed in a range of 4 to 70°C was 50°C. Thermal stability showed that the enzyme lost 40% of its activity at 35°C and

TABLE 2. Summary of purification of ABF3 Purification step

Total protein (mg)

Total activitya (U)

Sp act (U/mg)

Yield (%)

Crude extract Concentrate (NH4)2SO4 precipitate DEAE-Sephadex SP-Sepharose BioGel P-300

369 105 43.8 11.5 2.3 0.42

648 638 550 305 105 37.6

1.76 6.09 12.6 26.5 45.8 89.5

100 99 85 47 16 5.8

a

Activity was assayed with pNPAra.

FIG. 1. SDS-polyacrylamide gel electrophoresis results of ABF3. Lane St, molecular mass standards; lane 1, ABF3. (A) Gel stained with Coomassie brilliant blue R 250. (B) Glycoprotein staining with periodic acid-Schiff reagent.

5250

RAVANAL ET AL.

APPL. ENVIRON. MICROBIOL.

TABLE 3. Kinetic parameters of ABF3 Parameter

Km (mM) kcat (min⫺1) kcat/Km (min⫺1 ⫻ M⫺1)

pNPAra

0.65 ⫾ 0.08 164 ⫾ 5.9 2.5 ⫻ 105

TABLE 4. Action of ABFs on natural substrates % Arabinose liberated

pNPXyl

12 ⫾ 2.6 15,200 ⫾ 1,665 1.3 ⫻ 106

Natural substrate

Sugar beet arabinan Debranched sugar beet arabinan Wheat flour arabinoxylan a

90% at 50°C after 30 min of incubation. Activity of ABF3 against xylo- and arabinooligosaccharides was assayed. Figure 2 shows the TLC results for the reaction products detected using xylooligosaccharides and pNPXyl. pNPXyl was completely hydrolyzed, but only small amounts of xylose were released from the oligosaccharides. Smaller oligosaccharides were produced when X3 through X5 were used as substrates, suggesting a possible xylobiohydrolase activity. No transglycosylation (appearance of oligosaccharides with a higher degree of polymerization than the substrate) was detected. ABF1 and ABF2 showed no activity over xylooligosaccharides (data not shown). A similar experiment was performed with arabinooligosaccharides (di-, tri-, and hexasaccharides); no liberation of arabinose was detected either by TLC or by enzymatic assay of arabinose (data not shown), and no transglycosylation was observed, indicating that ABF3 does not act on short arabinooligosaccharides. Table 4 shows the results of the action of ABFs on arabinan, debranched arabinan, and arabinoxylan as a percentage of arabinose liberated. ABF1 was more active against all substrates, and the three enzymes showed low activity on debranched arabinan. ABF3 is most active on arabinoxylan. When the time course of arabinose liberation by ABF3 from arabinoxylan was followed, 2% was liberated after 6 h and 32.8% after 72 h. Figure 3 shows the liberation of oligosaccharides from arabinoxylan by ABF3; small amounts of xylose were found, while several oligosaccharides were apparent; ABF1, on the other hand, released only arabinose. Synergism between ABFs and endoxylanase A from P. purpurogenum in the liberation of arabinose. The effects of xylanase A on the release of arabinose from wheat arabinoxylan by

ABF1

ABF2a

ABF3

25.6 2.87 32.8

1.35 0.58 5.86

0.82 1.35 15.9

Data are from Fritz et al. (15). Incubation was for 72 h at 28°C.

the three ABFs were analyzed. The results (Fig. 4) indicated that a significant effect is observed only when xylanase A is combined with ABF1. Amino acid sequences of the amino-terminal end and of internal peptides. By means of Edman degradation, the following amino-terminal sequence of ABF3 was obtained: DNP IIQTIYTADPAXVVXDG (where X could not be determined). By mass spectrometry, the following internal peptides were identified: AWASQ, NEDMIN, and EEGPW. A BLAST search using the amino-terminal peptide gave an 85% identity with xylanase 7 from the fungus Gibberella zeae. Good alignment was also obtained with the internal peptide sequences. The amino-terminal sequence and the internal peptides NE DMIN and EEGPWV were used to design degenerate primers. No good PCR products were obtained, so a new alignment (using ClustalW) of all fungal proteins giving more than 75% identity to the amino-terminal peptide was performed. Two regions of 100% identity of 10 and 8 residues, respectively, were identified (data not shown). The one closest to the C terminal (sequence DYIKVKGV) was used to design degenerate primer JE-12C. Sequence of abf3 and its derived protein. abf3 has an open reading frame of 1,380 bp, which is identical to the cDNA sequence, indicating that abf3 has no introns. The gene codes for a protein of 459 amino acid residues. The signal peptide is 26 amino acids in length, so the mature protein includes 433 residues with a calculated molecular weight of 47,305. The identity of the protein sequence derived from the cDNA se-

FIG. 2. Thin-layer chromatography of the hydrolysis products of xylooligosaccharides by ABF3. Lane 1, pNPXyl; lane 2, pNPxyl plus ABF3; lane 3, xylose; lane 4, xylose plus ABF3; lane 5, xylobiose; lane 6, xylobiose plus ABF3; lane 7, xylotriose; lane 8, xylotriose plus ABF3; lane 9, xylotetraose; lane 10, xylotetraose plus ABF3; lane 11, xylopentaose; lane 12, xylopentaose plus ABF3; line 13, oligosaccharide standards. The incubation was for 21 h at 30°C.

VOL. 76, 2010

ARABINOFURANOSIDASE FROM PENICILLIUM PURPUROGENUM

5251

FIG. 3. Thin-layer chromatography of the hydrolysis products of arabinoxylan by ABF1 and ABF3. Lane 1, arabinoxylan plus buffer; lane 2, arabinoxylan plus ABF1; lane 3, arabinoxylan plus ABF3; lane 4, mixture of xylooligosaccharides (xylose, xylobiose, xylotriose, xylotetraose, and xylopentaose); lane 5, arabinose. Incubation conditions were those of the experiment presented in Table 4.

quence compared to that of ABF3 has been confirmed by means of extensive mass spectrometry analysis of the purified ABF3: 35% coverage of the sequence with perfect matching has been achieved (for details, see Fig. S1 in the supplemental material). In addition, a good match was obtained with the amino-terminal sequence obtained by Edman degradation. Several potential N-glycosylation sites can be identified at N58, -63, -193, -207, and -313. A similarity search was performed with the Pfam database (http://pfam.sanger.ac.uk/), and two conserved domains were identified in ABF3: one is similar in sequence to family 43 of the glycosyl hydrolases (CAZY [http://www.cazy.org/]), spanning from the amino-terminal end through residue 309. The other (from residue 332 through 456) is similar to family 6 carbohydrate binding modules. They are separated by a linker sequence of about 33 residues. By means of BLASTP, proteins similar to ABF3 were searched for in the databases. Fourteen proteins with over 60% identity were found, seven belonging to fungi and seven to bacteria (Table 5). None of these enzymes has been biochemically characterized; they are annotated based on sequence similarity. Analysis of the promoter sequence of ABF3. A sequence of 482 bp upstream of the starting codon was sequenced. A putative TATA box was found at ⫺140. Several putative binding sites for transcription factors were recognized: four CreA binding sequences (a negative-acting regulatory protein mediating carbon catabolyte repression) (9) at ⫺219, ⫺270, ⫺324, and ⫺393; one XlnR binding sequence (a transcriptional activator in the absence of glucose) (36) at ⫺174; three possible AreA binding sites at ⫺128, ⫺302, and ⫺445 (a nitrogen metabolism activator) (26), although the site at ⫺128 is unlikely to be functional because of its downstream location with respect to the TATA box. Two HAP-like CCAAT boxes, which are present in the promoters of filamentous fungi (5) and are involved in the modulation of transcription levels in eukaryotic cells, were observed at ⫺95 and at ⫺467.

FIG. 4. Synergistic effect of xylanase A (XYL) from P. purpurogenum on the liberation of arabinose from wheat arabinoxylan (AX) by the action of ABFs. Incubations were for 6 h at 28°C. Arabinose was quantified as described in Materials and Methods. The arabinose content indicated by the manufacturer was considered 100%. The asterisk indicates that the addition of XYL increases the liberation of arabinose significantly with 95% confidence (based on analysis of variance).

DISCUSSION In this work we present the purification, properties, and sequence of ABF3 from P. purpurogenum. The enzyme has been purified to homogeneity. It is a glycosylated monomer with a molecular weight of 50,700, as determined by SDSPAGE. The glycosylation may explain the difference when comparing the molecular weights obtained by SDS-PAGE and its calculated value from the amino acid sequence (47,305). Five putative N-glycosylation sites are found; however, treatment with endo H did not show a significant loss in molecular weight, indicating a negligible degree of N-glycosylation. The sequence shows the presence of six Cys residues, which may be forming disulfide bridges. P. purpurogenum secretes into the medium three different ABFs. All three are recognized by their activity toward pNPAra. However, when the substrate specificity is analyzed in more detail, they show significant differences. When their activity toward pNP derivatives is compared, ABF1 is only active with pNPAra (10), while ABF2 shows significant activity toward pNP-␤-D-galactopyranoside (15). ABF3, on the other hand, is active toward pNPXyl. None of these enzymes presents activity with pNP-␣-L-arabinopyranoside, showing strong specificity toward the furanose form of L-arabinose. Interestingly, ABF3 is almost inactive in the presence of o-NP-␤-Dxylopyranoside, suggesting that the active site requires the nitro group to be distant from the sugar residue to promote hydrolysis. ABF1 and -2 do not act on xylooligosaccharides. In the presence of arabinooligosaccharides, ABF2 is the most active, particularly toward the shorter ones (di- and trisaccharides) (15), while ABF3 proved inactive.

5252

RAVANAL ET AL.

APPL. ENVIRON. MICROBIOL. TABLE 5. BLAST search results for proteins with sequences similar to ABF3 GenBank accession no.

% Identity to ABF3

Fungi Talaromyces stipitatus Pyrenophora tritici-repentis Gibberella zeae* Podospora anserina Aspergillus terreus Phaeosphaeria nodorum Magnaporte griseae*

XP_002485437 XP_001931632 AAT84252 XP_001905179 XP_001209374 XP_001801338 XP_366403

66 64 65 64 62 62 60

Xylanase, putative Endo ␤-xylanase D precursor Putative xylanase 7 Unnamed protein product Hypothetical protein Hypothetical protein Hypothetical protein

Bacteria Streptomyces sviceus Streptomyces hygroscopicus Micromonospora sp. Actinosynnema mirus* Cellulomonas flavigena Clostridium acetobutylicum* Clostridium thermocellum*

ZP_05021274 ZP_05513737 ZP_04604373 YP_003100967 ZP_04364824 NP_149278 YP_001038591

62 65 64 61 61 61 63

Carbohydrate binding family 6 protein Glycoside hydrolase family 43 Endo 1,4-␤-xylanase Glycoside hydrolase family 43 ␤-Xylosidase Endo 1,4-␤-xylanase Carbohydrate binding family 6 protein

Microorganisma

a

Function annotation

Sequences belonging to family 43 (according to CAZY) are marked with an asterisk.

The three ABFs were assayed for the liberation of arabinose from natural polysaccharides, such as arabinan, debranched arabinan, and arabinoxylan (Table 4). Significant differences were detected. ABF1 is clearly a debranching enzyme, as shown by its high activity on arabinan and arabinoxylan. When incubated in the presence of endoxylanase (Fig. 4), a synergistic effect is observed: the products of endoxylanase (arabinoxylooligosaccharides of different lengths) are better substrates for ABF1. ABF2 shows less activity with arabinoxylan than the others; it is clearly an “exo” type of enzyme, and its physiological role may be related to the hydrolysis of short oligosaccharides. ABF3 is particularly active in arabinose liberation from arabinoxylan. It also shows xylobiohydrolase activity, since it releases short oligosaccharides from arabinoxlan. If the classification of Beldman (3) is applied to these enzymes, ABF1 (GH family 54) should be considered among those “active toward polymers” and ABF2 (GH family 51) as “active on oligosaccharides,” while ABF3 (GH family 43) is in the group of those “acting on arabinoxylan.” These results strongly support different roles for the three ABFs in the physiology of the fungus. All three abfs are subject to catabolite repression by glucose, since no ABF activity is detected in the supernatant of the fungus when grown on glucose (data not shown). This agrees with the finding that all three promoters have CreA binding sites: four in abf1 (8), two in abf2 (15), and four in abf3. The functionality of CreA and XlnR binding sites in P. purpurogenum has been demonstrated (11), but no information is available on the functionality of the potential AreA sites in this fungus. ABF3 may be considered a bifunctional ␣-L-arabinofuranosidase/␤-D-xylosidase based on its activity on pNPAra and pNPXyl. Results shown in Fig. 2 compare its activity against pNPXyl and several xylooligosaccharides. In the incubation period studied (21 h), complete hydrolysis with liberation of xylose was only observed for pNPXyl. However, only small amounts of xylose were liberated from the oligosaccharides, indicating that p-nitrophenol is a better leaving group than xylose. This is additional evidence that ABF3 may be a xylobiohydrolase rather than a ␤-xylosidase. The production of multiple ABFs has been reported for several filamentous fungi. Three ABFs have been found in Aspergillus

terreus (24, 25). However, they have all been classified in GH family 54 because of a similar amino-terminal sequence, and they show very similar substrate specificities. Two ABFs from Aspergillus niger have been studied (13, 14, 27). They differ considerably in size and substrate specificity; ABF A belongs to GH family 51, and ABF B belongs to GH family 54. A third enzyme (AXHA) from the same fungus, an arabinoxylan arabinofuranohydrolase, belonging to GH family 62 has been described (16). Several fungi with two characterized ABFs are described in the literature. Filho et al. (12) described the properties of two ABFs from Penicillium capsulatum; they showed similar sizes and substrate specificities, but their sequences have not been reported. Penicillium chrysogenum secretes two ABFs, both with broad substrate specificity, one belonging to family 51 (AFQ1) and the other to family 54 (AFS1) (29). A similar finding of two ABFs from family 51 and 54 in Aspergillus awamori is described elsewhere (22). So far the presence of three ABFs belonging to families 43, 51, and 54 has been found only in P. purpurogenum. Although a good number of sequences of putative fungal ABFs have been incorporated into the CAZY database, a lack of biochemical studies currently precludes a valid comparison. Family 43 of the glycosyl hydrolases is very heterogeneous. Enzymes belonging to this family include ␤-xylosidases (EC 3.2.1.37), ␤-1,3-xylosidases (EC 3.2.1.-), ␣-L-arabinofuranosidases (EC 3.2.1.55), arabinanases (EC 3.2.1.99), xylanases (EC 3.2.1.8), and galactan 1,3-␤-galactosidases (EC 3.2.1.145). ABF3 from P. purpurogenum can be assigned to this family based on sequence similarities. Enzymes belonging to GH family 43 with bifunctional ␣-L-arabinofuranosidase/␤-D-xylosidase activity have been described in several bacteria: Butyrivibrio fibrisolvens (35), Selenomonas ruminantium (20, 37), Geobacillus stearothermophilus (6, 33), Bacteroides ovatus (38), and Clostridium stercorarium (30). The only fungal bifunctional ABF/␤-xylosidase from family 43 identified so far is produced by Fusarium graminearum (7). This enzyme is active on pNPAra and pNPXyl, but it does not liberate either xylose or arabinose from oat spelt xylan; however, it hydrolyzes arabinose when incubated together with an endoxylanase. When the sequences of the bacterial or the F. graminareum bifunctional

VOL. 76, 2010

ARABINOFURANOSIDASE FROM PENICILLIUM PURPUROGENUM

enzymes were aligned to ABF3 using ClustalW, less than 37% identity was found for the bacterial enzyme and less than 30% for the F. graminarium enzyme. Only the availability of the three-dimensional structure may show the true differences and similarities between these proteins. On the other hand, none of the proteins that does align with more than 60% identity to ABF3 has a defined function (Table 5). Most are either hypothetical or have been assigned a putative function, particularly those from fungi, indicating the need for a biochemical study of their properties. These results show that considerably more work, particularly on the biochemical properties of family 43 enzymes, must be performed in order to better understand the structural and functional relationships between these enzymes. In conclusion, P. purpurogenum is the first fungus where the presence of ABFs from GH families 43, 51, and 54 has been described. ABF3, a bifunctional ␣-L-arabinofuranosidase/xylobiohydrolase, is a novel enzyme based on its amino acid sequence and catalytic properties. The determination of its three-dimensional structure would be a valuable addition for a good understanding of the catalytic properties of this enzyme. ACKNOWLEDGMENTS This work was supported by grants from FONDECYT (numbers 1040201, 1070368, and 1100084) and Universidad Andre´s Bello (numbers 03-05/R, 01-05/I, and 02-08/R). M.C.R. was a recipient of a MECESUP fellowship (project UAB0602). We thank Wladimir Mardones for his help in the preparation of the figures, Lee Meisel for careful revision of the manuscript, and the Proteomics Core Facility at SD-BRIN (NIH grant number 2 P20 RR016479 from the INBRE Program of the National Center for Research Resources) for the mass spectrometry analysis. REFERENCES 1. Bainbridge, B. W., C. L. Spreadbury, F. G. Scalise, and J. Cohen. 1990. Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi. FEMS Microbiol. Lett. 54:113– 117. 2. Belancic, A., J. Scarpa, A. Peirano, R. Díaz, J. Steiner, and J. Eyzaguirre. 1995. Penicillium purpurogenum produces several xylanases: purification and properties of two of the enzymes. J. Biotechnol. 41:71–79. 3. Beldman, G., H. A. Schols, S. M. Pitson, M. J. F. Searle-van Leeuwen, and A. G. J. Voragen. 1997. Arabinans and arabinan-degrading enzymes. Adv. Macromol. Carbohydr. Res. 1:1–64. 4. Bollag, D. M., and S. J. Edelstein. 1991. Protein methods. Wiley-Liss, New York, NY. 5. Brakhage, A. A., A. Andrianopoulos, M. Kato, S. Steidl, M. A. Davis, N. Tsukagoshi, and M. J. Hynes. 1999. HAP-like CCAAT-binding complexes in filamentous fungi: implications for biotechnology. Fungal Genet. Biol. 27:243–252. 6. Bru ¨x, C., A. Ben-David, D. Shallom-Shezifi, M. Leon, K. Niefind, G. Shoham, Y. Shoham, and D. Schomburg. 2006. The structure of an inverting GH43 ␤-xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues. J. Mol. Biol. 359:97–109. 7. Carapito, R., C. Carapito, J.-M. Jeltsch, and V. Phalip. 2009. Efficient hydrolysis of hemicellulose by a Fusarium graminearum xylanase blend produced at high levels in Escherichia coli. Bioresour. Technol. 100:845–850. 8. Carvallo, M., P. De Ioannes, C. Navarro, R. Cha ´vez, A. Peirano, P. Bull, and J. Eyzaguirre. 2003. Characterization of an ␣-L-arabinofuranosidase gene (abf1) from Penicillium purpurogenum and its expression. Mycol. Res. 107:388–394. 9. Cubero, B., and C. Scazzochio. 1994. Two different, adjacent and divergent zinc finger binding sites are necessary for CREA-mediated carbon catabolite repression in the proline gene cluster of Aspergillus nidulans. EMBO J. 13:407–415. 10. De Ioannes, P., A. Peirano, J. Steiner, and J. Eyzaguirre. 2000. An ␣-Larabinofuranosidase from Penicillium purpurogenum: production, purification and properties. J. Biotechnol. 76:253–258. 11. Díaz, J., R. Cha ´vez, L. F. Larrondo, J. Eyzaguirre, and P. Bull. 2008. Functional analysis of the endoxylanase B (xynB) promoter from Penicillium purpurogenum. Curr. Genet. 54:133–141. 12. Filho, E. X. F., J. Puls, and M. P. Coughlan. 1996. Purification and characterization of two arabinofuranosidases from solid-state cultures of the fungus Penicillium capsulatum. Appl. Environ. Microbiol. 62:168–1753.

5253

13. Flipphi, M. J. A., M. van Heuvel, P. van der Veen, J. Visser, and L. H. de Graaff. 1993. Cloning and characterization of the abfB gene coding for the major ␣-L-arabinofuranosidase (ABF B) of Aspergillus niger. Curr. Genet. 24:525–5323. 14. Flipphi, M. J. A., J. Visser, P. van der Veen, and L. de Graaff. 1994. Arabinase gene expression in Aspergillus niger: indications for coordinated regulation. Microbiology 140:2673–2682. 15. Fritz, M., M. C. Ravanal, C. Braet, and J. Eyzaguirre. 2008. A family 51 ␣-L-arabinofuranosidase from Penicillium purpurogenum: purification, properties and amino acid sequence. Mycol. Res. 112:933–942. 16. Gielkens, M. M. C., J. Visser, and L. H. de Graaff. 1997. Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis. Curr. Genet. 31:22–29. 17. Gordillo, F., V. Caputo, A. Peirano, R. Cha ´vez, J. Van Beeumen, I. Vandenberghe, M. Claeyssens, P. Bull, M. C. Ravanal, and J. Eyzaguirre. 2006. Penicillium purpurogenum produces a family 1 acetyl xylan esterase containing a carbohydrate-binding module: characterization of the protein and its gene. Mycol. Res. 110:1129–1139. 18. Henrissat, B., and G. Davies. 1997. Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7:637–644. 19. Hidalgo, M., J. Steiner, and J. Eyzaguirre. 1992. ␤-Glucosidase from Penicillium purpurogenum: purification and properties. Biotechnol. Appl. Biochem. 15:185–191. 20. Jordan, D. B., X.-L. Li, C. A. Dunlap, T. R. Whitehead, and M. A. Cotta. 2007. Structure-function relationships of a catalytically efficient ␤-D-xylosidase. Appl. Biochem. Biotechnol. 141:51–76. 21. Joseleau, J. P., J. Comptat, and K. Ruel. 1992. Chemical structure of xylans and their interaction in the plant cell walls. Prog. Biotechnol. 7:1–15. 22. Kaneko, S., M. Arimoto, M. Ohba, H. Kobayashi, T. Ishi, and I. Kusakabe. 1998. Purification and substrate specificities of two ␣-L-arabinofuranosidases from Aspergillus awamori IFO 4033. Appl. Environ. Microbiol. 64:4021–4027. 23. Laemmli, U. K. 1970. Cleavage of structural protein during the assembly of the head of bacteriophage T4. Nature 227:680–685. 24. Luonteri, E., M. Siika-aho, M. Tenkanen, and L. Viikari. 1995. Purification and characterization of three ␣-arabinofuranosidases from Aspergillus terreus. J. Biotechnol. 38:279–291. 25. Luonteri, E., G. Beldman, and M. Tenkanen. 1998. Substrate specificities of Aspergillus terreus ␣-arabinofuranosidases. Carbohydr. Polym. 37:131–141. 26. Merika, M., and S. H. Orkin. 1993. DNA-binding specificity of GATA family transcription factors. Mol. Cell. Biol. 13:3999–4010. 27. Rombouts, F. M., A. G. J. Voragen, M. F. Searle-van Leeuwen, C. C. J. M. Geraeds, H.A. Schols, and W. Pilnik. 1988. The arabinanases of Aspergillus niger: purification and characterization of two ␣-L-arabinofuranosidases and an endo-1,5-␣-L-arabinanase. Carbohydr. Polym. 9:25–47. 28. Saha, B. C. 2000. ␣-L-Arabinofuranosidases: biochemistry, molecular biology and application in biotechnology. Biotechnol. Adv. 18:403–423. 29. Sakamoto, T., and H. Kawasaki. 2003. Purification and properties of two type B ␣-L-arabinofuranosidases produced by Penicillium chrysogenum. Biochim. Biophys. Acta 1621:204–210. 30. Sakka, K., K. Yoshikawa, Y. Kojima, S. Karita, K. Ohmiya, and K. Shimada. 1993. Nucleotide sequence of the Clostridium stercorarium xylA gene encoding a bifunctional protein with ␤-D-xylosidase and ␣-L-arabinofuranosidase activities, and properties of the translated product. Biosci. Biotechnol. Biochem. 57:268–272. 31. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, New York, NY. 32. Scheller, H. V., J. K. Jensen, S. O. Sørensen, J. Harholt, and N. Geshi. 2007. Biosynthesis of pectin. Physiol. Plant 129:283–295. 33. Shallom, D., M. Leon, T. Bravman, A. Ben-David, G. Zaide, V. Belakhov, G. Shoham, D. Schomburg, T. Baasov, and Y. Shoham. 2005. Biochemical characterization and identification of the catalytic residues of a family 43 ␤-Dxylosidase from Geobacillus stearothermophilus T-6. Biochemistry 44:387–397. 34. Sinitsyna, O. A., F. E. Bukhtoyarov, A. V. Gusakov, O. N. Okunev, A. O. Bekkarevitch, Y. P. Vinetsky, and A. P. Sinitsyn. 2003. Isolation and properties of major components of Penicillium canescens extracellular enzyme complex. Biochemistry (Moscow) 68:1200–1209. 35. Utt, E. A., C. K. Eddy, K.F. Keshaw, and L. O. Ingram. 1991. Sequencing and expression of the Butyrivibrio fibrisolvens xylB gene encoding a novel bifunctional protein with ␤-D-xylosidase and ␣-L-arabinofuranosidase activities. Appl. Environ. Microbiol. 57:1227–1234. 36. van Peij, N. N., M. M. Gielkens, R. P. de Vries, J. Visser, and L. H. de Graaff. 1998. The transcriptional activator XlnR regulates both xylanolytic and endoglucanase gene expression in Aspergillus niger. Appl. Environ. Microbiol. 64:3615–3619. 37. Whitehead, T. R., and M. A. Cotta. 2001. Identification of a broad-specificity xylosidase/arabinosidase important for xylooligosaccharide fermentation by the ruminal anaerobe Selenomonas ruminantium GA192. Curr. Microbiol. 43:293–298. 38. Whitehead, T. R., and R. B. Hespell. 1990. The genes for three xylandegrading activities from Bacteroides ovatus are clustered in a 3.8-kilobase region. J. Bacteriol. 172:2408–2412.