Screening of a Novel Glycoside Hydrolase Family 51 α-L-Arabinofuranosidase from Paenibacillus polymyxa KF1 : Cloning , Expression , and Characterization

Paenibacillus polymyxa exhibits remarkable hemicellulolytic activity. In the present study, 13 hemicellulose-degrading enzymes were identified from the secreted proteome of P. polymyxa KF-1 by liquid chromatography-tandem mass spectrometry analysis. α-L-arabinofuranosidase is an important member of hemicellulose-degrading enzymes. A novel α-L-arabinofuranosidase (PpAbf51b), belonging to glycoside hydrolase family 51, was identified from P. polymyxa. Recombinant PpAbf51b was produced in Escherichia coli BL21 (DE3) and was found to be a tetramer using gel filtration chromatography. PpAbf51b hydrolyzed neutral arabinose-containing polysaccharides, including sugar beet arabinan, linear-1,5-α-L-arabinan, and wheat arabinoxylan, with L-arabinose as the main product. The products from hydrolysis indicate that PpAbf51b functions as an exo-α-L-arabinofuranosidase. Combining PpAbf51b and Trichoderma longibrachiatum endo-1,4-xylanase produced significant synergistic effects for the degradation of wheat arabinoxylan. The α-L-arabinofuranosidase identified from the secretome of P. polymyxa KF-1 is potentially suitable for application in biotechnological industries.


Introduction
Lignocellulosic biomass is the most abundant renewable resource on earth, and lignocellulose shows potential for the production of biofuels and valuable chemicals [1].Lignocellulose is composed of lignin, cellulose and hemicellulose [2].The efficient conversion of lignocellulose to desirable target chemicals is challenging due to the complex structure of lignocellulosic materials [3,4].Therefore, developing efficient lignocellulosic degradation approaches has attracted the attention of researchers.Many microorganisms exhibiting lignocellulosic biodegradation activities have been reported, with most species being filamentous fungi [5].Research has shown that bacteria will play an increasingly important role in the bioconversion of lignocellulose due to their strong environmental adaptability and rich biochemical diversity [4].However, compared with the numerous studies on fungi, there are only a few reports describing the degradation of lignocelluloses by bacteria [4].Bacterial degradation of lignocellulose is not well understood, and lignocellulose-degrading enzymes from bacteria and the associated degradation pathways remain to be discovered.
Paenibacillus polymyxa is a plant-growth-promoting rhizobacterium [11,12].P. polymyxa was reported to use the major components of hemicellulose biomass and exhibited remarkable endoglucanase and α-L-arabinofuranosidase activities [13].Genome sequencing of P. polymyxa revealed the presence of a diverse range of putative hemicellulose-degrading enzymes, signifying the potential of the bacterium for hemicellulose hydrolysis [11,14].A glycoside hydrolase family 51 enzyme (PpAFase-1) from the fermentation broth of P. polymyxa was partially purified, identified, and overexpressed in Escherichia coli [13].PpAFase-1 showed a synergistic effect with xylanase in hemicellulose degradation, but still has some disadvantages such as insufficient synergistic efficiency, which pushed us to explore robust α-L-arabinofuranosidases.In the present study, the secreted hemicellulose-degrading enzymes of P. polymyxa were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS).An α-L-arabinofuranosidase belonging to the glycoside hydrolase (GH) family 51 was identified, heterologously expressed, and characterized, and its potential applications were explored.

Measurement of the Extracellular Hemicellulytic Activities
The extracellular hemicellulytic activity of P. polymyxa KF-1 cultured in M9 minimal medium with arabinoxylan as the sole carbon source (10 g/L) at 30 • C for 36 h was measured.As shown in Table 1, P. polymyxa KF-1 showed high α-L-arabinofuranosidase activity and low levels of β-mannosidase, α-galactosidase, and β-xylosidase activities (Table 1).The specific activities against pNPαAraf, pNPβMan, pNPαGal, and pNPβXyl were 12.66, 1.22, 1.98, and 1.45 U/mg, respectively.P. polymyxa KF-1 secreted xylanase with a specific activity of 2.68 U/mg by using oat spelt xylan as a substrate.The hydrolytic activity of the enzyme extract toward wheat arabinoxylan was estimated by high-performance anion exchange chromatography (HPAEC).As shown in Figure 1, arabinoxylan was hydrolyzed by the enzyme extract of P. polymyxa with monosaccharides and oligosaccharides observed as products.The monosaccharide eluted at ~3.1 min was identified as L-arabinose.This result was confirmed by the reducing sugar assay.After 24 h, the reducing sugar released from arabinoxylan was 40.1 ± 3.7 mg/g.observed as products.The monosaccharide eluted at ~3.1 min was identified as L-arabinose.This result was confirmed by the reducing sugar assay.After 24 h, the reducing sugar released from arabinoxylan was 40.1 ± 3.7 mg/g.Figure 1.High-performance anion exchange chromatography (HPAEC) analysis of the hydrolysis of arabinoxylan by Paenibacillus polymyxa enzyme extract.Reaction mixture containing 0.1 mg/mL enzyme extract and 5 mg/mL arabinoxylan was incubated at 37 °C for 24 h.The reaction was terminated by boiling the sample for 5 min, and the product was detected by HPAEC using a CarboPac PA-200 analytical column (3 × 250 mm).The black dotted line represents the substrate (wheat arabinoxylan) and the blue line represents the transformed product.

Expression and Purification of PpAbf51b
The recombinant protein was purified by Ni-NTA column chromatography, and 15.3 mg of purified protein was obtained from a 200-mL culture.A specific activity of 57.5 U/mg was obtained with pNPαAraf (1 mM) as a substrate.A single protein band with an estimated molecular weight (Mw) of 56.4 kDa was observed by SDS-PAGE analysis, which is consistent with the calculated Mr (Figure 4a).The native Mr was determined by gel filtration chromatography performed on Superdex 200 10/300 GL column (Figure 4b).The native Mr was calculated to be approximately 229.5 kDa, which indicated PpAbf51b existed as a tetramer.

Characterization of Purified PpAbf51b
PpAbf51b was incubated at different pH values (2.0-11.0)and showed the highest activity at pH 6.5 (Figure 5a).PpAbf51b was quite stable over the pH range of 4.0 to 11.0.More than 75% activity was retained at pH 4.0 and 11.0 after preincubation of the enzyme for 1 hour at 40 °C (Figure 5c).The optimal temperature was determined to be 40 °C.Enzymatic activity was near maximal over a relatively wide temperature range of 30 to 70 °C (Figure 5b).The enzyme was stable at temperatures

Expression and Purification of PpAbf51b
The recombinant protein was purified by Ni-NTA column chromatography, and 15.3 mg of purified protein was obtained from a 200-mL culture.A specific activity of 57.5 U/mg was obtained with pNPαAraf (1 mM) as a substrate.A single protein band with an estimated molecular weight (Mw) of 56.4 kDa was observed by SDS-PAGE analysis, which is consistent with the calculated Mr (Figure 4a).The native Mr was determined by gel filtration chromatography performed on Superdex 200 10/300 GL column (Figure 4b).The native Mr was calculated to be approximately 229.5 kDa, which indicated PpAbf51b existed as a tetramer.

Expression and Purification of PpAbf51b
The recombinant protein was purified by Ni-NTA column chromatography, and 15.3 mg of purified protein was obtained from a 200-mL culture.A specific activity of 57.5 U/mg was obtained with pNPαAraf (1 mM) as a substrate.A single protein band with an estimated molecular weight (Mw) of 56.4 kDa was observed by SDS-PAGE analysis, which is consistent with the calculated Mr (Figure 4a).The native Mr was determined by gel filtration chromatography performed on Superdex 200 10/300 GL column (Figure 4b).The native Mr was calculated to be approximately 229.5 kDa, which indicated PpAbf51b existed as a tetramer.

Characterization of Purified PpAbf51b
PpAbf51b was incubated at different pH values (2.0-11.0)and showed the highest activity at pH 6.5 (Figure 5a).PpAbf51b was quite stable over the pH range of 4.0 to 11.0.More than 75% activity was retained at pH 4.0 and 11.0 after preincubation of the enzyme for 1 hour at 40 °C (Figure 5c).The optimal temperature was determined to be 40 °C.Enzymatic activity was near maximal over a relatively wide temperature range of 30 to 70 °C (Figure 5b).The enzyme was stable at temperatures

Characterization of Purified PpAbf51b
PpAbf51b was incubated at different pH values (2.0-11.0)and showed the highest activity at pH 6.5 (Figure 5a).PpAbf51b was quite stable over the pH range of 4.0 to 11.0.More than 75% activity was retained at pH 4.0 and 11.0 after preincubation of the enzyme for 1 hour at 40 • C (Figure 5c).The optimal temperature was determined to be 40 • C. Enzymatic activity was near maximal over a relatively wide temperature range of 30 to 70 • C (Figure 5b).The enzyme was stable at temperatures lower than 60 • C. At 60 • C, the activity was still greater than 70% after 1 hour's incubation (Figure 5d).In subsequent studies, the pH and temperature for hydrolyzing arabinose-containing polysaccharides by PpAbf51b were chosen to be pH 6.5 and 40 lower than 60 °C.At 60 °C, the activity was still greater than 70% after 1 hour's incubation (Figure 5d).In subsequent studies, the pH and temperature for hydrolyzing arabinose-containing polysaccharides by PpAbf51b were chosen to be pH 6.5 and 40 °C, respectively.The effect of metal ions and chemicals on the activity of PpAbf51b was examined.FeCl2, at a concentration of 10 mM, enhanced PpAbf51b activity by more than 30%.CuCl2 and Triton X-100 showed slightly positive effects, leading to an activity increase of more than 10%.AlCl3 inhibited the activity of PpAbf51b with 72.4% of activity retained.For other tested ions and chemicals, inhibitory effects of around 10-20% were detected.The chemicals DTT, SDS, and Tween 40/60/80 did not significantly affect the activity of PpAbf51b (Table 3).The effect of metal ions and chemicals on the activity of PpAbf51b was examined.FeCl 2 , at a concentration of 10 mM, enhanced PpAbf51b activity by more than 30%.CuCl 2 and Triton X-100 showed slightly positive effects, leading to an activity increase of more than 10%.AlCl 3 inhibited the activity of PpAbf51b with 72.4% of activity retained.For other tested ions and chemicals, inhibitory effects of around 10-20% were detected.The chemicals DTT, SDS, and Tween 40/60/80 did not significantly affect the activity of PpAbf51b (Table 3).
The effect of monosaccharides on the activity of PpAbf51b was determined.The residual activity of PpAbf51b was 85.4% and 78.8% when incubated with 100 mM L-arabinose or D-xylose, respectively.At a concentration of 500 mM, L-arabinose and D-xylose partially inhibited enzyme activity, with inhibition percentages of 26.1% and 36.9%,respectively (Figure 6).111.0 ± 3.2 1 The activity was determined with pNPAraf (1 mM) as a substrate.
The effect of monosaccharides on the activity of PpAbf51b was determined.The residual activity of PpAbf51b was 85.4% and 78.8% when incubated with 100 mM L-arabinose or D-xylose, respectively.At a concentration of 500 mM, L-arabinose and D-xylose partially inhibited enzyme activity, with inhibition percentages of 26.1% and 36.9%,respectively (Figure 6).

Substrate Specificity of PpAbf51b
Activities of PpAbf51b on 10 p-nitrophenyl-linked glycosides were determined.PpAbf51b exhibited high activity toward pNPαAraf and relative low activity toward pNPαGal.No activity was observed on other pNP glycosides (Table 4).These results demonstrate that PpAbf51b is an α-Larabinofuranosidase.Using pNPαAraf as a substrate, the kinetic values Km, Vmax, and kcat were determined to be 0.2 mM, 58.1 μmol/min/mg, and 54.7 s −1 , respectively.The catalytic efficiency kcat/Km was calculated to be 273.3s −1 mM −1 .

Substrate Specificity of PpAbf51b
Activities of PpAbf51b on 10 p-nitrophenyl-linked glycosides were determined.PpAbf51b exhibited high activity toward pNPαAraf and relative low activity toward pNPαGal.No activity was observed on other pNP glycosides (Table 4).These results demonstrate that PpAbf51b is an α-L-arabinofuranosidase.Using pNPαAraf as a substrate, the kinetic values K m , V max , and k cat were determined to be 0.2 mM, 58.1 µmol/min/mg, and 54.7 s −1 , respectively.The catalytic efficiency k cat /K m was calculated to be 273.3s −1 mM −1 .
The hydrolytic activity toward various arabinose-containing polysaccharides was examined.As shown in Figure 7, PpAbf51b was active against sugar beet arabinan, with L-arabinose detected by HPAEC after 16 h incubation.Similar enzyme activity was observed on linear-1,5-α-arabinan and wheat arabinoxylan.The degradation of sugar beet arabinan generated the highest amount of arabinose, followed by arabinoxylan and linear-1,5-α-arabinan.PpAbf51b showed no activity toward arabinogalactan or xylan.

Synergistic Hydrolysis of Wheat Arabinoxylan by PpAbf51b and Tl-Xyn
The complete degradation of arabinoxylan requires the synergistic catalysis of endoxylanase and arabinofuranosidase.The synergistic effect of Trichoderma longibrachiatum endo-1,4-xylanase (Tl-Xyn) and PpAbf51b was detected by HPAEC with wheat arabinoxylan as the substrate (Figure 8).Using Tl-Xyn or PpAbf51b alone, Tl-Xyn liberated xylose (45.5 mg/g) and xylobiose (117.9 mg/g), whereas PpAbf51b released only arabinose (29.4 mg/g).All the simultaneous reactions and sequential enzyme combinations had significant synergistic effects on the degradation of wheat arabinoxylan, releasing substantially more arabinose, xylose, and xylobiose (Table 5).The degree of synergy for the simultaneous reaction was 1.38.For sequential enzyme combinations, the degree of synergy was 1.43 for PpAbf51b/Tl-Xyn (first addition of PpAbf51b) and 1.67 for Tl-Xyn/PpAbf51b (first addition of Tl-Xyn).Initial addition of Tl-Xyn followed by PpAbf51b liberated the most reducing sugar (321.9 mg/g).The molecular weight distribution of the hydrolytic products was determined by high-performance gel permeation chromatography (HPGPC) (Figure 9).The wheat arabinoxylan (retention time 9.67 min, ~330 kDa) was degraded to smaller molecular weight species after the simultaneous and sequential enzyme combinations.

Synergistic Hydrolysis of Wheat Arabinoxylan by PpAbf51b and Tl-Xyn
The complete degradation of arabinoxylan requires the synergistic catalysis of endoxylanase and arabinofuranosidase.The synergistic effect of Trichoderma longibrachiatum endo-1,4-xylanase (Tl-Xyn) and PpAbf51b was detected by HPAEC with wheat arabinoxylan as the substrate (Figure 8).Using Tl-Xyn or PpAbf51b alone, Tl-Xyn liberated xylose (45.5 mg/g) and xylobiose (117.9 mg/g), whereas PpAbf51b released only arabinose (29.4 mg/g).All the simultaneous reactions and sequential enzyme combinations had significant synergistic effects on the degradation of wheat arabinoxylan, releasing substantially more arabinose, xylose, and xylobiose (Table 5).The degree of synergy for the simultaneous reaction was 1.38.For sequential enzyme combinations, the degree of synergy was 1.43 for PpAbf51b/Tl-Xyn (first addition of PpAbf51b) and 1.67 for Tl-Xyn/PpAbf51b (first addition of Tl-Xyn).Initial addition of Tl-Xyn followed by PpAbf51b liberated the most reducing sugar (321.9 mg/g).The molecular weight distribution of the hydrolytic products was determined by high-performance gel permeation chromatography (HPGPC) (Figure 9).The wheat arabinoxylan (retention time 9.67 min, ~330 kDa) was degraded to smaller molecular weight species after the simultaneous and sequential enzyme combinations.  The amounts of saccharides were determined by HPAEC. 2 Degree of synergy = the amounts of saccharides released from simultaneous or sequential enzyme combinations/the sum of saccharide released by the individual enzymes. 3Not detected.

Discussion
The degradation products of hemicellulose are used widely in the production of pulp, paper, chemicals, and biofuels [6].Degradation can be achieved using different types of processing, such as thermal, mechanical, chemical, and biological [6,7].Among these methods, the specific action of hemicellulytic-degrading enzymes produced by microorganisms is considered to be the most promising approach [4,5].
P. polymyxa, previously known as Bacillus polymyxa, has a rich xylanolytic machinery, which makes the bacterium a potentially suitable candidate for the hydrolysis of hemicellulose [13].Several alkaline endoxylanases, acidic endoxylanase, xyloglucanase, and α-L-arabinofuranosidase from this bacterium have been purified or cloned [13,27].Genome sequence analysis of P. polymyxa revealed a number of genes coding for a variety of hemicellulases, which signifies the potential use of this bacterium in the general hydrolysis of hemicellulose (Table S2) [11,14].A glycoside hydrolase family 51 enzyme (PpAFase-1) from the fermentation broth of P. polymyxa was partially purified, identified, and overexpressed in E. coli [13].PpAFase-1 showed a synergistic effect with xylanase in hemicellulose degradation, but still has some disadvantages, such as insufficient synergistic efficiency, which pushed us to explore robust α-L-arabinofuranosidases.
In this report, the secreted hemicellulose-degrading enzymes of P. polymyxa KF-1 were identified by LC-MS/MS.As shown in Table 2, 13 proteins identified from the secreted proteomics analysis were assigned to hemicellulose-degrading enzymes.According to the activity assays, P. polymyxa displayed endoxylanase, β-mannosidase, β-xylosidase, α-galactosidase, and α-L-arabinofuranosidase activities (Table 1), with the α-L-arabinofuranosidase activity being measured as the highest.Therefore, P. polymyxa is a good candidate for obtaining an α-L-arabinofuranosidase.
Most bacterial GH51 α-L-arabinofuranosidases are optimally active under neutral pH (pH 6.0-7.0) and mesophilic (30-50 • C) conditions [23].Similarly, PpAbf51b was most active at pH 6.5 and 40 • C. Previously, two GH51 α-L-arabinofuranosidases from Paenibacillus sp. were characterized, which had higher optimal temperatures, 75 • C for THSAbf from Paenibacillus sp.THS1 and 60 • C for rAbfA from Paenibacillus sp.DG-22 [24,32].PpAbf51b showed better stability than other published bacterial GH51 α-L-arabinofuranosidases.PpAbf51b showed stability over a broad pH range (pH 4.0-11.0),which was similar to that of the GH51 α-L-arabinofuranosidase Tx-Abf from T. xylanilyticus [33], but better than that of PpAFase-1.Since some endo-xylanases usually exhibit maximal activity at alkaline conditions, PpAbf51b, with excellent alkali tolerance, would be suitable for synergistic catalysis with endoxylanases and β-xylosidases.PpAbf51b showed good resistance to various metal ions and chemicals at a concentration of 10 mM, such as Co 2+ , Ag + , and Hg 2+ , which have been shown to inhibit GH51 enzymes [29,30].The resistance against metal ions and chemicals by PpAbf51b was stronger than that of PpAFase-1, which was significantly inhibited by Fe 2+ , Cu 2+ , Mg 2+ , EDTA, SDS, Tween-40, Tween-60, Tween-80, and TritonX-100 at 10 mM [13].For some glycoside hydrolases, the product inhibits the enzyme activity and becomes a limiting step in enzymatic technologies [34].PpAbf51b showed good tolerance toward L-arabinose and D-xylose even at 500 mM.Previously, the GH51 enzyme PpAFase-1 was found to show tolerance to L-arabinose and D-xylose at concentrations lower than 50 mM.The excellent tolerance to monosaccharides and good stability makes PpAbf51b more suitable for biotechnological applications.

Strains and Culture Conditions
The P. polymyxa KF-1 (CCTCC AB 2018146) strain was cultured on LB agar (10% tryptone, 5% yeast extract, 10% NaCl, 2% agar, pH 7.0) at 30 • C in darkness.After incubation for 12 h, bacterial colonies were inoculated in 100 mL M9 minimal medium with arabinoxylan as the sole carbon source (Na 2 HPO 4 •7H 2 O 12.8 g/L, KH 2 PO 4 3 g/L, NaCl 0.5 g/L, NH 4 Cl 1 g/L, MgSO 4 2 mM, CaCl 2 0.1 mM, wheat arabinoxylan 10 g/L, pH 7.2).The cells were grown at 30 • C and shaking at 180 rpm for 36 h.The fermentation broth was centrifuged at 5000 rpm and 4 • C for 10 min, the supernatant was filtrated by 0.22 µm microporous membrane to remove the bacteria, and used as the enzyme extract for the enzymatic activity assay and LC-MS/MS analysis.

Hemicellulytic Activities of the P. polymyxa KF-1 Enzyme Extract
The endo-1,4-xylanase and endo-1,4-mannanase activities of the P. polymyxa KF-1 enzyme extract were determined with oat spelt xylan and locust bean gum as the substrates, respectively.Reaction mixtures containing 20 µL enzyme extract (final concentration 0.1 mg/mL), 180 µL phosphate buffer (pH 6.5), and 1 mg/mL substrate were incubated at 37 • C for 1 h.The reactions were terminated by boiling samples for 5 min, and the released reducing sugar was measured by the DNS method [38].The activities of β-xylosidase, α-L-arabinofuranosidase, β-mannosidase, α-glucuronidase and α-galactosidase activities were measured with pNPβXyl, pNPαAraf, pNPβMan, pNPαGlcA, and pNPαGal as substrates, respectively.Reaction mixtures containing 20 µL enzyme extract (final concentration 0.1 mg/mL), 180 µL phosphate buffer (pH 6.5), and 1 mM substrate were incubated at 37 • C for 1 h, and the absorbance of the samples at 405 nm were read [13].One unit of enzyme activity was determined as the enzyme required to release 1 µmol of reducing sugar/p-nitrophenol from the substrate per minute under the above assay conditions.
The activity of the P. polymyxa KF-1 enzyme extract toward wheat arabinoxylan was detected by HPAEC [36].Reaction mixtures containing 100 µL enzyme extract (final concentration 0.1 mg/mL) and 5 mg/mL substrate in 1 mL of phosphate buffer (pH 6.5) were incubated at 37 • C for 24 h.The reaction was terminated by boiling samples for 5 min, and the product was detected by HPAEC using a CarboPac PA-200 analytical column (3 × 250 mm) [7,36].The reducing sugar released was determined by the DNS method, the experiment was done in triplicate [38].

Gene Cloning, Protein Expression, and Purification
The genomic DNA of P. polymyxa KF-1 was extracted using the E.Z.N.A. Bacterial DNA Kit (D3350-01, Omega Bio-tek).The α-L-arabinofuranosidase gene was amplified with genomic DNA as the template.The oligonucleotides used for PCR were: 5 -CGGGATCCATGGTGAAGGGTTCTATTAT-3 and 5 -CCGCTCGAGTTAAGGAGCGATTGTCAGCA-3 (BamHI and XhoI sites are underlined).The PCR product and vector pET-28a were digested with BamHI/XhoI, and ligated to form the recombinant plasmid pET-28a-ppabf51b.The recombinant plasmid was used to transform E. coli DH5α cells.The recombinant plasmid with the correct coding sequence was used to transform E. coli BL21 (DE3) cells [42].
E. coli BL21 (DE3) cells harboring the recombinant pET-28a-ppabf51b plasmid were inoculated in LB broth at 37 • C and 180 rpm for 3 h with an inoculum volume of 1%.Protein expression was initiated by the addition of isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM and cells were grown for a further 12 h at 25 • C. Cells were harvested by centrifugation and the intracellular recombinant protein was released from the cells by sonication.The recombinant protein, PpAbf51b, which included an N-terminal His6-tag, was purified by Ni-NTA agarose (Qiagen, Hilden, Germany) using a linear gradient of 10-200 mM imidazole (in 20 mM Tris-HCl, pH 7.0).The α-L-arabinofuranosidase activity of each fraction was determined with pNPαAraf (1 mM) as a substrate [13].The fractions with high α-L-arabinofuranosidase activity were combined, dialyzed to remove imidazole, and the protein concentration was measured by the BCA method [43].
The purified PpAbf51b was analyzed by SDS-PAGE using a 10% separation gel and 3.9% stacking gel [44].The native molecular mass was detected by gel filtration chromatography performed on Superdex 200 10/300 GL column (GE Healthcare, Little Chalfont, UK).The column was pre-equilibrated and eluted by 50 mM phosphate buffer (pH 7.0) at a flow rate of 0.4 mL/min.The elution was monitored by UV-vis detector at 280 nm.Apoferritin (443 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa) were used to draw the standard curve.

Sequence Analysis and Protein Structure Prediction
The amino acid sequence was analyzed by BLASTp and aligned with published GH51 enzymes by Clustal Omega [26].The phylogenetic tree was generated by MEGA6 [45].The signal peptide and non-classical signal were predicted by the SignalP 4.1 Server [15] and the SecretomeP 2.0 server [16].The protein structure was modeled by SWISS-MODEL [46] using GH51 α-L-arabinofuranosidase from T. xylanilyticus (PDB ID: 2VRQ, identity 69%) as the template [20].

Characterization of Recombinant PpAbf51b
The effect of pH on PpAbf51b activity was determined over the pH range of 2.0 to 11.0 using pNPαAraf (1 mM) as the substrate.The effect of pH on enzyme stability was determined by incubating samples at various pH values for 15 min at 40 • C. Residual arabinofuranosidase activity was measured under standard assay conditions.The effect of temperature on PpAbf51b activity was investigated by measuring the arabinofuranosidase activity at temperatures ranging from 20 to 80 • C at pH 6.5 for 15 min.The effect of temperature on enzyme stability was determined by incubating the enzyme at temperatures between 20 and 80 • C for up to 1 h.Residual arabinofuranosidase activity was measured under standard assay conditions (pH 6.5, 40 • C, reaction time 15 min).The initial activity measured prior to incubation was set to 100% [13].
The effects of metal ions and chemicals on the activity of PpAbf51b were determined by incubating 1 µg purified enzyme with each metal ion or chemical (10 mM) for 1 h at 40 • C and pH 6.5.The residual activity was then determined under standard assay conditions.The initial activity measured prior to incubation was set as 100% [13].
The effect of monosaccharides D-xylose and L-arabinose on PpAbf51b activity was assayed by preincubation of 1µg purified enzyme with each monosaccharide (10-500 mM) at 40 • C and pH 6.5 for 1 h.Enzymatic activity was then determined under standard assay conditions [13].
The standard assay conditions were as following: 2 µg purified enzyme and 1 mM pNPαAraf were incubated in 200 µL phosphate buffer (pH 6.5) at 40 • C for 15 min, then the absorbance at 405 nm were recorded [13].

Substrate Specificity of PpAbf51b
Activity of PpAbf51b against 10 p-nitrophenyl-linked glycosides was determined by reading the absorbance at 405 nm after 15-min reaction at 40 • C. The reaction mixture consisted of 2 µg recombinant enzyme, 200 µL phosphate buffer (pH 6.5), and 1 mM substrate.The kinetics values of PpAbf51b on pNPAraf were determined by the 15 min-incubation (substrate concentration 0.1-5 mM) with 2 µg purified enzyme at pH 6.5 and 40 • C. K m and V max values were calculated by Lineweaver-Burk plots [47].
The synergistic effect of PpAbf51b and T. longibrachiatum endo-1,4-β-xylanase (Tl-Xyn) on the degradation of wheat arabinoxylan was detected.The reaction mixture of 100 µL containing 50 µL 4 mg/mL arabinoxylan and 50 µL Tl-Xyn and/or PpAbf51b (1 U for each) was incubated in 50 mM phosphate buffer (pH 6.5) at 40 • C.After 12 h, the reaction mixture was precipitated by 80% ethanol, dried, and re-dissolved in 400 µL distilled water, and analyzed by HPAEC [7,36].For sequential reactions, the substrate was incubated with the first enzyme for 4 h and the second enzyme was added.The degree of synergy was calculated as the ratio between the reducing sugar released from the combination reaction (Tl-Xyn and PpAbf51b) and the sum of the reducing sugar released from each enzyme separately.The molecular weight distribution of the hydrolytic product was determined by HPGPC using a TSK-gel G-3000PWXL column (7.8 × 300 mm, TOSOH, Japan).The column was pre-calibrated using standard dextrans (1,5,12,25, and 50 kDa) [48].

Conclusions
Soil bacterium P. polymyxa KF-1 produces various CAZymes including hemicellulases.In this report, 13 hemicellulose-degrading enzymes were identified from LC-MS/MS analysis of secreted proteins by P. polymyxa.A novel α-L-arabinofuranosidase PpAbf51b identified from the secretome was heterologously expressed and characterized.The enzyme showed excellent pH (4.0-11.0)and temperature (≤60 • C) stability and was active in the presence of metal ions (10 mM), chemicals (10 mM), and monosaccharides (500 mM).The enzyme acted as an exo-type α-L-arabinofuranosidase and showed a synergistic effect with endo-1,4-β-xylanase.Thus, PpAbf51b is a hemicellulase potentially suitable for biotechnological applications.

Figure 1 .
Figure 1.High-performance anion exchange chromatography (HPAEC) analysis of the hydrolysis of arabinoxylan by Paenibacillus polymyxa enzyme extract.Reaction mixture containing 0.1 mg/mL enzyme extract and 5 mg/mL arabinoxylan was incubated at 37 • C for 24 h.The reaction was terminated by boiling the sample for 5 min, and the product was detected by HPAEC using a CarboPac PA-200 analytical column (3 × 250 mm).The black dotted line represents the substrate (wheat arabinoxylan) and the blue line represents the transformed product.

Catalysts 2018, 8 , 18 Figure 3 .
Figure 3.The three-dimensional structure model of PpAbf51b predicted by SWISS-MODEL.(a) Amino acids from position 5 to position 493 of PpAbf51b were covered by the model; (b) E174 and E296, labeled in light blue, are predicted to be putative catalytic residues.The root-mean-square deviation (RMSD) was determined to be 0.070 between the homology model of PpAbf51b and the α-L-arabinofuranosidase from T. xylanilyticus (PDB ID: 2VRQ).

Figure 3 .
Figure 3.The three-dimensional structure model of PpAbf51b predicted by SWISS-MODEL.(a) Amino acids from position 5 to position 493 of PpAbf51b were covered by the model; (b) E174 and E296, labeled in light blue, are predicted to be putative catalytic residues.The root-mean-square deviation (RMSD) was determined to be 0.070 between the homology model of PpAbf51b and the α-L-arabinofuranosidase from T. xylanilyticus (PDB ID: 2VRQ).

Catalysts 2018, 8 , 18 Figure 3 .
Figure 3.The three-dimensional structure model of PpAbf51b predicted by SWISS-MODEL.(a) Amino acids from position 5 to position 493 of PpAbf51b were covered by the model; (b) E174 and E296, labeled in light blue, are predicted to be putative catalytic residues.The root-mean-square deviation (RMSD) was determined to be 0.070 between the homology model of PpAbf51b and the α-L-arabinofuranosidase from T. xylanilyticus (PDB ID: 2VRQ).

Figure 5 .
Figure 5.Effect of pH and temperature on activity and stability of PpAbf51b.(a) Optimal pH and (c) pH stability were determined by incubating samples at various pH values for 15 min at 40 °C.(b)Optimal temperature and (d) temperature stability were determined by incubating samples at various temperatures at pH 6.5 for 15 min.The enzyme activity was detected by using pNPαAraf as a substrate (1 mM).The following buffers were used: 50 mM acetate buffer, pH 2.0-6.0;50 mM phosphate buffer, pH 6.0-8.0; and 50 mM glycine sodium buffer, pH 8.0-11.0.

Figure 5 .
Figure 5.Effect of pH and temperature on activity and stability of PpAbf51b.(a) Optimal pH and (c) pH stability were determined by incubating samples at various pH values for 15 min at 40 • C. (b) Optimal temperature and (d) temperature stability were determined by incubating samples at various temperatures at pH 6.5 for 15 min.The enzyme activity was detected by using pNPαAraf as a substrate (1 mM).The following buffers were used: 50 mM acetate buffer, pH 2.0-6.0;50 mM phosphate buffer, pH 6.0-8.0; and 50 mM glycine sodium buffer, pH 8.0-11.0.

Figure 6 .
Figure 6.Effect of L-arabinose and D-xylose on the activity of PpAbf51b.Values represent the mean ± SD (n = 3).

Figure 6 .
Figure 6.Effect of L-arabinose and D-xylose on the activity of PpAbf51b.Values represent the mean ± SD (n = 3).

Table 1 .
Hemicellulytic activities of the enzyme extract of Paenibacillus polymyxa KF-1 on different substrates.

Table 3 .
Effect of various metal ions and chemicals on the activity of PpAbf51b.

Table 3 .
Effect of various metal ions and chemicals on the activity of PpAbf51b.
1The activity was determined with pNPAraf (1 mM) as a substrate.

Table 4 .
Hydrolytic activity of PpAbf51b on different p-nitrophenyl linked glycosides.

Table 4 .
Hydrolytic activity of PpAbf51b on different p-nitrophenyl linked glycosides.