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Appl Microbiol Biotechnol (2007) 75:813–820 DOI 10.1007/s00253-007-0884-1

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Purification and characterization of a highly thermostable α-L-Arabinofuranosidase from Geobacillus caldoxylolyticus TK4 Sabriye Canakci & Ali Osman Belduz & Badal C. Saha & Ahmet Yasar & Faik Ahmet Ayaz & Nurettin Yayli

Received: 9 January 2007 / Revised: 7 February 2007 / Accepted: 8 February 2007 / Published online: 15 March 2007 # Springer-Verlag 2007

Abstract The gene encoding an α-L-arabinofuranosidase from Geobacillus caldoxylolyticus TK4, AbfATK4, was isolated, cloned, and sequenced. The deduced protein had a molecular mass of about 58 kDa, and analysis of its amino acid sequence revealed significant homology and conservation of different catalytic residues with α-L-arabinofuranosidases belonging to family 51 of the glycoside hydrolases. A histidine tag was introduced at the N-terminal end of AbfATK4, and the recombinant protein was expressed in Escherichia coli BL21, under control of isopropyl-β-Dthiogalactopyranoside-inducible T7 promoter. The enzyme was purified by nickel affinity chromatography. The molecular mass of the native protein, as determined by gel filtration, was about 236 kDa, suggesting a homotetrameric structure. AbfATK4 was active at a broad pH range (pH 5.0– 10.0) and at a broad temperature range (40–85°C), and it had an optimum pH of 6.0 and an optimum temperature of 75– 80°C. The enzyme was more thermostable than previously described arabinofuranosidases and did not lose any activity after 48 h incubation at 70°C. The protein exhibited a high level of activity with p-nitrophenyl-α-L-arabinofuranoside, S. Canakci (*) : A. Belduz : F. Ayaz Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey e-mail: [email protected] B. C. Saha Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA-ARS, Peoria, IL 61604, USA A. Yasar : N. Yayli Department of Chemistry, Faculty of Arts and Sciences, Karadeniz Technical University, 61080 Trabzon, Turkey

with apparent Km and Vmax values of 0.17 mM and 588.2 U/ mg, respectively. AbfATK4 also exhibited a low level of activity with p-nitrophenyl-β-D-xylopyranoside, with apparent Km and Vmax values of 1.57 mM and 151.5 U/mg, respectively. AbfATK4 released L-arabinose only from arabinan and arabinooligosaccharides. No endoarabinanase activity was detected. These findings suggest that AbfATK4 is an exo-acting enzyme. Keywords α-L-Arabinofuranosidase . Geobacillus caldoxylolyticus . Exo-acting . Thermostable . Xylosidase

Introduction Lignocelluloses of plant cell walls are composed of cellulose, hemicellulose, and lignin. Hemicelluloses, the second most common polysaccharide in plants, represent about 20–35% of lignocellulosic biomass (Wyman 1994). L-Arabinosyl residues are widely distributed in hemicelluloses, as they constitute monomeric and/or oligomeric side chains on the β-(1→4)-linked xylose or galactose backbones in xylans, arabinoxylans, and arabinogalactans and are the core in arabinans forming α-(1→5)-linkages (Manin et al. 1994; Ward and Moo-Young 1989). Microbial degradation of hemicellulose requires a wide variety of enzymes. Among them, endoxylanases and β-xylosidase are responsible for the backbone degradation, whereas a number of ‘accessory enzymes’ such as α-glucuronidases, acetylxylan esterases, phenolic acid esterases, and α-L-arabinofuranosidase are responsible for the cleavage of the side chains (Biely et al. 1986). α-L-Arabinofuranosidases (Abf, EC 3.2.1.55) are exo-type enzymes that hydrolyze terminal nonreducing α-L-arabinofuranosyl groups from Larabinose-containing polysaccharides. These enzymes can

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hydrolyze (1→3)- or (1→5)-α-L-arabinofuranosyl linkages of arabinan or both. The α-L-AFases are part of the microbial xylanolytic systems required for the complete breakdown of heteroxylans (Bachmann and McCarthy 1991; Greve et al. 1984; Lee and Forsberg 1987; Poutanen 1988). L-Arabinose is a major component of arabinan, which, although it has a sweet taste, is not readily adsorbed by the body (Seri et al. 1996). Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, and for the improvement of animal feedstock digestibility (Bezalel et al. 1993; Saha 2000; Suurnakki et al. 1997; Wong and Sanddler 1993). Arabinofuranosidases can be also used to increase the aroma of wines and fruit juices (Gunata et al. 1990; Spagna et al. 1998). As most of the industrial processes operate at high temperature, the use of thermostable enzymes appears to be ideal. The most recent classification scheme based on amino acid sequences, primary structure similarities, and hydrophobic cluster analysis has classified α-L-AFases into five glycosyl hydrolases families (GHs), i.e., GH3, GH43, GH51, GH54, and GH62 (Coutinho and Henrissat 1999; Henrissat and Davies 2000). This classification is useful to study evolutionary relationship, mechanistic information, and structural features of these enzymes (Davies and Henrissat 1995). The genus Geobacillus separated from the genus Bacillus according to Nazina et al. (2001). It is known that, to date, only two members of this genus (G. stearothermophilus and G. thermoleovorans) have thermostable Abf activity and the strains of the G. stearothermophilus T6 and L1 have two different Abf that have been well characterized (Bezalel et al. 1993; Gilead and Shoham 1995) In this study, we report the purification, characterization, substrate specificity, and gene sequence of an Abf from Geobacillus caldoxylolyticus TK4 showing significant differences from those previously reported.

Materials and methods Substrates and chemicals L-Arabinose, ethylenediamine tetraacetic acid (EDTA), β-mercaptoethanol, p-nitrophenyl (pNP) glycoside substrates, oat spelts xylan, and standard proteins for gel filtration were purchased from Sigma Chemical (St. Louis, MO). Sugar beet arabinan, wheat flour arabinoxylan, red debranched arabinan (RDA), arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose were obtained from Megazyme (Bray, Wirklow, Ireland). Wizard Genomic DNA Purification Kit, Wizard Plus SV Minipreps DNA Purification System, MagneHis

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Protein Purification System, Taq DNA Polymerase, deoxyribonucleotide triphosphate, and all of the restriction enzymes were purchased from Promega. DIG High Prime DNA Labeling and Detection Starter Kit 1 was obtained from Roche Applied Science (Mannheim, Germany). All chemicals were reagent grade, and all solutions were made with distilled and deionized water. Strains, vectors, and media The strain TK4 was isolated from the Kestanbol hot spring in the provinces of Canakkale in Turkey and identified as G. caldoxylolyticus based on morphological, physiological, and biochemical properties and DNA–DNA homology study. G. caldoxylolyticus strain TK4 has been deposited in the National Collections of Industrial Food and Marine Bacteria under the number 14283. The Escherichia coli strains used in this study were BL21 (DE3): pLysS (Novagen) and XL1-Blue (Stratagene). The plasmid vectors used were as follows: pUC18 (Promega), pGEM-TEasy (Promega), and pET28a+ (Novagen). G. caldoxylolyticus TK4 was grown aerobically at 60°C in Luria–Bertani medium (LB) and minimal medium with the addition of 2 g l−1 casamino acids (M9CA) (Sambrook et al. 1989). All E. coli strains containing recombinant plasmids were cultured in LB and M9CA supplemented with 50 μg/ml ampicillin or kanamycin, as appropriate, at 37°C, unless otherwise stated. Detection of the α-L-arabinofuranosidase gene of G. caldoxylolyticus TK4 G. caldoxylolyticus TK4 was found to possess both intra- and extracellular Abf activity when grown on arabinose as carbon source. To clone the gene, firstly, we designed two degenerate primers against conserved regions of arabinofuranosidase genes after aligning the sequences of the other GH51 family members. By using these primers (af TK4 F, 5′-MGN TAY CCN GGN GGN AAY TTY-3′ and af TK4 R, 5′-CAT YTC RTT NCC NAR RCA C-3′), about 300-bp polymerase chain reaction (PCR) fragment was amplified. The fragment was cloned into a pGEM-T vector system and sequenced by Taq DyeDeoxy Terminator Cycle Sequencing Kit and analyzed with an Applied Biosystems Model 370A automatic sequencer (Davis Sequencing LLC, California). Similarity analyses of the sequence were carried out with the Advanced Blast Program of GenBank (NCBI, NIH, Washington, DC). As a result of this study, we found that this sequence was a part of the α-L-arabinofuranosidase gene, and then this fragment was used as a probe in Southern blotting. Cloning of the abf gene from G. caldoxylolyticus TK4 The genomic DNA isolated from G. caldoxylolyticus TK4 by the genomic DNA isolation Kit (Promega) was digested with EcoRV blotted to a nitrocellulose membrane and hybridized with the G. caldoxylolyticus TK4 arabinofu-

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ranosidase gene probe, and the band that has a positive signal was ligated into SmaI-digested pUC18. The positive clones were tested with G. caldoxylolyticus TK4 arabinofuranosidase probe again. These positive clones were sequenced, and similarity analyses were carried out with the Advanced Blast Program of GenBank. The similarity analyses showed that the cloned gene is not complete, and it is missing the 3′ end of the gene. To clone the rest of the gene, genomic DNA was digested with EcoRI and then self-ligated, and then ligation products were used as a template for PCR amplification by af Ara1, 5′-gCC TCC CAT TCC gCA AAg gTT gg-3′,and af Ara3, 5′-GGC AGA GGT GTA GCC TTA CAT CC-3′, oligonucleotides. Each of the 35 amplification cycles consisted of a denaturation step at 95°C for 1 min followed by annealing at 55°C for 1 min and a primer extension step at 72°C for 4 min. These inverse-PCR product were cloned into the pGEM-T easy vector and was sequenced by using the Taq DyeDeoxy Terminator Cycle Sequencing Kit (Davis Sequencing LLC). Then when we analyzed the resulting sequences, we have found the missing part of the gene and combined the sequences to have the full sequences of the arabinouranosidase gene of G. caldoxylolyticus TK4. The abf TK4 gene was amplified from the genomic DNA by using 5′-CAA gCT TC C ATA Tg A AAA CCA TgA ACA C-3′ and 5′-Cgg gAT CCC TAT TAT TTC TTA gCC AAA Cg-3′ primers, and the PCR product was digested with NdeI and BamHI and then ligated with pET28a+ digested with NdeI and BamHI. The ligation products were transformed into E. coli BL21 (DE3):pLysS. The recombinant plasmid was designated as pAbfATK4 and expressed in the same cell. Expression products contained His Tag at their N-terminal and were designated as AbfATK4. Overexpression and purification of the enzyme Transformed cells of E. coli BL21 were grown to an optical density at 600 nm of about 0.6. The expression of the recombinant proteins was induced with 1 mM isopropyl-βD-thiogalactopyranoside (IPTG) and the strain was grown for 3 h. The protein was purified by MagneHis™ Protein Purification System (Promega). Determination of the molecular mass The apparent molecular mass of the native enzyme was determined by highperformance liquid chromatography (HPLC) under the following conditions: column, Superdex™ 200 10/300 GL (Amersham) Gel filtration column; mobile phase, 50 mM phosphate buffer; flow rate, 0.5 ml/min. The eluted compound was detected by UV index. Carbonic anhydrase (29 kDa), bovine serum albumin (66 kDa), Alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), and apoferritin (443 kDa) were used as the molecular mass marker proteins.

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Determination of enzyme activities The activities of Abf and β-xylosidase were assayed at pH 6.0 and 75°C in a reaction mixture (0.5 ml) containing 0.2 mM pNP-α-L-arabinofuranoside and 1 mM pNP-β-D-xyloside, respectively, 50 mM potassium phosphate buffer, and appropriately diluted enzyme solution. Enzyme activities with all pNP-glycosides were performed as described previously (Margolles and de los Reyes-Gavilan 2003). The ability of Abf to release arabinose from sugar beet arabinan (3%, w/v) was tested by using the appropriate diluted enzyme solution. After incubation at pH 6.0 and 75°C, the reaction was stopped by cooling the reaction mixture on ice. The reducing sugar content in the reaction mixture was determined by the dinitrosalicyclic acid method (Miller 1959), with L-arabinose as the standard. One unit of activity was defined as the amount of enzyme which produces 1 μmol of arabinose equivalents per minute. The endoarabinanase activity of AbfATK4 was tested by using RDA as a substrate and following the manufacturer’s instructions. After incubation, unhydrolysed substrate was precipitated by addition of 4 volumes of ethanol, and the absorbance of the supernatant was measured at 520 nm. Effect of pH and temperature on Abf activity and stability The optimum pH for the activity of α-L-arabinofuranosidase was determined by incubation at 75°C for 10 min in the pH range from 4 to 10. Intervals of 1-pH unit were used between pH 4.0 and 5.0, whereas 0.5-pH unit intervals were used for the remaining pH range. The following buffers were used: 50 mM sodium acetate, pH 4.0–5.5; 50 mM potassium phosphate, pH 5.5–7.5; 25 mM Tris–HCl, pH 7.5–9.0; and 50 mM Glisin–NaOH, pH 10.0. The maximum temperature for enzymatic activity was determined in 50 mM potassium phosphate buffer (pH 6.0) by using temperatures ranging from 40 to 95°C. The results were expressed as percentages of the activity obtained at either the optimum pH or the optimum temperature. The thermostability of the purified enzyme was determined in microcentrifuge tubes (0.5 ml) containing 0.5 U of Abf in 50 mM potassium phosphate buffer (pH 6.0). The tubes were incubated at 65, 70, 75, and 80°C for different time periods. Samples were removed at the indicated times, cooled on ice bath, and assayed for the residual enzyme activities at 75°C and pH 6. To determine the effect of pH on the stability of AbfATK4, the enzyme solution was kept for 5 h at the optimum temperature (75°C) in buffers at various pH values and then the residual Abf activity was determined. Effects of chemical agents and metal cations The effects of several metals (HgCl2, CuSO4, ZnSO4, MgCl2, MnCl2, CaCl2, CoCl2, and NaCl, each at 1 mM), chelating agent

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(EDTA, 10 mM), and reducing agents (dithiothreitol [DTT] and β-mercaptoethanol, each at 10 mM) on the Abf activity of the purified enzyme were determined as described above and expressed as a percentage of the activity obtained in the absence of the compound. Substrate specificity The substrate specificity of the enzyme was tested by using the following pNP-glycosides: pNP-α-L-arabinopyranoside, pNP-β-L-arabinopyranoside, pNP-β- D -galactopyranoside, pNP-β- D -xylopyranoside, pNP-N-acetyl-β-D-glucosaminide, pNP-β-D-fucopyranoside, pNP-α-D-galactopyranoside, pNP-α-D-glucopyranoside, pNP-β-D-glucopyranoside, pNP-α-L-rhamnopyranoside, pNP-α-D-xylopyranoside, and pNP-α-L-arabinofuranoside. For nonchromogenic substrates, a solution was prepared containing 0.1 ml of α-L-Abf solution (1 U) and 0.5 ml of 2% (w/v) substrate larch wood arabinogalactan, oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, sugar beet arabinan, azurine-cross-linked arabinan (debranched), and arabinooligosaccharides (arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose). After inccubation for 6 h at 75°C, the reaction was stopped by boiling for 5 min. The released L-arabinose was determined by the dinitrosalicylic acid method (Miller 1959). Sugars from arabinan and arabinooligosaccharides were analyzed by thin-layer chromatography (TLC). Chromatography was performed by the ascending method on silica gel 60 F254 TLC plates (Merck) with a solvent system consisting of ethyl acetate, acetic acid, and water (2:1:1). Sugars on the plates were detected by heating the plates at 120°C for about 10 min after they were sprayed with 5% (v/v) sulfuric acid in ethanol. Sugars from arabinan were analyzed by HPLC. One ionmoderated partition chromatography column (Aminex HPX-87P with De-ashing) was used. The column was maintained at 85°C, and the sugars are eluted with Milli-Qfiltered water at a flow rate of 0.6 ml/min. Peaks are detected by the refractive index and are identified and quantified by comparison to retention times of authentic standards (glucose, xylose, galactose, and arabinose). Nucleotide sequence accession number The nucleotide sequence of the G. caldoxylolyticus TK4 gene cluster containing the α-L-arabinofuranosidase gene has been deposited in the GenBank database under accession number DQ883641.

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conserved amino acid regions. We used this region and designed two degenerate primers. PCR fragments amplified by using these primers were cloned into the pGEM-T Easy vector system and then sequenced. Blast search showed that the cloned fragment is a part of a α-L-arabinofuranosidase. Southern blotting using the clone as a probe identified a positive signal, and the excised fragment containing the most of the gene except 265 nucleotides from the 3′ end was cloned into the pUC18 vector. The rest of the gene sequence was obtained by using inverse PCR. The analysis of the whole gene revealed the presence of a 1,509-bp open reading frame (ORF) encoding a hypothetical 502 amino acid protein with a molecular mass of 57.7 kDa (calculated by ProtParam, http://www.expasy.org) and identified by a database enquiry (Blast program) as a putative family 51 α-L-arabinofuranosidase. A putative ribosome binding sequence, 5′-AGGA, was found 8 bp upstream of the potential ATG initiation codon. No signal sequence was detected with SIGNALP (Nielsen et al. 1997), indicating that the protein may not be secreted. However, we determined extracellular enzyme activity in G. caldoxylolyticus TK4 cultures indicating the secretion of the enzyme from the bacteria in addition to intracellular enzyme activity indicating the presence of soluble cellular enzyme in G. caldoxylolyticus TK4. Extracellular and intracellular crude extracts were prepared from the G. caldoxylolyticus TK4 culture, and an aliquot of each were subjected to native gel electrophoresis, followed by incubation with methylumbelliferyl-α-L-arabinofuranoside. The results of viewing under UV light are shown in Fig. 1. As shown on Fig. 1, extracellular and intracellular AbfATK4 have the same molecular mass. Overexpression of AbfATK4 in E. coli BL21 and purification of the enzyme To investigate the biochemical properties of AbfATK4, cell extracts from E. coli BL21 harboring pTAbfTK4 that were collected after IPTG induction showed a very high level of activity against pNP-α-L-Abf. Control cells, harboring the empty vector pET28a+, did not show any activity. The AbfATK4 was purified from cell extracts of E. coli BL21 harboring pTAbfTK4 by MagneHis™ Protein Purification System (Promega; Fig. 2). Characterization of the enzyme The following items present the characteristics of the enzymes:

Results In this work, two degenerate primers were designed based on the amino acid sequence and DNA similarity of GH51 members. We know that this family has three highly

(a) Molecular mass. The molecular mass and the polymeric state of the histidine-tagged AbfATK4 were estimated by permeation chromatography; the molecular mass of the enzyme was estimated to be around 236 kDa, and from sodium dodecyl sulfate–polyacryl-

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Fig. 1 Native gel electrophoretic analysis of arabinofuranosidase. 1 Pure enzyme from pAbfATK4 expression, 2 the crude cell extracts from G. caldoxylosilyticus TK4, 3 the cell free supernatant from G. caldoxylosilyticus TK4

Fig. 2 Purification of AbfATK4 by Ni column. 1 Protein marker; 2 pure enzyme from pAbfATK4 expression; 3 crude extract from E coli BL21 DE3 including pAbfATK4; 4 crude extract from E coli BL21 DE3 including only pET28a(+) vector

ods”). The dependence of the rate of the enzymatic reaction on the pNP-α-L-Abf concentration followed Michaelis–Menten kinetics, with apparent Km and Vmax values of 0.17 mM and 588.2 U/mg, respectively. The apparent Km and Vmax values of the enzyme for pNP-β-D-xylopyranoside are 1.57 mM and 151.5 U/ mg, respectively. To assure whether AbfATK4 could hydrolyze arabinosecontaining oligosaccharides and polysaccharides and to define the mode of action of the enzyme (endo-acting versus exo-acting), hydrolysis of α-1,5-linked arabinooligosaccharides (containing from two to five arabinose residues) and some arabinose-containing polysaccharides was studied. The enzyme did not display activity against

100 Residual Activity (%)

amide gel electrophoresis, the molecular mass was estimated to be around 59 kDa (Fig. 2). These results indicated that the native form of the enzyme is a polymeric structure, probably a homotetramer. (b) Effects of pH and temperature on activity and stability. The purified enzyme was active at a broad pH range (pH 4.0–10.0), and the optimum pH was pH 6.0 in the potassium phosphate buffer. The optimal temperature for AbfATK4 activity was 75–80°C. The enzymatic activity decreased significantly at temperatures below 40°C and above 90°C. The enzyme retained full activity at pH values from 5.5 to 6.5. No residual activity was detected at pH values 4.0 and 5.0 and pH values above 7.0. The thermostability of AbfATK4 fitted to the first-order reaction. The enzyme exhibit full activity at 60–65°C for 96 h, at 70°C for 48 h, and 75°C for 12 h. There is no activity loss after 10 min at 80°C, but the residual activity was 90% after 30 min at 80°C, and the enzyme lost all its activity after 80 min at 80°C (Fig. 3). (c) Effects of chemical agents and metal cations on the activity. The effects of various metals at 1 mM concentration on the activity of AbfATK4 were determined by using pNP-α-L-arabinofuranoside as a substrate. No effect on activity was detected with Zn2+, Ca2+, or Mg2+ whereas Cu2+ and Hg2+ caused complete inhibition of activity. The metals with a small stimulating effect on the activity were Mn2+ and Co2+. Enzymatic activity was not affected by the chelating agent EDTA (10 mM) or by the reducing agents DTT and β-mercaptoethanol (10 mM). (d) Substrate specificity and kinetic analysis. In a previous experiment, the substrate specificity of the enzyme was tested at pH 6.0 and 75°C by using several pNPglycosides. AbfATK4 was able to hydrolyze pNP-α-LAbf and pNP-β-D-xylopyranoside, whereas no activity was detected with a variety of other pNP-glycosides tested, including those with β-linkages and pyranoside conformations of arabinose (see “Materials and meth-

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Fig. 3 Stability of the α-L-arabinofuranosidase at different temperatures. Residual AbfATK4 activity was monitored after different times of incubation at 60, 65, 70, 75, and 80°C. The initial activity was defined as 100%

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debranched linear sugar beet arabinan dyed with Procoin Red dye, indicating that there was no endoarabinanase activity. AbfATK4 was active with sugar beet arabinan (Fig. 4). It also exhibited activity against α-1,5-linked arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose (Fig. 5). The apparent Km and Vmax values for degradation of arabinan were 67.56 mg/ml and 0.0148 μmol, respectively. In all cases, arabinose was detected as the final product. These results indicated that AbfATK4 is an exo-acting enzyme that hydrolyzes the nonreducing terminal of L-arabinofuranose residues. Activity staining of the purified enzyme after native gel electrophoresis showed that both α-L-arabinofuranosidase and β-xylosidase activities reside in the same protein band (Fig. 6).

Discussion Several arabinofuranosidases from different microorganisms, including yeast, fungi, and bacteria, have been documented and characterized to date. Among the genus of the Geobacillus, two different arabinofuranosidases from G. stearothermophilus have been characterized (Bezalel et al. 1993; Gilead and Shoham 1995). When the sequence of AbfATK4 was compared with the sequences in GenBank, 82% DNA identity, 96% amino acid similarity, and 90% amino acid identity has been seen with G. streathermophilus T6 Abf (AF159625). In addition, AbfATK4 has the highly conserved PGG (P74), LGNE (E174), and DEW (E297) loci, which is conserved in most members of family 51. These data strongly support the conclusion that AbfATK4 should be included in family 51 of the glycoside hydrolases. G. steorothermophilus L1 produced an arabinofuranosidase that had a temperature optimum of 70°C, a native molecular mass of 110 kDa, and which consisted of 52.5and 57.5-kDa subunits. G. stearothermophilus T6 produced a mostly cell-associated arabinofuranosidase with a native

Fig. 4 Thin-layer chromatography of hydrolysis products obtained from sugar beet arabinan. Lane 1, arabinose; lane 2, arabinan (2%, w/ v) incubated with purified enzyme (1 U) for 6 h; lane 3, nontreated arabinan

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Fig. 5 Thin-layer chromatography of hydrolysis products obtained from arabinooligosaccharides. Lane 1, arabinose; lane 2, arabinobiose; lane 3, arabinotriose; lane 4, arabinotetraose; lane 5, arabinopentaose; lanes 6, 7, 8, and 9, arabinobiose, arabinotriose, arabinotetraose, arabinopentaose, respectively, after incubation with 1 U of enzyme for 6 h

molecular mass of 256 kDa that was composed of four identical 64-kDa subunits (Gilead and Shoham 1995), and the temperature optimum for the T6 arabinofuranosidase was the same as that from G. steorothermophilus L1. As mentioned above, AbfATK4 has a native molecular mass of 236 kDa that was composed of four identical 59-kDa subunits, and the temperature optimum for the AbfATK4 was 75–80°C; as shown, the temperature optimum of the AbfATK4 was higher than the other Geobacillus arabinofuranosidases. G. caldoxyloslyticus TK4 arabinofuranosidase activity was found both in the cell extract and the cell-free supernatant. Extracellular and intracellular AbfATK4 have shown the same molecular weight in the native gel (Fig. 1), indicating that the protein does not have any signal peptide. SIGNALP (Nielsen et al. 1997) analysis has not detected any signal sequence, supporting the result from the native gel. Arabinofuranosidase activity in the cell-free supernatant could be the result from cell lysis or the secretion of the enzyme by the nonclassical way (Bendtsen et al. 2005). However, these need to be tested. The optimal temperature for AbfATK4 activity (75–80°C) is the same as Thermobacillus xylanilyticus (75°C) Fig. 6 Native gel electrophoretic analysis of arabinofuranosidase and xylosidase activities in purified enzyme from pAbfATK4 expression. After electrophoresis, the gels were incubated in the presence of 4-methylumbelliferyl-α-L-arabinofuranoside (1) or 4-methylumbelliferyl-β-Dxylopyranoside (2) and examined under UV light

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although higher than the optimal temperatures for the Abf of G. stearothermophilus T6 and L1 but lower than Thermotoga maritima (90°C; Bezalel et al. 1993; Debeche et al. 2000; Gilead and Shoham 1995; Miyazaki 2005). When AbfATK4 is compared with the T. maritima and T. xylanilyticus Abf’s in terms of temperature stability, T. maritima and T. xylanilyticus Abf’s have better temperature stabilities (Debeche et al. 2000). Based on these data, we conclude that AbfATK4 is the third best enzyme among the known arabinofuranosidases with respect to thermal stability and the best among the Geobacillus arabinofuranosidases with very high thermal stability. The purified native enzyme had a multimeric conformation; it was probably a tetramer. The multimeric quaternary structures of several arabinofuranosidases have been reported previously (Margolles and de los ReyesGavilan 2003). The optimal pH of AbfATK4 (pH 5.5–6.0) falls in the same range as the optimal pHs found for G. stearothermophilus T6. Slightly acid pHs, between pH 5.5– 7.0, have also been reported to be optimal for most bacterial arabinofuranosidases, although fungal enzymes had lower optimal pHs (Saha 2000). AbfATK4 showed great stability during prolonged (5 h) incubation at pHs ranging from 5.5 to 6.5 and temperatures lower than 75°C, and the enzyme lost all of its activity at pH 5.0 and higher than pH 7.0. The activity of AbfATK4 was completely inhibited by Cu2+ and Hg2+, but no effect on activity was detected in the presence of known inhibitors (Zn2+, Ca2+, or Mg2+). Cu2+, Hg2+, and Zn2+ are known inhibitors of other enzymes of this family (Margolles and de los Reyes-Gavilan 2003). Addition of the chelating agent EDTA and the reducing agents DTT and β-mercaptoethanol did not affect enzymatic activity, so the enzyme does not seem to need any metal ion or disulfide bonds to carry out the hydrolysis reaction. With respect to substrate specificity, AbfATK4 was not able to hydrolyze other pNP-glycosides other than pNP-αL-Abf and pNP-β-D-xylopyranoside (pNP-β-D-xyloside; Fig. 6). The purified enzyme exhibited activity against sugar beet arabinan and α-1,5-linked oligosaccharides, yielding arabinose as the sole hydrolysis end product. However, AbfATK4 also was not able to hydrolyze oat spelt xylan, wheat arabinoxylan, arabinogalactan, gum arabic, and carboxymethyl cellulose. AbfATK4 also was not able to hydrolyze RDA, a substrate that is cleaved only by endo-1,5-arabinanase activity (McCleary 1988), and dye molecules attached to the arabinose residues prevent the release of arabinosyl residues from the nonreducing end. On the other hand, single arabinose residues are predominantly linked to the main chain in sugar beet arabinan through α-1,3-linkages. Therefore, we conclude from these results that AbfATK4 of G. caldoxyloslyticus TK4 is an exo-acting enzyme that exhibits hydrolytic activity against α-1,3- and α-1,5-linked nonreducing terminal L-arabinofu-

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ranose residues but not against internal α-L-arabinosyl linkages. The substrate specificity of AbfATK4 is narrow when compared with Abf’s of T. maritima, G. stearothermophilus, and T. xylanilyticus. On the basis of sequence homology, AbfATK4 belongs to family 51, but it has a simply broader specificity than the other GH51 enzymes because of it acting on pNP-β-D-xylopyranoside and 4-methylumbelliferyl-β-D-xylopyranoside. This broader substrate specificity assigns a second activity, xylosidase activity, to this enzyme (Fig. 6). As only one ORF was evident, we suggest that both activities are associated by the same enzyme. Whitehead and Cotta (2001) has also shown that both xylosidase and arabinofuranosidase activities are associated with only one gene in Selemonas ruminantium GA192. Whereas β-xylosidase activity in S. ruminantium GA192 is higher than arabinofuranosidase activity, on the contrary, AbfATK4 has a higher arabinofuranosidase activity than xylosidase activity. In conclusion, AbfATK4 is a highly stable, highly active α-L-arabinofuranosidase that, based upon sequence similarities, would appear to belong to family 51 of the glycosyl hydrolase classification system. As AbfATK4 has not only arabinofuranosidase but also xylosidase activity, it is an arabinofuranosidase with a broader substrate specificity than the other family GH51 enzymes.

Acknowledgments We are grateful to the Karadeniz Technical University Research Foundation (grant no. 24.111.004.8) for financial support.

References Bachmann SL, McCarthy AJ (1991) Purification and cooperative activity of enzymes constituting the xylan-degrading system of Thermomonospora fusca. Appl Microbiol 57:2121–2130 Bendtsen JD, Kiemer L, Fausboll A, Brunak S (2005) Non-classical protein secretion in bacteria. BMC Microbiology 5: art. no. 58 Bezalel L, Shoham Y, Rosenberg E (1993) Characterization and delignification activity of a thermostable α-L-arabinofuranosidase from Bacillus stearothermophilus. Appl Microbiol Biotechnol 40:57–62 Biely P, Mackenzie CR, Puls J, Schneider H (1986) Cooperativity of esterases and xylanases in the enzymatic degradation of acetyl xylan. Bio/Technology 4:731–733 Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes server at http://www.afmb.cnrs-mrs.fr/_/cazy/CAZY/index.html Davies G, Henrissat B (1995) Structures and mechanisms of glycosyl hydrolases. Structure 3:853–859 Debeche TN, Cummings N, Connerton I, Debeire P, O’Donohue MJ (2000) Genetic and Biochemical characterization of a highly thermostable of α-L-arabinofuranosidase from Thermobacillus xylanilyticus. Appl Environ Microbiol 66:1734–1736 Gilead S, Shoham Y (1995) Purification and characterization of α-Larabinofuranosidase from Bacillus stearothermophilus T-6. Appl Environ Microbiol 61:170–174

820 Greve LC, Labavitch JM, Hungate RE (1984) α-L-arabinofuranosidase from Rumionococcus albus 8: purification and possible roles in hydrolysis of alfalfa cell wall. Appl Environ Microbiol 47:1135–1140 Gunata ZY, Brillouet JM, Voirin S, Baume R, Cordonnier R (1990) Purification and some properties of an α-L-arabinofuranosidase from Aspergillus niger. Action on grape monoterpenyl arabinofuranosylglucosides. J Agric Food Chem 38:772–776 Henrissat B, Davies GJ (2000) Glycoside hydrolases and glycosyltransferases families, modules and implications for genomics. Plant Physiol 124:1515–1519 Lee SF, Forsberg CW (1987) Purification and characterization of an α-L-arabinofuranosidase from Clostridium acetobutylicum ATCC 824. Can J Microbiol 33:1011–1016 Manin C, Shareek F, Morosoli R, Kluepfel D (1994) Purification and characterization of an α-L-arabinofuranosidase from Streptomyces lividans 66 and DNA sequence of the gene (abfA). Biochem J 302:443–449 Margolles A, de los Reyes-Gavilan CG (2003) Purification of a novel of α-L-arabinofuranosidase from Bifidobacterium longum B667. Appl Environ Microbiol 69:5096–5103 McCleary BV (1988) Novel and selective substrates for the assay of endo-arabinanase. In Phillips GO, Wedlock DJ, Williams PA (eds) Gums and stabilisers for the food industry, vol 5. IRL, Oxford, UK, pp 291–300 Miller GL (1959) Use of dinitrosalicyclic acid reagent for determination of reducing sugars. Anal Chem 31:426–428 Miyazaki K (2005) Hyperthermophilic α-L-arabinofuranosidase from Thermotoga maritima MSB8: molecular cloning, gene expression, and characterization of the recombinant protein. Extremophiles 9:399–406 Nazina TN, Tourova TP, Poltaraus A.B, Novikova EV, Grigoryan AA, Ivanova AE, Lysenko A.M, Petrunyaka VV, Osipov GA, Belyaev SS, Ivanov MV (2001) Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermoglucosidasius and Bacillus thermodeni-

Appl Microbiol Biotechnol (2007) 75:813–820 trificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int J Syst Evol Microbiol 51:433–446 Nielsen H, Engelbrecht J, Brunak S, von Heijne G (1997) Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10:1–6 Poutanen K (1988) An α-L-arabinofuranosidase of Trichoderma reesei. J Biotechnol 7:271–282 Saha BC (2000) α-L-arabinofuranosidase: biochemistry, molecular biology and application in biotechnology. Biotechnol Adv 18:403–423 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY Seri K, Sanai K, Matsuo N, Kawakubo K, Xue C, Inoue S (1996) Arabinose selectively inhibits intestinal sucrase in uncompetitive manner and reduces glycemic response after sucrose ingestion in animals. Metabolism 45:1368–1374 Spagna G, Romagnoli D, Angela M, Bianchi G, Pifferi PG (1998) A simple method for purifying glycosidases: α-L-arabinofuranosidase and β-D-glucopyranosidase from Aspergillus niger to increase the aroma of wine. Enzyme Microb Technol 22:298–304 Suurnakki A, Tenkanen M, Buchert J, Viikari L (1997) Hemicellulases in the bleaching of chemical pulps. Adv Biochem Eng Biotechnol 57:261–287 Ward OP, Moo-Young M (1989) Degradation of cell wall and related plant polysaccharides. Crit Rev Biotechnol 8:237–274 Whitehead TR, Cotta MA (2001) Identification of a broad-specificity xylosidase/arabinosidase important for xylooligosaccharide fermentation by the ruminal anaerobe Selenomonas ruminantium GA192. Curr Microbiol 43:293–298 Wong KKY, Sanddler JN (1993) In: Coughlan MP, Hazlewood GP (eds) Hemicellulose and Hemic°ellulases. Portland Press, London, pp 127–143 Wyman CE (1994) Ethanol from lignocellulosic biomas: technology, economics, and opportunities. Bioresour Technol 50:3–16