Hydrolytic properties of a thermostable ... - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Hydrolytic properties of a thermostable a-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus Y.-R. Lim, R.-Y. Yoon, E.-S. Seo, Y.-S. Kim, C.-S. Park and D.-K. Oh Department of Bioscience and Biotechnology, Konkuk University, Seoul, Korea

Abstract

Keywords a-L-arabinofuranosidase, arabinooligosaccharides, Caldicellulosiruptor saccharolyticus, hydrolytic properties, thermostable enzyme. Correspondence Deok-Kun Oh, Department of Bioscience and Biotechnology, Konkuk University, 1 Hwayang-Dong Gwangjin-Gu, Seoul 143-701, Korea. E-mail: [email protected]

2009 ⁄ 1852: received 22 October 2009, revised 19 March 2010 and accepted 30 March 2010 doi:10.1111/j.1365-2672.2010.04744.x

Aims: To characterize of a thermostable recombinant a-l-arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabinooligosaccharides to l-arabinose. Methods and Results: A recombinant a-l-arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi-Trap anion exchange chromatography with a specific activity of 28Æ2 U mg)1. The native enzyme was a 58-kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of a-l-arabinofuranosidases were completely conserved in a-l-arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5Æ5 and 80C with a half-life of 49 h at 75C. Among aryl-glycoside substrates, the enzyme displayed activity only for p-nitrophenyla-l-arabinofuranoside [maximum kcat ⁄ Km of 220 m(mol l)1))1 s)1] and p-nitrophenyl-a-l-arabinopyranoside. This substrate specificity differs from those of other a-l-arabinofuranosidases. In a 1 mmol l)1 solution of each sugar, arabino-oligosaccharides with 2–5 monomer units were completely hydrolysed to l-arabinose within 13 h in the presence of 30 U ml)1 of enzyme at 75C. Conclusions: The novel substrate specificity and hydrolytic properties for arabino-oligosaccharides of a-l-arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of l-arabinose in concert with endoarabinanase and ⁄ or xylanase. Significance and Impact of the Study: The findings of this work contribute to the knowledge of hydrolytic properties for arabino-oligosaccharides performed by thermostable a-l-arabinofuranosidase.

Introduction Hemicellulose is one of the most abundant biomass materials on Earth. It has recently attracted much attention as a renewable alternative energy resource. However, to use hemicellulose as energy resource, it must be hydrolysed to monosaccharides by the treatment of various glycosidic hydrolases (GHs). Among these, a-l-arabinofuranosidases (EC 3.2.1.55) in concert with endoarabinanases and ⁄ or xylanases, which hydrolyse xylan, are required for the complete degradation of hemicellulose polysaccharides 1188

such as arabinan, arabinoxylan and arabinogalactan (Brice and Morrison 1982). a-l-Arabinofuranosidases hydrolyse the nonreducing termini of the a-l-arabinofuranosyl residues as side chains of arabinoxylan, arabinan and arabinogalactan (Kaji 1984; Taylor et al. 2006). According to the carbohydrate-active enzyme server CAZY (http://www.cazy.org/ Glycoside-Hydrolases.html), these enzymes belong to GH families 3, 10, 43, 51, 54 and 64. a-l-Arabinofuranosidases have been reported in various micro-organisms, including the bacteria Anoxybacillus kestanbolensis

ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 1188–1197

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(Canakci et al. 2008), Bacillus subtilis (Inacio et al. 2008), Bacillus pumilus (Pei and Shao 2008), Bifidobacterium longum (Gueimonde et al. 2007), Clostridium thermocellum (Taylor et al. 2006), Clostridium stercorarium (Schwarz et al. 1995), Geobacillus caldoxylolyticus (Canakci et al. 2007), Geobacillus stearothermophilus (Hovel et al. 2003a), Thermobacillus xylanilyticus (Debeche et al. 2002) and Thermomicrobia sp. (Birgisson et al. 2004); the archae Rhodothermus marinus (Gomes et al. 2000), Sulfolobus solfataricus (Morana et al. 2007) and Thermotoga maritima (Miyazaki 2005); the actinomyces Streptomyces avermitilis (Ichinose et al. 2008) and Streptomyces thermoviolaceus (Tsujibo et al. 2002); the yeasts Aureobasidium pullulans (de Wet et al. 2008) and Pichia capsulata (Yanai and Sato 2000); and the fungi Aspergillus awamori (Wood and McCrae 1996), Aspergillus kawachii (Koseki et al. 2006), Aspergillus nidulans (FernandezEspinar et al. 1994), Aspergillus niger (Crous et al. 1996), Aspergillus oryzae (Hashimoto and Nakata 2003), Aspergillus sojae (Kimura et al. 2000), Penicillum purpurogenum (De Ioannes et al. 2000), Rhizomucor pusillus (Rahman et al. 2003), Talaromyces thermophilus (Guerfali et al. 2009), Trichoderma reesei (Kaneko et al. 1998) and Trichoderma koningii (Wan et al. 2007). The enzymes work synergistically with endo-1,5-a-l-arabinanases for converting polysaccharides or oligosaccharides containing l-arabinose residues to l-arabinose, which is used commercially as a low-calorie sweetener. l-Arabinose inhibits sucrase and prevents the elevation of blood glucose levels induced by sucrose intake (Seri et al. 1996; Osaki et al. 2001). As a result, it can potentially inhibit obesity and prevent or treat diseases associated with hyperglycaemia. Moreover, this enzyme is one of the so-called accessory enzymes, which display a key role in the release of simple sugars (glucose, xylose, mannose, arabinose and others) from lignocellulosic biomass. These simple sugars may be used for the production of biofuels, as bioethanol, and building blocks for the chemical industry (biorefinery). In general, the enzymes originating from thermophilic micro organisms are more active and stable at higher temperatures than those obtained from mesophilic microorganisms. In addition, thermostable enzymes have many advantageous characteristics in industrial applications, such as high reaction velocities, resistance to chemical denaturation, reduced risk of contamination and high substrate solubility (Beguin and Aubert 1994). Therefore, thermostable a-l-arabinofuranosidases have considerable industrial potential. In this study, the gene that encodes putative a-l-arabinofuranosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The biochemical properties of the recombinant enzyme, such as optimum pH and temperature,

a-L-Arabinofuranosidase from C. saccharolyticus

thermostability and substrate specificity, were investigated. The ability of the recombinant enzyme to hydrolyse arabino-oligosaccharides to l-arabinose was assessed. Materials and methods Bacterial strains, plasmid and culture conditions Caldicellulosiruptor saccharolyticus DSM 8903 (DSMZ, Brauschweig, Germany), E. coli ER2566 (New England Biolabs, Herfordshire, UK) and plasmid pTrc99A (GE Healthcare Bioscience AB, Uppsala, Sweden) were used as the source of a-l-arabinofuranosidase gene, host cells and expression vector, respectively. Caldicellulosiruptor saccharolyticus was cultivated in ‘Caldicellulosiruptor’ medium (DSM Media Formulation No. 640) and grown at 70C under anaerobic conditions with 100% N2 gas in a 3-l anaerobic jar (Difco, Sparks, MD, USA) for 5 days. The recombinant E. coli for protein expression of the enzyme was cultivated in a 2-l flask containing 500 ml of LB medium and 50 lg ml)1 of ampicillin at 37C with agitation at 200 rev min)1. When the optical density of bacteria reached 0Æ5 at 600 nm, isopropyl-b-d-thiogalactopyranoside (IPTG) was added to a final concentration of 0Æ1 mmol l)1 to induce a-l-arabinofuranosidase expression, and then the culture was incubated with shaking at 200 rev min)1 at 37C for 4 h. Cloning and gene expression The genomic DNA from C. saccharolyticus was extracted using the genomic DNA extraction kit (Qiagen, Hilden, Germany). The gene (1518 bp) encoding putative a-l-arabinofuranosidase was amplified from the genomic DNA as a template by polymerase chain reaction (PCR) using Pfu DNA polymerase (Solgent, Daejeon, Korea). The sequence of the oligonucleotide primers used for gene cloning was based on the DNA sequence of putative a-l-arabinofuranosidase gene from C. saccharolyticus (GenBank accession number, CP000679). Forward (5¢-TTTGGATCCATGAAAAAAGCAAAAGTCATC TA-3¢) and reverse primers (5¢-TTTCTGCAGTTAATTT TCTCTCTTCTTCAATCTG-3¢) were designed to introduce the BamHI and PstI (New England Biolabs) restriction sites (underlined). The amplified DNA fragment obtained by PCR was purified and digested with both BamHI and PstI restriction endonucleases. The digested DNA fragment was extracted from gel using the QIA quick gel extraction kit (Qiagen) and then inserted into the pTrc99A vector digested with the same restriction enzymes. E. coli ER2566 strain was transformed with the ligation mixture and plated on LB agar containing 50 lg ml)1 of ampicillin. Ampicillin-resistant colonies


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were selected, and plasmid DNA from the transformants was isolated using a plasmid purification kit (Promega, Madison, WI). DNA sequencing was performed at the Macrogen facility (Seoul, Korea). The expression of a-l-arabinofuranosidase gene was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and by enzyme activity assay. Enzyme purification Cultured E. coli cells were harvested and disrupted by sonication using Sonic Dismembrator (Fisher Scientific Model 100, Pittsburgh, PA, USA) at 18 watts on ice for 2 min in 50 mmol l)1 Tris–HCl buffer (pH 7Æ0) containing 0Æ1 mmol l)1 phenylmethylsulfonyl fluoride as protease inhibitor. The unbroken cells and cell debris were removed by centrifugation at 13 000 g for 20 min at 4C, and the supernatant obtained was used as a crude extract. The crude extract was heated at 75C for 10 min to remove denatured E. coli proteins, and the suspension was centrifuged at 13 000 g for 20 min. The enzyme solution of the supernatant was applied onto a Hi-Trap Q HP column (Amersham Biosciences, Uppsala, Sweden) equilibrated with Tris-HCl buffer (pH 7Æ0). The column was eluted with 50 mmol l)1 Tris-HCl buffer (pH 7Æ0) with a linear gradient of water and 1 mol l)1 NaCl. Each fraction was analysed in SDSPAGE, and the fractions containing a-l-arabinofuranosidase were collected. The active fractions were dialysed at 4C for 16 h against 50 mmol l)1 citric acid buffer (pH 5Æ5). The resulting solution was used as a purified enzyme. The purification step using the column was carried out by a fast protein liquid chromatography (FPLC) system (Bio-Rad, Hercules, CA, USA) in a cold room at 4C. Enzyme assay One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 lmol of pNP per min at 80C and pH 5Æ5. Unless otherwise stated, the reaction was performed as follows: 1 ml of enzyme solution with a concentration of 0Æ01 mg ml)1 in 50 mmol l)1 citric acid buffer (pH 5Æ5) was mixed with 1 ml of substrate solution with a concentration of 2 mmol l)1 p-nitrophenyla-l-arabinofuranoside (pNP-Araf) in 50 mmol l)1 citric acid buffer (pH 5Æ5), and then the mixture was used as the reaction solution. The enzyme reaction was performed at 80C for 10 min with the reaction solution, and the activity was determined by the release of p-nitrophenol (pNP). The absorbance at 415 nm was measured after quenching the reactions by adding 0Æ2 mol l)1 Na2CO3 (Margolles and de los Reyes-Gavilan 2003). 1190

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Molecular mass determination The subunit molecular mass of a-l-arabinofuranosidase was examined by SDS-PAGE, using the proteins of a prestained ladder (MBI Fermentas, Hanover, MD, USA) as reference proteins. Twenty microlitres of enzyme solution with a concentration of 0Æ5 mg ml)1 was loaded on SDS-PAGE gel after boiling for 5 min. Electrophoresis was performed at 150 V using mini polyacrylamide gel system (Bio-Rad). All protein bands were stained with Coomassie blue for visualization. The molecular mass of native enzyme was determined by gel filtration chromatography using a Sephacryl S-300 HR 16 ⁄ 60 preparativegrade column (Amersham Biosciences). The enzyme solution was applied to the column and eluted with 50 mmol l)1 citric acid buffer (pH 5Æ5) containing 150 mmol l)1 NaCl at a flow rate of 1 ml min)1. The column was calibrated with thyroglobulin (669 kDa), apoferritin (443 kDa), albumin (66 kDa) and ovalbumin (43 kDa) as reference proteins (Amersham Biosciences), and the molecular mass of the native enzyme was calculated by comparing the migration length of the reference proteins. Effects of metal ions, pH and temperature To investigate the effect of metal ions on a-l-arabinofuranosidase activity, enzyme assays were carried out after treatment with ethylenediaminetetraacetic acid (EDTA) at 4C for 1 h or after addition of each metal ion such as Ba2+, Co2+, Mn2+, Mg2+, Ca2+, Zn2+, Cu2+ or Fe2+. The reactions were performed in 50 mmol l)1 citric acid buffer (pH 5Æ5) containing 1 mmol l)1 of each metal ion at 80C. To examine the effect of pH on a-l-arabinofuranosidase activity, pH was varied from 4Æ5 to 7Æ5 using 50 mmol l)1 sodium acetate buffer (pH 4Æ5)5Æ5), 50 mmol l)1 maleic acid buffer (pH 5Æ5)6Æ5) and 50 mmol l)1 piperazineN,N¢-bis(2-ethanesulfonic acid) (PIPES) buffer (pH 6Æ5)7Æ5) at 80C. The effect of temperature on the enzyme activity was evaluated in 50 mmol l)1 citric acid buffer (pH 5Æ5) at temperatures ranging from 60 to 90C. The influence of temperature on enzyme stability was monitored as a function of incubation time by placing the enzyme solution at four different temperatures (65, 70, 75 and 80C) in 50 mmol l)1 citric acid buffer (pH 5Æ5). The incubation time was 132 h for 65 and 70C, 83 h for 75C and 1 h for 80C. The samples were withdrawn and were assayed in 2 ml of reaction volume with 50 mmol l)1 citric acid buffer (pH 5Æ5) at 80C for 10 min. The best-fitting line expressing the linear decline in logarithmic relative activity with incubation time at each temperature was obtained by the least square



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method program (Sigma Plot 10.0 software; Systat Software, San Jose, CA, USA). The half-life of the enzyme was the period of time it took for the relative activity on the best-fitting line to decrease by half. Hydrolytic reactions of various substrates The hydrolytic reactions for aryl-glycosides including p-nitrophenyl-a-l-arabinofuranoside (pNP-Araf), pNP-a-larabinopyranoside (pNP-Arap), pNP-a-d-galactopyranoside, pNP-a-d-glucopyranoside, pNP-b-d-fucopyranoside, pNP-b-d-galactopyranoside, pNP-b-d-glucopyranoside, pNP-b-d-lactopyranoside, pNP-b-d-maltopyranoside, pNPb-d-xylopyranoside, pNP-b-l-arabinopyranoside, o-nitrophenyl-b-d-fucopyranoside, oNP-b-d-galactopyranoside and oNP-b-d-glucopyranoside were performed in 50 mmol l)1 citric acid buffer (pH 5Æ5) containing 1 mmol l)1 arylglycoside and 0Æ1 U ml)1 of enzyme at 80C for 10 min, and the activity was determined by release of pNP or oNP. The hydrolytic reactions for arabino-oligosaccharides, such as arabinobiose, arabinotriose, arabinotetraose and arabinopentaose, and polysaccharides, such as starch, arabinan, xylan, debranched arabinan and wheat arabinoxylan, were performed in 50 mmol l)1 citric acid buffer (pH 5Æ5) containing 1 mmol l)1 arabino-oligosaccharide or 1% (w ⁄ v) polysaccharide and 10 U ml)1 of enzyme at 80C for 30 min, and the activity was determined by the released amount of l-arabinose. The time courses of the hydrolytic reaction for arabino-oligosaccharides were performed with 30 U ml)1 of enzyme at 75C for 13 h. The reactions were stopped by adding HCl with a final concentration of 0Æ2 mol l)1. Determination of kinetic parameters Various concentrations (from 0Æ5 to 3 mmol l)1) of pNPAraf and pNP-Arap were used to determine kinetic parameters of the enzyme. The enzyme kinetic parameters, Km (mmol l)1) and kcat (s)1), were calculated from the Lineweaver–Burk plot of Michaelis–Menten equation. Analytical methods The concentrations of l-arabinose (Sigma-Aldrich, St Louis, MO, USA) and arabino-oligosaccharides from n = 2 to 5 (Megazyme, Wicklow, Ireland) were determined by a Bio-LC system (Dionex ICS-3000, Sunnylvale, CA, USA) with an electrochemical detector using a CarboPac PA1 column (4 · 250 mm; Dionex) and elution with 100 mmol l)1 NaOH (0–5 min), followed by a linear gradient (5–35 min) of sodium acetate (0–200 mmol l)1) at a flow rate of 1 ml min)1 at 30C (Ichinose et al. 2008).

Table 1 Purification of Caldicellulosiruptor saccharolyticus a-L-arabinofuranosidase

Step

Total protein (mg)

Total activity (U)

Specific activity (U mg)1)

Yield (%)

Purification (fold)

Crude extract Heat treatment Hi-Trap Q HP

1094 63 5

1203 951 141

1Æ1 15Æ1 28Æ2

100 79 12

1Æ0 13Æ7 25Æ6

kDa

1

2

3

4

170 130 95 72 58 kDa

55 43

34

26

17 Figure 1 SDS-PAGE analysis of each purification step. Lane 1 marker proteins; lane 2 crude extract; lane 3 supernatant after heat treatment at 75C for 10 min; lane 4 Hi-Trap Q HP chromatography column product (purified enzyme).

Table 2 Effect of metal ions on the activity for of thermostable a-L-arabinofuranosidase Metal ions

Relative activity (%)

None EDTA MnCl2 MgSO4 CaCl2 BaCl2 CuCl2 FeSO4 ZnSO4 CoSO4

100 90 97 90 92 102 79 83 94 90

± ± ± ± ± ± ± ± ± ±

2Æ1 1Æ6 0Æ1 1Æ9 2Æ2 1Æ9 0Æ3 1Æ8 2Æ4 3Æ4

Data were obtained from three separate experiments.


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100

(a) 120

80 Relative activity (%)


100 80 60 40

60

40

20 20 0

4

5

(b)

6 pH

7

8


100 80 60 40 20

60

65

70 75 80 Temperature (°C)

85

90

95

Figure 2 Effects of pH and temperature on the activity of Caldicellulosiruptor saccharolyticus a-L-arabinofuranosidase. (a) pH effect: The reactions were performed in 50 mmol l)1 sodium acetate buffer (d, pH 4Æ5–5Æ5), 50 mmol l)1 maleic acid buffer (s, pH 5Æ5–6Æ5) or 50 mmol l)1 PIPES buffer ( , pH 6Æ5–7Æ5) containing 1 mmol l)1 pNPa-L-arabinofuranoside (pNP-Araf) and 0Æ1 U ml)1 of enzyme at 80C for 10 min. (b) Temperature effect: the reactions were performed in 50 mmol l)1 citric acid buffer (pH 5Æ5) containing 1 mmol l)1 pNPAraf and 0Æ1 U ml)1 of enzyme for 10 min. Data represent the means of three separate experiments, and error bars represent standard deviation. PIPES, piperazine-N,N¢-bis(2-ethanesulfonic acid).

Results Cloning, purification and molecular mass of a-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus A gene encoding a putative a-l-arabinofuranosidase from C. saccharolyticus, with the same sequence as the gene reported in GenBank (accession number CP000679), was cloned and expressed in E. coli. The recombinant enzyme was purified as a soluble protein from crude extract via 1192

20

40

60 80 Time (h)

100

120

140

Figure 3 Thermal inactivation of Caldicellulosiruptor saccharolyticus a-L-arabinofuranosidase. The enzymes were incubated at 65 (d), 70 (h), 75 ( ) and 80C (s) for varying periods of time. A sample was withdrawn at each time interval, and the relative activity was determined. Data represent the means of three experiments, and error bars represent standard deviation.

120

0 55

0

heat treatment and Hi-Trap Q HP anion exchange chromatography with a purification factor of 25Æ6-fold, a yield of 11% and a specific activity of 28Æ2 U mg)1 (Table 1). The purified a-l-arabinofuranosidase from C. saccharolyticus showed a single band, as detected by SDS-PAGE, with a molecular mass of approx. 58 kDa (Fig. 1), which is consistent with the calculated value of 57 882 Da determined with the Compute pI ⁄ Mw tool using the 505 amino acid residues. The native enzyme existed as an octamer with a molecular mass of 460 kDa, as determined by Sephacryl S-300 gel filtration chromatography (data not shown). Effects of metal ions, pH and temperature on the activity of a-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus The addition of monovalent or divalent cations did not significantly activate or inhibit the activity of a-l-arabinofuranosidase from C. saccharolyticus, resulting in 79–103% activity relative to that without added metal ions (Table 2). The enzyme has no metal dependence as 90% of the enzyme activity remained after the removal of metal ions by treatment with EDTA. The enzyme activity was examined over a pH range of 4Æ5–7Æ5 at 80C (Fig. 2a). Maximum activity was observed at pH 5Æ5. The effect of temperature on enzyme activity was also investigated, and maximum activity was recorded at 80C (Fig. 2b). The thermostability was examined by measuring the activity over time (Fig. 3). Recombinant a-l-arabinofuranosidase from C. saccharolyticus displayed first-order kinetics for thermal inactivation, and the half-lives of the


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0·8

4

0·6

3

0·4

2

0·2

1

0·0 4

Arabinobiose, Arabinotriose, Arabinotetraose (mmol l–1)

(c)

6 8 Time (h)

10

12

1·0

5

0·8

4

0·6

3

0·4

2

0·2

1

2

4

6 8 Time (h)

1·0

5

0·8

4

0·6

3

0·4

2

0·2

1

0·0 0

2

4

(d) 6

0

6

0 14

1·2

0·0

1·2

10

12

0 14

Arabinobiose, Arabinotriose, Arabinotetraose, Arabinopentaose (mmol l–1)

2

(mmol l–1)

0

(b)

6 8 Time (h)

10

12

(mmol l–1)

5

The hydrolytic reactions of a-l-arabinofuranosidase from C. saccharolyticus on arabino-oligosaccharides, such as arabinobiose, arabinotriose, arabinotetraose and arabinopentaose, were investigated (Fig. 4). Hydrolysis of

L-Arabinose

1·0

Hydrolysis of arabino-oligosaccharides to L-arabinose using a-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus

0 14

1·2

6

1·0

5

0·8

4

0·6

3

0·4

2

0·2

1

0·0 0

2

4

6 8 Time (h)

10

12

(mmol l–1)

6

L-Arabinose

1·2

L-Arabinose

Arabinobiose (mmol l–1)

(a)

(mmol l–1)

a-l-Arabinofuranosidase from C. saccharolyticus exhibited hydrolytic activity for pNP-Araf, pNP-Arap and arabinooligosaccharides with 2–5 monomer units, whereas there was no activity by the analytical methods used in this study for pNP-a-d-galactopyranoside, pNP-a-d-glucopyranoside, pNP-b-d-fucopyranoside, pNP-b-d-galactopyranoside, pNP-b-d-glucopyranoside, pNP-b-d-lactopyranoside, pNP-b-d-maltopyranoside, pNP-b-d-xylopyranoside, pNP-b-l-arabinopyranoside, o-nitrophenyl-b-d-fucopyranoside, oNP-b-d-galactopyranoside, oNP-b-d-glucopyranoside, starch, arabinan, xylan, debranched arabinan or wheat arabinoxylan. The specific hydrolytic activity of the enzyme for arabino-oligosaccharides as a substrate followed the order arabinotetraose (1Æ75 U mg)1) > arabinopen-

L-Arabinose

Substrate specificity of a-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus

taose (0Æ76 U mg)1) > arabinotriose (0Æ75 U mg)1) > arabinobiose (0Æ5 U mg)1). The Michaelis–Menten constants (Km), turnover numbers (kcat) and catalytic efficiencies (kcat ⁄ Km) of the enzyme were 1Æ29 mmol l)1, 285 s)1 and 221 m (mol l)1))1 s)1 for pNP-Araf and 1Æ10 mmol l)1, 226 s)1 and 205 m(mol l)1))1 s)1 for pNP-Arap, respectively. The kcat ⁄ Km value for pNP-Araf was 1Æ08-fold compared to the kcat ⁄ Km for pNP-Arap. These results indicate that the enzyme is an a-l-arabinofuranosidase that exhibits narrow substrate specificity.

Arabinobiose, Arabinotriose (mmol l–1)

enzyme at 65, 70, 75 and 80C were 1700, 390, 49 and 0Æ4 h, respectively.

0 14

Figure 4 Hydrolysis for arabino-oligosaccharides from n = 2 to 5 of Caldicellulosiruptor saccharolyticus a-L-arabinofuranosidase. (a) Arabinobiose. (b) Arabinotriose. (c) Arabinotetraose. (d) Arabinopentaose. The used concentrations of arabino-oligosaccharides from n = 2 to 5 were 1 mmol l)1. Symbols: L-arabinose (d), arabinobiose (s), arabinotriose ( ), arabinotetraose (h) and arabinopentaose ( ). ª 2010 The Authors Journal compilation ª 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 1188–1197

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Figure 5 Alignment of the amino acid sequences of Caldicellulosiruptor saccharolyticus a-L-arabinofuranosidase with GH family 51 a-L-arabinofuranosidases. CCAraf, C. saccharolyticus a-L-arabinofuranosidase (GenBank accession number, CP000679); GSAraf, Geobacillus caldoxylosilyticus a-L-arabinofuranosidase (DQ883641); BSAraf, Bacillus stearothermophilus a-L-arabinofuranosidase (AAD45520); AKAraf, Anoxybacillus kestanbolensis a-L-arabinofuranosidase (EU563946); and BLAraf, Bifidobacterium longum a-L-arabinofuranosidase (AAO84266). Asterisks mean identity. The catalytic residues (E173 and E292) and consensus sequences of GH family 51 are highlighted with black and grey backgrounds, respectively. GH, glycosidic hydrolase.

arabinopentaose resulted initially in l-arabinose and arabinotetraose, with the latter being further hydrolysed to l-arabinose and arabinotriose. The arabinotriose was converted to l-arabinose and arabinobiose, and the latter was hydrolysed finally to l-arabinose. As a result, each arabino-oligosaccharide was completely hydrolysed to l-arabinose within 13 h. The complete hydrolysis for the substrate followed the order arabinotetraose > arabinotriose > arabinobiose > arabinopentaose. The enzyme converted 1 mmol l)1 concentration of arabinobiose, arabinotriose, arabinotetraose or arabinopentaose to approx. 2, 3, 4 or 5 mmol l)1 l-arabinose, respectively. Discussion The amino acid sequence of a-l-arabinofuranosidase from C. saccharolyticus exhibited 96, 70, 69 and 66% identities with a-l-arabinofuranosidases from Anaerocellum thermophilum (YP002572977), Dictyoglomus thermophilum (YP002251436), Dictyoglomus turgidum (YP002353621) and G. stearothermophilus (ACE73682), respectively. Although the crystal structure of G. stearothermophilus a-l-arabinofuranosidase is known (Hovel et al. 2003b), these enzymes have not been fully characterized. The consensus sequences with the GH family 51 of a-l-arabinofuranosidases consist of a PGG locus with ArgTyr-Pro-Gly-Gly (RYPGG), an acid–base catalytic locus with Ile-Gly-Glu-Asn (IGEN) and a nucleophilic catalytic 1194

locus with Asp-Glu-Trp (DEW) (Margolles and de los Reyes-Gavilan 2003). Two Glu residues may act as acid ⁄ base and nucleophilic catalytic residues and participate in the hydrolysis of glycosidic bonds (Shallom et al. 2002). The consensus sequences and two catalytic residues of the GH family 51 of a-l-arabinofuranosidases from Geobacillus caldoxylosilyticus (DQ883641), Bacillus stearothermophilus (AAD45520), A. kestanbolensis (EU563946) and Bif. longum (AAO84266) were completely conserved in a-l-arabinofuranosidase from C. saccharolyticus (Fig. 5). These results indicate that a-l-arabinofuranosidase from C. saccharolyticus belongs to GH family 51. The biochemical properties of thermostable a-l-arabinofuranosidases are summarized in Table 3. The activity of thermostable a-l-arabinofuranosidases is maximal at pH 5Æ0–7Æ0. The maximal activities of thermostable a-l-arabinofuranosidases from T. maritima (Miyazaki 2005), S. solfataricus (Morana et al. 2007), Cl. thermocellum (Taylor et al. 2006) and C. saccharolyticus have been observed at temperatures above 80C. The half-lives of thermostable a-l-arabinofuranosidases from T. maritima (Miyazaki 2005), S. solfataricus (Morana et al. 2007) and Th. xylanilyticus (Debeche et al. 2002) have been reported to be 2Æ7, 2 and 2 h at 90C, respectively. The kcat ⁄ Km of a-l-arabinofuranosidase from C. saccharolyticus for pNP-Araf was 4Æ2-fold higher than that from T. maritima and 1Æ9-fold lower than that from Cl. thermocellum (Miyazaki 2005; Taylor et al. 2006).



Y.-R. Lim et al.

Table 3 Biochemical properties of thermostable a-L-arabinofuranosidases

pH

Temperature (C)

Half-life (h)

Vmax (U mg)1)

Km (mmol l)1)

Bacillus pumilus Anoxybacillus kestanbolensis Bacillus stearothermophilus Clostridium stercorarium Thermomicrobia sp. Geobacillus caldoxylolyticus Thermobacillus xylanilyticus Caldicellulosiruptor saccharolyticus Sulfolobus solfataricus Clostridium thermocellum Thermotoga maritima

6Æ4 5Æ5 5Æ5 5Æ0 6Æ0 6Æ0 6Æ0 5Æ5 6Æ5 7Æ0 7Æ0

60 65 70 70 70 75 75 80 80 82 90

1Æ0 60 1Æ0 0Æ7 8Æ3 57 2Æ0 49 2Æ0

240 1019 749

1Æ05 0Æ14 0Æ42

122 588 555 186 286

0Æ60 0Æ17 0Æ50 1Æ29

285

220

0Æ25 0Æ41

103 22

412 52

(70C) (70C) (70C) (90C) (70C) (75C) (90C) (75C) (90C)

2Æ7 (90C)

Several of the a-l-arabinofuranosidases from Bif. longum (Margolles and de los Reyes-Gavilan 2003), Hordeum vulgare (Lee et al. 2003) and S. solfataricus (Morana et al. 2007) that exhibit broad substrate specificity were active for pNP-Arap. In contrast, many a-l-arabinofuranosidases with broad substrate specificity were not active for pNPArap. Only two a-l-arabinofuranosidases, those isolated from Bifidobacterium breve (Shin et al. 2003) and A. kestanbolensis (Canakci et al. 2008), both of which exhibit narrow substrate specificity, displayed activity only for pNP-Araf. Neither of these, however, showed any activity for pNP-Arap. a-l-Arabinofuranosidase from C. saccharolyticus exhibited narrow substrate specificity for not only pNP-Araf, which is representative of substrates for exo-type arabinan-hydrolysing enzymes, but also for pNP-Arap. No activity was observed by the analytical methods used in this study for the other aryl-glycosides, arabinan or debranched arabinan. Thus, as opposed to other a-l-arabinofuranosidases, a-l-arabinofuranosidase from C. saccharolyticus exhibited novel substrate specificity. The activity of a-l-arabinofuranosidase from C. saccharolyticus with both pNP-Araf and pNP-Arap would allow this enzyme to be used to cleave l-arabinose residues from polysaccharides containing side-chain l-arabinose, such as arabinan, arabinogalactan and arabinoxylan, in concert with endoarabinanase and ⁄ or xylanase. The hydrolytic ability of the enzyme is demonstrated in Fig. 4, where arabino-oligosaccharides with 2–5 monomer units were completely hydrolysed to l-arabinose. Thus, the enzyme could be applied in the production of l-arabinose because of its hydrolytic properties. Acknowledgements This study was supported by a grant (20090054) from Agricultural R&D Promotion Center, Korea Rural

kcat (s)1)

kcat ⁄ Km [m(mol l)1))1 s)1]

Bacteria

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