Identification and enzymatic characterization of an endo-1,3-β-glucanase from Euglena gracilis

Phytochemistry 116 (2015) 21–27

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Identification and enzymatic characterization of an endo-1,3-b-glucanase from Euglena gracilis Takumi Takeda a,⇑, Yuki Nakano a, Machiko Takahashi a, Naotake Konno a, Yuichi Sakamoto a Ryo Arashida b, Yuka Marukawa b, Eriko Yoshida b, Takahiro Ishikawa c, Kengo Suzuki b a b c

Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan euglena Co., Ltd., 2-6-1, Koraku, Bunkyo-ku, Tokyo 112-0004, Japan Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu, Matsue, Shimane 690-8504, Japan

a r t i c l e

i n f o

Article history: Received 29 September 2014 Received in revised form 8 May 2015 Accepted 18 May 2015 Available online 28 May 2015 Keywords: Euglena Euglenaceae Endo-1,3-b-glucanase Paramylon Hydrolysis Transglycosylation Glycoside hydrolase family 17

a b s t r a c t Euglena produces paramylon as a storage polysaccharide, and is thought to require b-1,3-glucan degrading enzymes to release and utilize the accumulated carbohydrate. To investigate b-1,3-glucan degradation in Euglena, endo-1,3-b-glucanases were partially purified from Euglena gracilis by hydrophobic, gel filtration and anion-exchange chromatography. Tryptic digests and mass-spectrometric analysis identified three proteins in the purified fraction as a member of glycoside hydrolase family (GH) 17 and two members of GH81. These genes were cloned from an Euglena cDNA pool by PCR. EgCel17A fused with a histidine-tag at the carboxy terminus was heterologously produced by Aspergillus oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A had a molecular weight of about 40 kDa by SDS–PAGE, which was identical to that deduced from its amino acid sequence. The enzyme showed hydrolytic activity towards b-1,3-glucans such as laminarin and paramylon. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0–5.5 and 60 °C after 1 h incubation or 50 °C after 20 h incubation. The enzyme had a Km of 0.21 mg/ml and a Vmax of 40.5 units/mg protein for laminarin degradation at pH 5.0 and 50 °C. Furthermore, EgCel17A catalyzed a transglycosylation reaction by which reaction products with a higher molecular weight than the supplied substrates were initially generated; however, ultimately the substrates were degraded into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble b-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-b-glucanase to be identified from E. gracilis. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction b-1,3-Glucans, composed of b-1,3-linked glucose residues, are found as major structural components of fungal cell walls and extracellular sheaths in fungal cultures (Sietsma and Wessels, 1981; Ruel and Joseleau, 1991). Plants also produce a form of cell wall-associated b-1,3-glucan, called callose, which is involved in pollen development, cell plate formation and responses to biotic or abiotic stress (McCormick, 1993; Hong et al., 2001; Chen and Kim, 2009). Some algae accumulate b-1,3-glucan as a storage polysaccharide (Michel et al., 2010). Degradation of b-1,3-glucan involves the actions of endo-1,3-bglucanases (EC 3.2.1.6 and EC 3.2.1.39) and exo-1,3-b-glucanases (EC 3.2.1.58), which are found in glycoside hydrolase families

⇑ Corresponding author. Tel.: +81 197 682911. E-mail address: [email protected] (T. Takeda). http://dx.doi.org/10.1016/j.phytochem.2015.05.010 0031-9422/Ó 2015 Elsevier Ltd. All rights reserved.

(GHs) 5, 16, 17, 55, 64 and 81, and GH 3, 5, 17 and 55, respectively, based on the amino acid similarity (Henrissat, 1991; Henrissat and Bairoch, 1996; Henrissat and Davies, 1997). Endo-1,3-b-glucanases hydrolyze internal b-1,3-glucosidic bonds at random sites to generate oligosaccharides, and exo-1,3-b-glucanases release glucose from the generated oligosaccharides (Takahashi et al., 2011). b-1,3Glucanosyltransferase from Aspergillus fumigatus, a member of GH 72, catalyzes the hydrolysis of b-1,3-glucan and transglycosylation reaction to produce water-insoluble b-1,3-glucans from oligosaccharides (Hartland et al., 1996). Some endo-1,3-b-glucanases possess carbohydrate-binding domains or immunoglobulin like domains, which enhance the ability of the enzymes to bind waterinsoluble substrate (van Bueren et al., 2005; Cheng et al., 2013). Glycosylphosphatidylinositol-anchored endo-1,3-b-glucanase in the ascomycete fungus Ustilago esculenta is localized in the plasma membrane and functions to modify the inner cell wall structure (de Groot et al., 2003; Nakajima et al., 2012). Localization via such binding domains or lipids is thought to influence the efficiency of

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the catalytic domain in hydrolyzing the appropriate substrate. The actions of 1,3-b-glucanases are thought to play key roles in cell wall morphogenesis and in acquiring carbon source. Euglena gracilis is a phototrophic alga that synthesizes highly crystalline b-1,3-glucan, called paramylon, as a storage polysaccharide under photoautotrophic conditions. The breakdown of paramylon is induced by light treatment in dark-grown Euglena cells and dark treatment in light-grown cells, and is enhanced in the presence of a nitrogen source (Sumida et al., 1987). Synthesized paramylon is enzymatically degraded by the action of endo- and exo-1,3-bglucanases in Euglena cells (Barras and Stone, 1969a,b). An E. gracilis exo-1,3-b-glucanase participating in paramylon degradation has been purified and characterized (Barras and Stone, 1969a,b). However, enzymes that endo-lytically hydrolyze paramylon have not been identified yet. In this study, three endo-1,3-b-glucanases from E. gracilis were identified by partial purification based on hydrolytic activity towards b-1,3-linked laminarin by hydrophobic, gel-permeation and strong-anion exchange chromatography, followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS), and cDNA cloning by PCR. Furthermore, enzymatic properties of the recombinant endo-1,3-b-glucanase belonging to GH 17 (EgCel17A) prepared in Aspergillus oryzae and efficient degradation of palamylon using EgCel17A were investigated. 2. Results 2.1. b-Glucan degrading activity in Euglena Fractions obtained by (NH4)2SO4 precipitation of the Euglena extract with sodium phosphate buffer containing 0.05% Tween 20 were assayed for b-glucan degrading activities using 1,3–1,4-bglucan, phosphoric acid-swollen cellulose (PSC), laminarin and paramylon (Supplementary Fig. 1). The enzyme preparation had low activities towards PSC and 1,3–1,4-b-glucan, whereas fractions of the 20–40% and 40–60% (NH4)2SO4 precipitates contained high activities towards laminarin and paramylon. This result indicated that the Euglena extract contained enzymes that may be responsible for degrading b-1,3-glucans. 2.2. Partial purification of proteins involved in b-1,3-glucan degradation Proteins obtained by (NH4)2SO4 precipitation (20–60%) from Euglena were fractionated on a hydrophobic interaction chromatography column and hydrolytic activity towards laminarin was examined (Supplementary Fig. 2A). Fractions 26–36 exhibited high activity, where the concentration of (NH4)2SO4 was almost zero. These fractions, even though 2 peaks appeared, were mixed and further fractionated by gel permeation chromatography (GPC) (Supplementary Fig. 2B). Fractions 28–38 showed high hydrolytic activity for laminarin. Finally, the fractions were mixed, desalted, equilibrated with sodium phosphate buffer (10 mM, pH 8.0) and separated on a strong anion-exchange column (Supplementary Fig. 2C). Fractions 22–36, where the concentration of NaCl was 275–450 mM, showed high hydrolytic activity for laminarin. Fractions 29–33 were mixed, concentrated and subjected to SDS–PAGE followed by silver staining (Fig. 1A). There were four visible protein bands in which top three bands look like double bands, and a few minor bands. 2.3. Sequence analyses of proteins responsible for b-1,3-glucan degradation Protein bands in the active fraction obtained after separation by SDS–PAGE were digested with trypsin, and the hydrolyzates were

(A)

1

(B)

2

1

2

kDa 100 75

(d) (c) (b)

50

kDa 100 75 50

37

(a) 37

25 20

25 20

Fig. 1. SDS–PAGE of Euglena proteins responsible for b-1,3-glucan degradation. A protein solution containing b-1,3-glucan degrading activity after partial purification on three columns was separated on an SDS–polyacrylamide gel followed by silver staining (A). Four protein bands indicated with arrows were subjected to LC/MS/MS. Recombinant EgCel17A that was produced in A. oryzae and purified by immobilized metal affinity chromatography was loaded on an SDS–polyacrylamide gel followed by staining with Coomassie Brilliant Blue R-250 (B).

analyzed by LC/MS/MS (Fig. 1A, Supplementary Tables 1 and 2, and Supplementary Fig. 3). For protein (a), 4 peptides obtained by LC/MS/MS matched a putative GH17 endo-1,3-b-glucanase with 26% sequence coverage of amino acid sequence translated from the Euglena EST database; for protein (b), 35 peptides matched a putative GH81 endo-1,3-b-glucanase with 56% sequence coverage; for protein (c), 33 peptides were found to match a putative GH81 endo-1,3-b-glucanase with 54% sequence coverage. For protein (d), 12 peptides matched a putative malate synthase with 13% sequence coverage of full amino acid sequence. From these results, proteins (a), (b) and (c) are predicted to be endo-1,3-b-glucanases. Partial DNA sequences of protein (a), (b) and (c) were extracted from the E. gracilis EST database. Unidentified DNA regions were amplified by PCR. DNA open reading frames of proteins (a), (b) and (c) consisted of 1233 bp, 2616 bp and 2940 bp, respectively. The deduced amino acid sequences showed that protein (a) was classified into the plant GH17 family of endoglucanases, and proteins (b) and (c) were classified into the GH 81 family of endoglucanases. Hence, proteins (a), (b) and (c) were designated as EgCel17A, EgCel81B and EgCel81A, respectively. EgCel17A has a putative signal peptide consisting of 16 amino acids, calculated molecular mass of 40.8 kDa with pI 4.3, and 3 positions of predicted N-glycosilation sites. EgCel81A and EgCel81B have a putative signal peptide consisting of 18 and 31 amino acids, calculated molecular mass of 107.6 kDa and 92.3 kDa with pI value of 6.4 and 6.2, and 10 and 5 positions of predicted N-glycosylation sites, respectively. None of the proteins has a typical carbohydrate-binding module.

2.4. Production of recombinant EgCel17A Recombinant EgCel81A and EgCel81B could not be produced by recombinant expression in Escherichia coli, Brevibacillus choshinensis, A. oryzae and Nicotiana benthamiana (data not shown). However, recombinant EgCel17A with a histidine-tag at the carboxy-terminus was produced in A. oryzae and purified by immobilized metal affinity chromatography. The purified protein consisted of a single major band on an SDS–polyacrylamide gel after Coomassie Brilliant Blue R-250 staining (Fig. 1B). Hereafter, the purified recombinant protein was used for determining enzymatic properties.

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T. Takeda et al. / Phytochemistry 116 (2015) 21–27

Substrate

Relative activity (%)

Km (mg/ml)

Vmax (units/mg protein)

Laminarin Paramylon Alkaline-swollen 1,3–1,4-b-Glucan Xyloglucan CMC Xylan Cellulose

100.0 ± 9.8 23.4 ± 3.0 78.6 ± 3.0 6.2 ± 2.0 2.2 ± 0.9 3.6 ± 3.0 4.2 ± 2.7 2.0 ± 0.6

0.21

40.5

40 35 30 25 20 15 10 5 0 0

0.2

0.4 0.6

0.8 1.0 1.2 1.4

Laminarin content, mg 1/Glucose equivalents, µmol/min/mg protein

(B)

30 Glucose equivalents, nmol

(A) Glucose equivalents, µmol/min/mg protein

Table 1 Substrate specificity of EgCel17A. EgCel17A was incubated with water-soluble and water-insoluble polysaccharides in 100 mM sodium phosphate buffer (pH 5.5) at 30 °C for 1 h. The activities were determined by measuring the increase in reducing power in the reaction mixtures. When water-insoluble substrates were used, the supernatants obtained from the reaction mixtures by centrifugation were assayed for hydrolytic activities. The activity towards laminarin was taken as 100%. Data are the means ± SE of three determinations. Values of a Km and a Vmax of EgCel17A were calculated based on the double reciprocal plot of initial reaction velocity as shown in Fig. 3.

0.12 0.10 0.08 0.06 0.04 0.02

25 0

20

5

10

15

20

1/S

15 10 5 0

0

0 5 101520 0 5 10 15 20 0 5 101520 0 5 10 15 20 0 5 1015 20 (h) 30 ˚C

40 ˚C

50 ˚C

60 ˚C

Fig. 3. Effects of varying concentrations of laminarin as a substrate on EgCel17A activity. After incubation of reaction mixtures (25 ll) containing laminarin (0.5– 30 lg), sodium phosphate buffer (100 mM, pH 5.0) and EgCel17A (0.1 lg) at 60 °C for 5 min, increased reducing power was measured (A). Double reciprocal plot of initial reaction velocity was calculated (B).

70 ˚C

Fig. 2. Effects of temperature on EgCel17A activity. The laminarin degrading activity of EgCel17A was determined at 30–70 °C in the presence of sodium phosphate buffer (pH 5.5) after 0.5–20 h incubation. The activities were determined by measuring the increase in reducing power. Data are the means ± SE of three determinations.

2.5. Optimum conditions for EgCel17A action The hydrolytic activity of EgCel17A was examined using watersoluble and water-insoluble b-glucans (Table 1). EgCel17A preferentially degraded laminarin, alkaline-treated paramylon, and had slight hydrolytic activity towards water-insoluble paramylon. This result indicates that EgCel17A is capable of degrading water-soluble and water-insoluble b-1,3-glucans. Laminarin was the best substrate among the polysaccharides tested with EgCel17A, and maximum hydrolysis was attained at pH 4.0–5.5 (Supplementary Fig. 4). At pH 3.5 and greater than pH 7.5, most EgCel17A activity was inhibited. EgCel17A effectively degraded laminarin at 50–70 °C by a short incubation (less than 1 h), but 40–50 °C by a long incubation (more than 1 h) (Fig. 2). The effects of various concentrations of laminarin as a substrate on EgCel17A activity were examined. The activity increased linearly with the increase in laminarin concentrations up to 0.14 mg/ml and increased gradually at higher concentrations (Fig. 3A). Based on the result, a double reciprocal plot was made (Fig. 3B). Values of a Km and a Vmax of EgCel17A were calculated as 0.21 mg ml1 and 40.5 units/mg protein, respectively. 2.6. Effects of metals on EgCel17A activity The effects of metals on the degradation of laminarin by EgCel17A was examined (Table 2). EgCel17A activity was not affected by NaCl (less than 100 mM), nor by 1 mM MgCl2, KCl, CaCl2 and ZnSO4. In contrast, 1 mM FeSO4, MnCl2, CuSO4 and

CoCl2 inhibited EgCel17A activity by about 40–50%. Markedly, 1 mM of NiCl2 inhibited EgCel17A activity to 15% of the control. None of the metals was found to increase the enzymatic activity in this study. 2.7. Transglycosylation activity of EgCel17A The endotransglycosylation activity of EgCel17A was examined using laminarioligosaccharides as substrates (Fig. 4A). EgCel17A did not generate a reaction product from laminaribiose. Reaction products with higher and lower molecular weights were observed after 0.5–4 h incubation, when laminarioligosaccharides with a degree of polymerization (DP) greater than 3 were used. These results indicate that EgCel17A catalyzes a transglycosylation reaction when PDP3 laminarioligosaccharides are used; however, glucose, laminaribiose and laminaritriose accumulated as reaction products after 18 h incubation. When polymeric laminarin was incubated with EgCel17A, oligosaccharides of various DPs were observed after 1 h incubation, and glucose, laminaribiose and laminaritriose accumulated after 18 h incubation (Fig. 4B). These results suggest that EgCel17A catalyzes hydrolytic and transglycosylation reactions but primarily functions to depolymerize b-1,3linked oligosaccharides and polymers. 2.8. Optimum conditions for paramylon degradation by EgCel17A To determine the conditions for efficient hydrolysis of paramylon present in Euglena freeze-dried powder, the effects of NaOH, sodium acetate and NaCl were investigated. Euglena freeze-dried powder was suspended in NaOH at final concentrations of 0– 1.5 M and then adjusted to pH 5.5 with AcOH. The suspensions were incubated with EgCel17A and the amounts of reaction products were determined. Reaction products generated by EgCel17A increased after treatment with increasing NaOH concentrations

T. Takeda et al. / Phytochemistry 116 (2015) 21–27

Additives

Relative activity (%)

No additive NaCl (50 mM) NaCl (100 mM) MgCl2 KCl CaCl2 FeSO4 MnCl2 ZnSO4 NiCl2 CuSO4 CoCl2

100.0 ± 1.6 106.1 ± 1.5 100.4 ± 1.4 99.7 ± 2.4 107.0 ± 1.6 96.4 ± 1.0 67.9 ± 2.4 49.2 ± 1.8 109.0 ± 1.7 15.5 ± 2.6 64.0 ± 1.7 61.4 ± 1.8

(A)

(A) Glucose equivalents, nmol

Table 2 Effects of metals on EgCel17A activity. EgCel17A was incubated with laminarin in 100 mM sodium phosphate buffer (pH 5.5) in the presence of metals at 30 °C for 1 h. The activities were determined by measuring the increase in reducing power in the reaction mixtures. The activity towards laminarin without additive was taken as 100%. Data are the means ± SE of three determinations.

250 200 150 100 50 0

0 NaOH concentration, M

(B) Glucose equivalents, nmol

24

350 300 250 200 150 100 50 0

0 0.1 0.5 1.0 2.0 Sodium acetate concentration, M

1 23456 1 234561 23456 123456 123456 12345 6

Substrate

L2

L3

L4

L5

L6

L7

(B) Glc L2 L3 L4 L5 L6 L7 1

2

Laminarin

Fig. 4. Transglycosylation reaction by EgCel17A. EgCel17A was incubated with DP 2–7 laminarioligosaccharides in sodium phosphate buffer (pH 5.5) for various periods (1, 0 h; 2, 0.5 h; 3, 1 h; 4, 2 h; 5, 4 h; 6, 10 h) at 30 °C (A). When laminarin was used as a substrate, incubation was conducted for 1 h (1) and 18 h (2) (B). The reaction mixtures were developed by TLC on silica gel plates in n-BuOH–AcOH–H2O (3:1:1, by vol.) and stained with 0.5% (w/v) thymol in EtOH/H2SO4. The position of laminarioligosaccharides is indicated on the left side.

up to 0.6 M (Fig. 5A). However, levels of reaction products decreased after treatment with more than 0.9 M NaOH compared to reaction mixtures pretreated with 0.6 M NaOH. The levels of reaction products by EgCel17A decreased with increasing concentration of added sodium acetate or NaCl in the reaction mixture (Fig. 5B and C). The presence of 2.0 M sodium acetate or NaCl in the reaction mixture reduced the level of reaction products by 27% or 31% compared to reactions omitting salts. These results imply that pretreatment with NaOH modifies the paramylon structure to increase the levels of reaction products by EgCel17A, whereas increased concentrations of sodium acetate or NaCl gradually inhibit EgCel17A activity. 3. Discussion 3.1. b-1,3-Glucan degradation by endo-1,3-b-glucanase in Euglena Euglena synthesizes paramylon as a storage polysaccharide. Endo-1,3-b-glucanases and exo-1,3-b-glucanases are thought to

Glucose equivalents, nmol

(C)

Glc L2 L3 L4 L5 L6 L7

350 300 250 200 150 100 50 0

0

0.1 0.5 1.0 2.0 NaCl concentration, M

Fig. 5. Efficient degradation of paramylon in Euglena freeze-dried powder by EgCel17A. Euglena freeze-dried powder was suspended in various concentrations of NaOH (0–1.5 M) for 15 min, after which the solution pH was adjusted with AcOH to pH 5.5. The NaOH-treated Euglena suspensions were incubated with EgCel17A at 30 °C for 18 h (A). The Euglena suspension treated with 1 M NaOH followed by washing with 100 mM sodium phosphate buffer (pH 5.5) was incubated with EgCel17A in the presence of various concentrations of sodium acetate (B) and NaCl (C) (0–2.0 M) at 30 °C for 18 h. The supernatant obtained by centrifugation was used to determine the increase in reducing power. Data are the means ± SE of three determinations.

participate in paramylon degradation to generate glucose as a carbon source. An E. gracilis exo-1,3-b-glucanase was purified and characterized previously (Barras and Stone, 1969a,b); however, an endo-1,3-b-glucanase has not been identified yet. Putative endo-1,3-b-glucanase genes, Egcel17A, Egcel81A and Egcel81B, were identified after partial purification, LC/MS/MS and cDNA cloning. To determine the enzymatic properties, recombinant EgCel17A with a his7-tag at the carboxy-terminus was prepared in A. oryze and purified by immobilized metal affinity chromatography (Fig. 1B). EgCel17A preferentially hydrolyzed water-soluble b-1,3-glucan and to a lesser extent hydrolyzed 1,3–1,4-b-glucan, hydroxyl ethyl cellulose and PSC (Table 1). Paramylon, a water-insoluble b-1,3glucan, was also degraded by the enzyme although alkalinetreated paramylon was more digestible. From these results, EgCel17A is considered to be an endo-1,3-b-glucanase. EgCel17A exhibited high activity at pH 4.0–6.0 and 50–70 °C after a short incubation, and at 40–50 °C after a long incubation (Supplementary Fig. 4 and Fig. 2). This finding is probably because the enzyme acts vigorously but is inactivated at high temperature. Comparing that endo-1,3-b-glucanases from soybean and banana fruit showed high activities at pH 5.0–5.5 and pH 4.0–5.0, respectively (Cline and Albersheim 1981; Peumans et al., 2000), EgCel17A has a broad optimum pH. In addition, endo-1,3-b-glucanase from banana fruit was heat-stable up to 70 °C (Peumans et al., 2000). To our

T. Takeda et al. / Phytochemistry 116 (2015) 21–27

knowledge, EgCel17A is the first heat-stable endo-1,3-b-glucanase found in algae. By contrast, exo-glucanses (b-glucosidase, b-mannosidase and galactosidase) and endotransglucosylases for xyloglucan, b-1,4-glucan and xylan have been reported to catalyze transglycosylation reaction (Nishitani and Tominaga, 1992; Fry et al., 1992; Shi et al., 2011; Gupta et al., 2012; Bhatia et al., 2002; Ducret et al., 2002; Johnstone et al., 2013; Takahashi et al., 2013), EgCel17A catalyzed hydrolysis and transglycosylation of b1,3-glucan (Fig. 4). However, b-1,3-linked oligosaccharides as well as polymeric laminarin were finally degraded into glucose, laminaribiose and laminaritriose by EgCel17A. This suggests that EgCel17A functions to depolymerize b-1,3-glucans. Furthermore, EgCel17A was eluted together with EgCel81A and EgCel81B on GPC (Supplementary Fig. 2) even though a molecular weight of EgCel17A is about 50 kDa smaller than those of EgCel81A and EgCel81B. This result may imply complex formation between EgCel17A and EgCel81A or EgCel81B. b-1,3-Gluco-oligosaccharides generated by the actions of endo-1,3-b-glucanases could be efficiently converted to glucose by the action of b-glucosidases (Barras and Stone, 1969a,b; Takahashi et al., 2011). These results suggest that EgCel17A is involved in the production of b-1,3-linked oligosaccharides and glucose from polymeric b-1,3-glucans, in which EgCel81A, EgCel81B and b-glucosidases might cooperatively act in vivo. Although EgCel17A hydrolyzes paramylon (Table 1), no reaction product was obtained from untreated Euglena freeze-dried powder containing paramylon. However, structural changes of paramylon resulting from NaOH treatment of Euglena freeze-dried powder increased the level of reaction products generated by EgCel17A even though sodium acetate, which was generated by neutralization of NaOH, and NaCl inhibited the activity (Fig. 5A–C). The results suggest that native paramylon in Euglena freeze-dried powder is structurally protected from enzymatic hydrolysis and that pretreatment with NaOH enables Euglena freeze-dried powder to be hydrolyzed efficiently by EgCel17A. Thus, EgCel17A can be applied to produce glucose and b-1,3-linked oligosaccharides from alkaline-treated Euglena freeze-dried powder. Endo-1,3-b-glucanases are believed to play a significant role in degradation and modification of b-1,3-glucan of fungal cell walls, plant callose and storage polysaccharides. EgCel17A is the first identified endo-1,3-b-glucanase from Euglena that catalyzes hydrolytic and transglycosylation reactions of water-soluble and waterinsoluble b-1,3-glucans, resulting in the accumulation of glucose and oligosaccharides. So far, however, there is no direct evidence that EgCel17A contributes to in vivo degradation of paramylon or cell wall component. Determining the localization of the enzyme, analysis of Euglena with gene deletions and gene overexpression will provide significant information on the function of EgCel17A in vivo. Furthermore, controlling the endo-1,3-b-glucanase level in Euglena may contribute to efficient production of oil, which is accompanied by paramylon degradation under anaerobic conditions.

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20 h incubation. The enzyme had a Km of 0.21 mg/ml and a Vmax of 40.5 units/mg protein for laminarin degradation at pH 5.0 and 50 °C. However, the enzyme generated reaction products at 70 °C equivalent to 68% of those at 60 °C, indicating a thermal stability enzyme. In addition, EgCel17A generated reaction products with a higher molecular weight than the supplied substrates, indicating EgCel17A catalyzes a transglycosylation reaction. However, EgCel17A ultimately degraded b-1,3-linked oligosaccharides and polymers into glucose, laminaribiose and laminaritriose. EgCel17A effectively produced soluble b-1,3-glucans from alkaline-treated Euglena freeze-dried powder containing paramylon. Thus, EgCel17 is the first functional endo-1,3-b-glucanase to be identified from E. gracilis that degrades water-soluble and waterinsoluble b-1,3-linked glucans. 5. Experimental 5.1. Cell culture and growth condition E. gracilis was maintained by regular subculturing and was grown in Koren–Hutner medium (Koren and Hunter, 1967) under continuous illumination (24 lmol m2 s1) at 26 °C for 6 days. A. oryzae (NBRC 4075) used for recombinant protein production was grown on Czapec-Dox (pH 6.0) agar plates (1% (w/v) glucose, 30 mM Na2CO3, 7 mM KCl, 4 mM K2HPO4, 4 mM MgSO4, 0.04 mM FeSO4, 1.5% (w/v) agar) and cultured in YPM (1% yeast extract, 2% peptone, and 2% maltose, w/v) medium at 30 and 28 °C, respectively. 5.2. Hydrolytic activities in Euglena protein extract Euglena was ground in liq. N2 using a mortar and pestle, homogenized in 10 mM sodium phosphate buffer (pH 7.0) containing 0.05% (v/v) polyoxyethylene sorbitan monolaurate (Tween 20) and sonicated 3 times for 1 min each. After centrifugation at 22,000g for 20 min, the supernatant was (NH4)2SO4 precipitated at a final concentration of 20, 20–40, 40–60 and 60–80% saturation, respectively. Each precipitate was suspended in 100 mM sodium phosphate buffer (pH 5.5) and the supernatant obtained by centrifugation at 22,000g for 10 min was used to assay hydrolytic activity. Reaction mixtures (100 ll) containing protein preparation derived from 50 mg of Euglena cells and 1,3–1,4-b-glucan (0.1%, w/v, Megazyme), PSC, (0.05%, w/v) made from cellulose (Sigmacell 20, Sigma–Aldrich), laminarin (0.1%, w/v, Sigma– Aldrich) or paramylon (0.05%, w/v) prepared from Euglena cells were incubated at 30 °C for 1 h. Hydrolytic activity was determined by measuring the increase in reducing power using p-hydroxybenzoic hydrazide-HCl as described previously (Miller, 1972). The supernatant (20 ll) obtained by centrifugation at 22,000g for 5 min was mixed with 60 ll of p-hydroxybenzoic hydrazide-HCl, heated in boiling H2O for 10 min and the absorbance of the solution was measured at 410 nm.

4. Conclusions Three endo-1,3-b-glucanase genes, Egcel17A, Egcel81A and Egcel81B, were identified after partial purification of the enzymes responsible for b-1,3-glucan degradation, peptide identification by LC/MS/MS and DNA amplification by PCR. EgCel17A with histidine-tag at the carboxy terminus was heterologously produced by A. oryzae and purified by immobilized metal affinity chromatography. Purified EgCel17A exhibited high activity on laminarin and alkaline-swollen paramylon, and to a lesser extent on crystalline paramylon, indicating EgCel17A is an endo-1,3-b-glucanase. Maximum activity of laminarin degradation by EgCel17A was attained at pH 4.0–5.5 and 60 °C after 1 h incubation or 50 °C after

5.3. Partial purification of proteins responsible for b-1,3-glucan degradation The 20–60% (w/v) (NH4)2SO4 precipitate was suspended in 10 mM sodium phosphate buffer (pH 7.0) containing 15% (w/v) (NH4)2SO4 and loaded on a hydrophobic column (PhenylToyopearl, Tosoh Co., Ltd.). The column was washed with the same buffer and bound proteins were eluted with a decreasing linear gradient of (NH4)2SO4 (0–20%, w/v) in 10 mM sodium phosphate buffer (pH 7.0) for 20 min at a flow rate of 4 ml/min. Fractions with b-1,3-glucan degrading activity were concentrated and equilibrated with 10 mM sodium phosphate buffer (pH 7.0) using an

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ultrafiltration spin column (10 kDa cut-off, Amicon Ultra-4, Millipore). The solution was fractionated by gel permeation chromatography (GPC, Superdex 75 10/300 GL, GE Healthcare) using 10 mM sodium phosphate buffer (pH 7.0) containing 100 mM NaCl at a flow rate of 0.4 ml/min. Fractions having b-1,3-glucan degrading activity were concentrated and equilibrated with 10 mM sodium phosphate buffer (pH 8.0) using ultrafiltration spin column (10 kDa cut off), and loaded on a strong anion-exchange column (MonoQ, GE Healthcare). Proteins bound to the column were eluted with an increasing linear gradient of NaCl (0–0.5 M) in 10 mM sodium phosphate buffer (pH 8.0) for 40 min at a flow rate of 0.4 ml/min. The eluate was subjected to SDS–PAGE followed by silver staining. 5.4. Peptide mass analysis of b-1,3-glucan degrading proteins Protein bands separated by SDS–PAGE from the MonoQ fractions were excised, treated with iodoacetamide and digested with trypsin as previously described (Kawamura and Uemura, 2003). Digested peptides were applied to a Magic C18 AQ Nano column (0.1  150 mm, MICHROM Bioresources, Inc.) in an ADVANCE UHPLC system (MICHROM Bioresources, Inc.) equilibrated with 0.1% (v/v) HCO2H and eluted with an increasing linear gradient at 5–45% (v/v) CH3CN at a flow rate of 500 nl/min. Mass analysis was performed using an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific) operating Xcalibur software Ver. 2.0.7 (Thermo Fisher Scientific). Peptides were identified using a MASCOT MS/MS ion search algorithm (http://www.matrixscience.com/home.html) in error tolerance mode (one amino acid substitution allowed) using the Euglena peptide mass database. Search parameters were as follows: max missed cleavages, 0; fixed modifications, carbamidomethyl; peptide tolerance, ±5 ppm, fragment mass tolerance, ±0.6 Da. The Euglena database was prepared from the E. gracilis expressed sequence tags (EST) and was downloaded from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov/) and annotated using the Blast2go program (http://www.blast2go.com/b2ghome). 5.5. cDNA cloning Partial DNA sequences encoding matched peptides from LC/MS/MS results were extracted from the Euglena EST database. cDNA pools were synthesized from total RNA using Superscript III reverse transcriptase (Invitrogen) and an oligo (dT20) primer for cloning the 30 end or random primers for cloning the 50 end using a 50 RACE system (Invitrogen). DNAs were amplified by PCR using PrimeStar GXL DNA polymerase (Takara Bio), a cDNA pool and DNA primers. The gene specific primers used were designed from the coding regions of the Euglena EST; 50 -GCCTCTGTCAGGACTGG GTCCTGGTGC-30 and 50 -CGACTGGAGCACGAGGACACTGA-30 for Egcel17A, 50 -GAGTGCCCTGTTCTGCGGGACATGAATG-30 and 50 -CATT CATGTCCCGCAGAACAGGGCACTC-30 for Egcel81A, and 50 -GATCGCT TCAGTCGGAACACTACGCTG-30 and 50 -CAGCGTAGTGTTCCGACTGA AGCGATC-30 for Egcel81B. DNA sequences were determined by a 3130 Genetic Analyzer (Applied Biosystems). BLASTP programs were used to search for conserved domains via the NCBI. 5.6. Sequence analysis Protein information, molecular weight and pI, was predicted using GENETYX ver. 12. Catalytic domain and carbohydrate-binding module were searched using a conserved domain search at National Center of Biotechnology Information website (http:// www.ncbi.nlm.nih.gov/). N-terminal signal peptide was predicted by the SignalP 4.0 Server (http://www.cbs.dtu.dk/services/

SignalP/). N-Glycosylation sites were predicted by the Net-NGlyc 1.0 Server (http:www.cbs.dtu.dk.service/NetNGlyc/). 5.7. Preparation of a recombinant EgCel17A To aid protein purification and immunoblot analysis of the recombinant enzyme, the coding sequence for seven contiguous histidine residues, 50 -TTAGTGATGGTGATGGTGGTGATGGCTAGG30 , was fused in frame to the 30 end of Egcel17A by PCR. The DNA fragment obtained by PCR using primers, 50 GGCCGCGCCCTCGCCGGCCACAAGCCACGCCAGGAGCTGGGATG-30 and 50 -CAGCCTTAAGTTGCCTTAGTGATGGTGATGGTGGTGATG-30 was cloned into a pPPamyBSP expression vector driven by the PamyB promoter (Yano et al., 2009) using an In-Fusion HD cloning kit (Clontech). The reaction mixture was transformed into E. coli (DH5a) and transformants were selected on LB agar plates supplemented with 50 lg/ml ampicillin. Prepared pPPamyBSP DNA (5 lg) containing the Egcel17A gene was transformed into A. oryzae according to the method described previously (Gomi et al., 1987), and the transformants were screened on Czapek-Dox agar plates containing 0.1 lg/ml pyrithiamine. The A. oryzae transformants were cultured in YPM medium at 28 °C for 3 days. 5.8. Purification of recombinant enzyme Culture medium (1 L) of the A. oryzae transformant was filtered through cheesecloth, concentrated to 100–200 ml using an ultrafiltration spin column (30 kDa cut off). A portion of the sample (5 ml) was added 15 ml of Wash/Equilibration buffer (50 mM sodium phosphate, pH 7.0, 50 mM NaCl) and ultrafiltrated to about 1 ml. The solution was added 20 ml of Wash/Equilibration and ultrafiltrated again to 1 ml. The solution was loaded onto a polyhistidine-binding resin (TALON metal affinity resin, Clontech) equilibrated with Wash/Equilibration buffer, and the resin was washed with the same buffer. Protein bound to the resin was eluted with 1 Elution buffer (50 mM sodium phosphate, pH 7.0, 50 mM NaCl, 200 mM imidazole). The eluate (about 10 ml) was added 10 ml of sodium phosphate buffer (10 mM, pH 6.0) and concentrated to 100–150 ll using an ultrafiltration spin column (10 kDa cut-off). The solution was added 10 ml of sodium phosphate buffer and concentrated to 100–150 ll again. The purity of the proteins was confirmed by electrophoresis on a 12.5% SDS– polyacrylamide gel followed by Coomassie Brilliant Blue R-250 staining. 5.9. Protein assay The protein concentration was determined using a Bradford protein assay kit (Thermo Scientific) with bovine serum albumin (BSA, Sigma–Aldrich) as a standard. 5.10. Hydrolytic activity of recombinant EgCel17A Reaction mixtures (50 lL) containing water-soluble substrate [0.1% (w/v) laminarin, 0.1% (w/v) 1,3–1,4-b-glucan, or 5 mM laminarioligosaccharides (Megazyme)] or water-insoluble substrate [0.05% (w/v) paramylon, 0.05% (w/v) cellulose or 0.05% (w/v) PSC], 100 mM sodium phosphate buffer (pH 5.5) and purified EgCel17A (0.1 lg for water-soluble substrates and 1.0 lg for water-insoluble substrates except for substrate specificity assay shown in Table 1) were incubated. Hydrolytic activity was determined using p-hydroxybenzoic hydrazide-HCl as described above. A unit of activity was defined as the amount of enzyme required to produce reducing power equivalent to 1 lmol of glucose per min. Reaction products generated from laminarin and laminarioligosaccharides were developed by thin layer chromatography (TLC) on

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silica gel plates (60 F254, Merck) in n-BuOH–AcOH–H2O (3:1:1, by vol.) and stained with 0.5% (w/v) thymol in EtOH/H2SO4 (19:1, by vol.). The effect of pH, temperature or metals on hydrolytic activities of EgCel17A was determined to assay laminarin hydrolysis as described above. Kinetic parameters of EgCel17A were determined by double reciprocal plot of initial reaction velocity. 5.11. Effects of Alkaline treatment of Euglena cells and salts on EgCel17A activity Euglena freeze-dried powder was suspended in NaOH at final concentrations of 0–1.5 M and kept for 15 min at room temperature. The suspension pH was adjusted with AcOH to pH 5.5. The NaOH-treated Euglena suspensions equivalent to 1 mg Euglena freeze-dried powder were incubated with EgCel17A (1.0 lg) in 100 ll of 100 mM sodium phosphate buffer (pH 5.5) at 30 °C for 18 h. The Euglena suspension treated with 1 M NaOH followed by washing with 100 mM sodium phosphate buffer (pH 5.5) was incubated with EgCel17A (1.0 lg) in the presence of various concentrations of 0–2.0 M NaCl or sodium acetate (pH 5.5) at 30 °C for 18 h. The supernatants obtained by centrifugation at 22,000g from reaction mixtures were used to determine the increase in reducing power. 5.12. Nucleotide sequence accession numbers DNA sequences of Egcel17A, Egcel81A and Egcel81B have been deposited in the DNA Data Bank of Japan (DDBJ) under accession numbers of AB969741, AB969742 and AB969742, respectively. Acknowledgement This work was supported by Iwate Prefecture. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2015. 05.010. References Barras, D.R., Stone, B.A., 1969a. b-1,3-Glucan hydrolases from Euglena gracilis. I. The nature of the hydrolases. Biochim. Biophys. Acta 191, 329–341. Barras, D.R., Stone, B.A., 1969b. b-1,3-Glucan hydrolases from Euglena gracilis. II. Purification and properties of the b-1,3-glucan exo-hydrolase. Biochim. Biophys. Acta 191, 342–353. Bhatia, Y., Mishra, S., Bisaria, V.S., 2002. Microbial b-glucosidases: cloning, properties, and applications. Crit. Rev. Biotechnol. 22, 375–407. Chen, X.Y., Kim, J.Y., 2009. Callose synthesis in higher plants. Plant Signal Behav. 4, 489–492. Cheng, R., Chen, J., Yu, X., Wang, S., Zhang, J., 2013. Recombinant production and characterization of full-length and truncated b-1,3-glucanase PglA from Paenibacillus sp. S09. BMC Biotechnol. 13, 105–125. Cline, K., Albersheim, P., 1981. Host-pathogen interactions: XVI. Purification and characterization of a beta-glucosyl hydrolase/transferase present in the walls of soybean cells. Plant Physiol. 68, 207–220. de Groot, P.W., Hellingwerf, K.J., Klis, F.M., 2003. Genome-wide identification of fungal GPI proteins. Yeast 20, 781–796. Ducret, A., Trani, M., Lortie, R., 2002. Screening of various glycosidases for the synthesis of octyl glucoside. Biotechnol. Bioeng. 77, 752–757.

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Fry, S.C., Smith, R.C., Renwick, K.F., Martin, D.J., Hodge, S.K., Matthews, K.J., 1992. Xyloglucan endotransglycosylase, a new wall-loosening enzyme activity from plants. Biochem. J. 282, 821–828. Gomi, K., Imura, Y., Hara, S., 1987. Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene. Agric. Biol. Chem. 51, 2549–2555. Gupta, R., Govil, T., Capalash, N., Sharma, P., 2012. Characterization of a glycoside hydrolase family 1b-galactosidase from hot spring metagenome with transglycosylation activity. Appl. Biochem. Biotechnol. 168, 1681–1693. Hartland, R.P., Fontaine, T., Debeaupuis, J.P., Simenel, C., Delepierre, M., Latg, J.P., 1996. A novel b-(1,3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus. J. Biol. Chem. 271, 26843–26849. Henrissat, B., 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280, 309–316. Henrissat, B., Bairoch, A., 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316, 695–696. Henrissat, B., Davies, G., 1997. Structural and sequence-based classification of glycoside hydrolases. Curr. Opin. Struct. Biol. 7, 637–644. Hong, Z., Delauney, A.J., Verma, D.P.S., 2001. A cell plate-specific callose synthase and its interaction with phragmoplastin. Plant Cell 13, 755–768. Johnstone, S.L., Prakash, R., Chen, N.J., Kumagai, M.H., Turano, H.M., Cooney, J.M., Atkinson, R.G., Paull, R.E., Cheetamun, R., Basic, A., Brummell, D.A., Schröder, R., 2013. An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls. Planta 237, 173–187. Kawamura, Y., Uemura, M., 2003. Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation. Plant J. 36, 141–154. Koren, L.E., Hunter, S.H., 1967. High-yield medium for photosynthesizing Euglena gracilis Z. J. Protozool. 14, 17. McCormick, S., 1993. Male gametophyte development. Plant Cell 5, 1265–1275. Michel, G., Tonon, T., Scornet, D., Cock, J.M., Kloareg, B., 2010. Central and storage carbon metabolism of the brown alga Ectocarpus siliculosus: insights into the origin and evolution of storage carbohydrates in eukaryotes. New Phytol. 188, 67–81. Miller, M., 1972. A new reaction for colorimetric determination of carbohydrates. Anal. Biochem. 47, 273–279. Nakajima, M., Yamashita, T., Takahashi, M., Nakano, Y., Takeda, T., 2012. A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in b-1,3-glucan degradation. Appl. Environ. Microbiol. 78, 5682–5689. Nishitani, K., Tominaga, R., 1992. Endo-xyloglucan transferase, a novel class of glycosyltransferase that catalyzes transfer of a segment of xyloglucan molecule to another xyloglucan molecule. J. Biol. Chem. 267, 21058–21064. Peumans, J.W., Barre, A., Derycke, V., Rougé, P., Zhang, W., May, D.G., Delcour, A.J., Van Leuven, F., Van Damme, J.M.E., 2000. Purification, characterization and structural analysis of an abundant b-1,3-glucanase from banana fruit. Eur. J. Biochem. 267, 1188–1195. Ruel, K., Joseleau, J.P., 1991. Involvement of an extracellular glucan sheath during degradation of populous wood by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 57, 374–384. Shi, P., Yao, G., Cao, Y., Yang, P., Yuan, T., Huang, H., Bai, Y., Yao, B., 2011. Cloning and characterization of a new b-mannosidase from Streptomyces sp. S27. Enzyme Microb. Technol. 49, 277–283. Sietsma, J.H., Wessels, J.G.H., 1981. Solubility of (1 ? 3)-b/(1 ? 6)-b-D-glucan in fungal walls: importance of presumed linkages between glucan and chitin. J. Gen. Microbiol. 125, 209–212. Sumida, S., Ehara, T., Osafune, T., Hase, E., 1987. Ammonia- and light-induced degradation of paramylum in Euglena gracilis. Plant Cell Physiol. 28, 1587–1592. Takahashi, M., Konishi, T., Takeda, T., 2011. Biochemical characterization of Magnaporthe oryzae b-glucosidases for efficient b-glucan hydrolysis. Appl. Microbiol. Biotechnol. 91, 1073–1082. Takahashi, M., Yoshioka, K., Imai, T., Miyoshi, Y., Nakano, Y., Yoshida, K., Yamashita, T., Furuta, Y., Watanabe, T., Sugiyama, J., Takeda, T., 2013. Degradation of synthesis of b-glucans by a Magnaporthe oryzae endotransglucosylase, a member of the glycoside hydrolase 7 family. J. Biol. Chem. 288, 13821–13830. Van Bueren, A.L., Morland, C., Gilbert, H.J., Boraston, A.B., 2005. Family 6 carbohydrate binding modules recognize the non-reducing end of b-1,3linked glucanas by presenting a unique ligand binding surface. J. Biol. Chem. 280, 530–537. Yano, A., Kikuchi, S., Nakagawa, Y., Sakamoto, Y., Sato, T., 2009. Secretory expression of the non-secretory-type Lentinula edodes laccase by Aspergillus oryzae. Microbiol. Res. 164, 642–649.