Lentinan Degradation in the Lentinula edodes Fruiting Body during

Jul 17, 2014 - Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable β-glucan for medical purposes based on its anticance...
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Lentinan Degradation in the Lentinula edodes Fruiting Body during Postharvest Preservation Is Reduced by Downregulation of the exoβ-1,3-Glucanase EXG2 Naotake Konno,*,†,‡ Keiko Nakade,† Yosuke Nishitani,§ Masashi Mizuno,§ and Yuichi Sakamoto*,† †

Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami-shi, Iwate 024-0003, Japan Department of Applied Biological Chemistry, Utsunomiya University, 350 mine-machi, Utsunomiya, Tochigi 321-8505, Japan § Department of Agrobioscience, Graduate School of Agricultural Science, Kobe University, 1-1, Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan ‡

S Supporting Information *

ABSTRACT: Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable β-glucan for medical purposes based on its anticancer activity and immunomodulating activity. However, lentinan content in fruiting bodies decreases after harvesting and storage due to an increase in glucanase activity. In this study, we downregulated the expression of an exo-β-1,3glucanase, exg2, in L. edodes using RNA interference. In the wild-type strain, β-1,3-glucanase activity in fruiting bodies remarkably increased after harvesting, and 41.7% of the lentinan content was lost after 4 days of preservation. The EXG2 downregulated strain showed significantly lower lentinan degrading activity (60−70% of the wild-type strain) in the fruiting bodies 2−4 days after harvesting. The lentinan content of fresh fruiting bodies was similar in the wild-type and EXG2 downregulated strains, but in the downregulated strain, only 25.4% of the lentinan was lost after 4 days, indicating that downregulation of EXG2 enables keeping the lentinan content high longer. KEYWORDS: shiitake mushroom, lentinan, β-glucan, β-glucanase

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have the ability to degrade the cell-wall β-glucans in L. edodes fruiting bodies, suggesting they are involved in fruiting body autolysis.14 Among these β-glucanases, EXG2 shows the highest activity in vitro toward lentinan in reaction tests.13 The complementary DNA of EXG2, exg2, includes an open reading frame of 2343 bp encoding a 21 amino acid signal peptide and a 759 amino acid mature protein. Sequence analysis indicated that EXG2 is a member of glycoside hydrolase (GH) family 55, and GH 55 exo-β-1,3-glucanases are found only in filamentous fungi (CAZy: http://www.cazy.org). The expression of exg2 increases after harvesting, and this pattern correlates with a decrease in lentinan in L. edodes fruiting bodies. From these characteristics of EXG2, we deduced that the enzyme strongly enhances lentinan degradation in L. edodes fruiting bodies after harvesting. Downregulation of gene expression in fruiting bodies has been reported in some basidiomycetes such as Agaricus bisporus and Pleurotus ostreatus.21,22 Previously, we showed that RNA interference (RNAi) could be used for gene silencing in L. edodes.23 RNAi is a post-transcriptional gene silencing phenomenon in which double-stranded RNA (dsRNA) triggers degradation of cognate mRNA in a sequence-specific manner. In this study, we downregulated the expression of the exg2 gene in an L. edodes strain using RNAi and raised fruiting bodies of the exg2 downregulated transformant (ivr-exg2#51). Analysis of

INTRODUCTION The cell wall of fungi is typically composed of chitin, β-1,3-, and β-1,6-glucans. Among these cell-wall polysaccharides, several βglucans from basidiomycetes have been reported to possess physiological activities.1−3 Lentinula edodes (shiitake mushroom) is one of the most widely cultivated edible mushrooms and is highly valued for its medical applications. The watersoluble β-1,3-/1,6-glucans from L. edodes fruiting bodies are called lentinan4 and have been reported to have anticancer and immunomodulating activity.5−8 Lentinan is used clinically as an antitumor and antiallergy agent. Lentinan has a high molecular weight (degree of polymerization range 5400−8900) and a chemical structure of linear β-1,3-glucans with two β-1,6glucosyl side chains every five residues.9,10 It has been suggested that these features of lentinan, the main chain length and the extent of side chains, contribute to its physiological activities.11 Basidiomycetes such as L. edodes form fruiting bodies for sporulation, and the cell wall components of fruiting bodies undergo autolysis after sporulation or artificial harvesting.12−15 Although lentinan can be purified from fresh L. edodes fruiting bodies, it is rapidly degraded during storage as a result of an increase in glucanase activity.16,17 Minato et al. (1999) have reported that lentinan content decreases to less than half of the original during 5 days of storage at 20 °C. Previously, we isolated four β-glucanases, an exo-β-1,3-glucanase, EXG2, an endo-β-1,3-glucanase, TLG1, an endo-β-1,3-glucanase, GLU1, and an endo-β-1,6-glucanase, LePus30A, from L. edodes fruiting bodies 4 days after harvest.13,18−20 These enzymes are produced in particular during fruiting body senescence and © 2014 American Chemical Society

Received: April 7, 2014 Accepted: July 10, 2014 Published: July 17, 2014 8153

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pNP was determined spectrophotometrically at 405 nm. The extinction coefficient of pNP was assumed to be 17 100 M−1 cm−1.28 One unit (U) of enzyme activity was defined as the amount of enzyme that produces 1 μmol of reducing sugar per minute. Measurement of Lentinan Content in Fruiting Bodies. The L. edodes fruiting body samples prepared as above (approximately 15 g) were freeze-dried and crushed. A water-soluble cell wall polysaccharide mixture was extracted by autoclaving in 20 mL of water (120 °C, 20 min). The hot-water extract was recovered from the supernatant by ethanol precipitation, and the dried precipitate was used for measurement of lentinan content. The lentinan content of the samples was determined by an ELISA inhibition assay with antilentinan antibodies prepared previously.16,29

the phenotype of ivr-exg2#51 indicated that EXG2 is a key enzyme in lentinan degradation in L. edodes fruiting bodies after harvest.

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MATERIALS AND METHODS

Strains and Culture Conditions. A dikaryotic strain of L. edodes SR-1 was used as the recipient host in all transformation experiments. Mycelia were maintained on 0.25 × MYPG medium containing 0.25% Bacto malt extract (Difco, Detroit, MI, USA), 0.1% Bacto yeast extract (Difco), 0.1% tryptone peptone (Difco), and 0.5% glucose. For postharvest preservation, fruiting bodies were prepared following a previously described method.13 The harvested mature fruiting bodies were immediately transferred to a room at 25 °C with 80% humidity.24 After harvest, fruiting bodies were sampled every day for 4 days, with day 0 being defined as corresponding to a fresh mature fruiting body just prior to postharvest preservation. After postharvest preservation, stipes were removed from samples and then immediately frozen in liquid nitrogen. For following enzymatic activity and lentinan content assays, three separate samples were prepared per data. Construction of the pChG′ and pChG′-ivrexg2 Vectors. The pChG′-Gateway vector containing a multicloning site was constructed by Nakade et al. from vector pChG.23,25 pChG′ was digested by EheI, and the Gateway cassette (Gateway Vector Conversion System; Invitrogen, CA, USA) was ligated into the digested pChG′ vector (pChG′-Gateway), then used to transform Escherichia coli competent cells (One Shot ccdB Survival cells, Invitrogen). To construct the RNAi vectors, we synthesized the 250-bp short homologous hairpin dsRNA sequences of the exg2,13 separated by a 60-bp spacer sequence of lcc1 intron 2 (Figure S1, Supporting Information) (MediBIC Group, Co., Ltd., Tokyo, Japan). Synthesized DNA was inserted into vector pDONR221 (Invitrogen) and then transformed into pChG′-Gateway by an LR reaction (Invitrogen). L. edodes Transformation. The SR-1 strain was transformed with 2.5 μg of DNA of the above vectors by the restriction enzymemediated integration (REMI) method using 5.0 U SphI.26 Transformants were selected on 0.25 × MYPG containing 20 μg mL−1 of hygromycin B. Protein Extraction. The frozen pileus and gill parts of the fruiting bodies (0−4 days after harvest, 2 g) were suspended in 0.5 mL of 100 mM sodium acetate buffer (pH 4.2) and mixed for 60 min at room temperature. The extracts containing protein were collected by centrifugation (10 000g, 20 min) and filtration (Ultrafree MC HV 0.45 μm filter, Merck Millipore, Darmstadt, Germany). The extracts were desalted and concentrated using an Amicon Ultra 10,000 NMWL filter (Merck Millipore), and 2 μg of the samples was used for Western blotting as described below. Protein Assays. Protein concentration was measured by the Bradford method using the Bio-Rad Protein Assay kit (Bio-Rad Laboratory, CA USA) with a bovine serum albumin (BSA) (Pierce, Rockford, IL, USA) standard. Protein concentration was monitored by absorbance at 595 nm. Western Blotting. For Western blot analysis, fruiting bodies after postharvest preservation and aging fruiting bodies were sampled as described above. Samples were frozen and crushed in liquid nitrogen, suspended in extraction buffer (200 mM sodium acetate, pH 4.2), and incubated by rotation for 15 min at room temperature. Electrophoresis and blotting were performed as described previously.24 For Western blot analysis, EXG2 antiserum was used as the primary antibody, and antirabbit Ig conjugated with horseradish peroxidase was used as the secondary antibody. ECL detection reagents were used for detection (GE Healthcare, Little Chalfont, UK).13 Measurement of Enzymatic Activity. To measure enzymatic activity in the fruiting bodies, extracts prepared as above were assayed using lentinan (0.5%), pustulan (0.5%), Curdlan (1%), and pnitrophenyl-N-acetyl-β-D-glucosaminide (pNP-GlcNAc, 0.5 mM, Sigma-Aldrich Inc., St. Louis, MO, USA) as substrates in 50 mM sodium acetate buffer (pH 4.2) at 37 °C. Reducing sugars liberated from lentinan, pustulan, and Curdlan were analyzed according to the Somogyi−Nelson method.27 To assay pNP-GlcNAc, the amount of

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RESULTS AND DISCUSSION Constructing Downregulated Transformants of exg2. An exg2 homologous hairpin dsRNA expression vector (pChG′-ivr-exg2) was constructed (Figure S1, Supporting Information) and introduced to L. edodes using REMI-based transformation, allowing integration in the genome.23,26 We obtained 67 transformants of pChG′-ivr-exg2; 6 transformants were selected and tested for EXG2 expression after harvesting of fruiting bodies. Expression of EXG2 in wild type increased after harvesting.13 Expression of EXG2 in ivr-exg2#7 was higher, but its expression in #26, #39, and #51 was significantly suppressed (Figure 1). Expression of the β-glucanase TLG1

Figure 1. Western blot analysis of the crude enzyme from fruiting bodies 4 days after harvest. Selected transformants of pChG′-ivr-exg2 (ivr-exg2 #7, #22, #26, #39,#50, #51) were tested. Western blot analysis was performed using (A) EXG2 antiserum and (B) TLG1 antiserum.

was also suppressed in #26 and #39 for unknown reasons but was similar to wild-type levels in #51.18 These results suggest that expression of exg2 is specifically suppressed in ivr-exg2#51. Analyzing the Phenotype of ivr-exg2#51. Fruiting bodies of the EXG2 downregulated transformant (ivrexg2#51), the pChG′ vector transformant (pChG′), and the control (SR-1) were cultivated, harvested, and preserved for 4 days (Figure S2, Supporting Information). As shown in Figure 2, EXG2 production of ivr-exg2#51 in fruiting bodies preserved for 2−4 days was significantly suppressed compared to pChG′ and SR-1. The EXG2 suppression was also observed in the transcription level (Figure S3, Supporting Information). Sakamoto et al. reported that EXG2 is involved in selfdegradation of cell-wall β-1,3-glucans, causing lentinan degradation during postharvest preservation.13 To analyze enzyme activity in the fruiting bodies after harvest, crude enzymes were extracted from fruiting bodies and tested for activity on substrates related to fungal cell-wall components. As shown in Figure 3A and 3B, β-1,3-glucanase activities of pChG′ 8154

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Table 1. Changes in Lentinan Content in the Fruiting Bodies of SR-1 (Original Strain), pChG′ (Control Strain), and an EXG2 Downregulated Strain (ivr-exg2#51) after Harvestinga SR-1 pChG′ ivr-exg2#51

Figure 2. Western blotting pattern of extracts from the fruiting bodies of SR-1 (original strain), PchG′ (control strain), and an EXG2 downregulated strain (ivr-exg2#51) using EXG2 antibody. Fruiting bodies were preserved for 0−4 days after harvesting, and the extracts (2 μg of protein) were separated by SDS-PAGE.

a

D0 (mg/g dw)

D4 (mg/g dw)

residual ratio (%)

4.16 ± 0.69 4.70 ± 0.89 5.45 ± 0.35

1.64 ± 1.07 1.80 ± 1.00 3.95 ± 1.30

39.4 38.1 72.5

The data indicate means ± SD (n = 3 samples).

only 27.5%, demonstrating that ivr-exg2#51 preserves the lentinan content over a longer time than the original strain. Previously, we purified two other β-1,3-glucanases, TLG1 and GLU1, next to EXG2 from harvested L. edodes fruiting bodies.18,19 These two enzymes are also expressed in fruiting bodies after harvest, suggesting that they are involved in fruiting body senescence. The β-1,3-glucanase activity and the decrease in lentinan content in harvested ivr-exg2#51 fruiting bodies might be caused primarily by the actions of TLG1 and GLU1. However, their β-1,3-glucanase activities are weaker than that of EXG2. Moreover, TLG1 and GLU1 have been suggested to have strict substrate specificity for β-1,3-glucan polymers; β-1,6linkages within β-1,3/1,6-glucan would prevent hydrolysis of the β-1,3-linkages, and the enzymes showed low activity toward β-1,3-glucan oligomers. Therefore, TLG1 and GLU1 might only perform a partial degradation of cell-wall β-1,3-glucans at the fruiting body senescence stage. On the other hand, EXG2 can cleave almost all β-1,3-linkages within β-1,3-glucans and β1,3/1,6-glucans regardless of their length and branching structure.30 Indeed, lentinan is completely degraded to glucose and gentiobiose by the exotype manner of action of EXG2.15 EXG2 belongs to GH 55, and the crystal structure of a GH 55 exo-β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium, homologous to EXG2 from L. edodes, has been reported.30 This P. chrysosporium enzyme appears to be able to accept a gentiobiose unit in the cleavage site owing to its substrate-binding pocket structure, suggesting that GH 55 exo-

and SR-1 remarkably increased after harvesting of fruiting bodies, whereas the activity of ivr-exg2#51 toward a watersoluble β-1,3/1,6-glucan (lentinan) was approximately 60−70% lower than that of pChG′ and SR-1. Moreover, the enzymatic activity against a water-insoluble β-1,3-glucan (Curdlan) was also decreased in the EXG2 downregulated strain; the activity of ivr-exg2#51 at day 2 was approximately 80% lower than that of SR-1 and pChG′, and the activity at day 4 was approximately 60−75% lower (Figure 3B). When β-1,6-glucan (pustulan) and artificial chitin oligosaccharide (pNP-GlcNAc) were used as substrates, no significant difference was observed in enzymatic activity (Figure 3C and 3D). These results indicate that downregulation of EXG2 specifically caused the reduction of β1,3-glucanase activity in the fruiting bodies after harvest. The lentinan content of hot-water extracts prepared from L. edodes fruiting bodies was determined using antibody-based detection.16,29 The lentinan content of fresh fruiting bodies (D0) was almost similar, 4.1−5.5 mg/g dry weight (dw) in SR1, pChG′, and ivr-exg2#51. Table 1 shows the changes in lentinan content in the fruiting bodies after harvest; 60.6% of the lentinan content was lost in SR-1, and 61.9% was lost in pChG′ after 4 days of preservation. However, the loss in lentinan content of ivr-exg2#51 after 4 days of preservation was

Figure 3. Changes in enzymatic activities in the fruiting bodies of SR-1 (original strain, white), pChG′ (control strain, gray), and an EXG2 downregulated strain (ivr-exg2#51, black) after harvesting. Lentinan (A), Curdlan (B), pustulan (C), and pNP-GlcNAc (D) were used as substrates. The data indicate means ± SD (n = 3 samples). 8155

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β-1,3-glucanases can bypass the β-1,6-glucosyl side chains in lentinan. In this study, approximately 75% of the lentinan content in L. edodes was preserved for 4 days by the downregulation of only EXG2. This suggests that EXG2 is the most effective β-1,3-glucanase in cell-wall autolysis of fruiting bodies after artificial harvesting. Lentinan from L. edodes fruiting bodies is a very valuable polysaccharide for medical applications because of its anticancer activity and immunomodulating activity. Lentinan is obtained only from fresh fruiting bodies and decreases rapidly during postharvest storage due to the actions of β-1,3-glucanases. This study indicates that a knockout or downregulation of EXG2 in L. edodes enables a significantly more stable supply of lentinan.

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(6) Mizuno, M.; Nishitani, Y.; Hashimoto, T.; Kanazawa, K. Different suppressive effects of fucoidan and lentinan on IL-8 mRNA expression in in vitro gut inflammation. Biosci. Biotechnol. Biochem. 2009, 73, 2324−2325. (7) Xu, X.; Pan, C.; Zhang, L.; Ashida, H. Immunomodulatory βglucan from Lentinus edodes activates mitogen-activated protein kinases and nuclear factor-kappaB in murine RAW 264.7 macrophages. J. Biol. Chem. 2011, 286, 31194−31198. (8) Xu, X.; Yasuda, M.; Nakamura-Tsuruta, S.; Mizuno, M.; Ashida, H. β-Glucan from Lentinus edodes inhibits nitric oxide and tumor necrosis factor-α production and phosphorylation of mitogen-activated protein kinases in lipopolysaccharide-stimulated murine RAW 264.7 macrophages. J. Biol. Chem. 2012, 287, 871−878. (9) Chizhov, A. O.; Dell, A.; Morris, H. R.; Reason, A. J.; Haslam, S. M.; McDowell, R. A.; Chizhov, O. S.; Usov, A. I. Structural analysis of laminarans by MALDI and FAB mass spectrometry. Carbohydr. Res. 1998, 310, 203−210. (10) Kim, Y. T.; Kim, E. H.; Cheong, C.; Williams, D. L.; Kim, C. W.; Lim, S. T. Structural characterization of β-D-(1−>3, 1−>6)-linked glucans using NMR spectroscopy. Carbohydr. Res. 2000, 328, 31−41. (11) Adams, E. L.; Rice, P. J.; Graves, B.; Ensley, H. E.; Yu, H.; Brown, G. D.; Gordon, S.; Monteiro, M. A.; Papp-Szabo, E.; Lowman, D. W.; Power, T. D.; Wempe, M. F.; Williams, D. L. Differential highaffinity interaction of dectin-1 with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and side-chain branching. J. Pharmacol. Exp. Ther. 2008, 325, 115−123. (12) Kües, U. Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiol. Mol. Biol. Rev. 2000, 64, 316−353. (13) Sakamoto, Y.; Minato, K.; Nagai, M.; Kawakami, S.; Mizuno, M.; Sato, T. Characterization of the Lentinula edodes exg2 gene encoding a lentinan-degrading exo-β-1,3-glucanase. Curr. Genet. 2005, 48, 195− 203. (14) Sakamoto, Y.; Nakade, K.; Sato, T. Characterization of the postharvest changes in gene transcription in the gill of the Lentinula edodes fruiting body. Curr. Genet. 2009, 55, 409−423. (15) Sakamoto, Y.; Nakade, K.; Konno, N.; Sato, T. Senescence of the Lentinula edodes fruiting body after harvesting. Food Quality; 2012; Chapter 6, pp 83−110, ISBN: 978-953-51-0560-2. (16) Minato, K.; Mizuno, M.; Terai, H.; Tsuchida, H. Autolysis of lentinan, an antitumor polysaccharide, during storage of Lentinus edodes, Shiitake mushroom. J. Agric. Food Chem. 1999, 47, 1530−1532. (17) Minato, K.; Kakawami, S.; Nomura, K.; Tsuchida, H.; Mizuno, M. An exo-β-1,3 glucanase synthesized de novo dgrades lentinan during storage of Lentinula edodes and diminishes immunomodulationg activity of the mushroom. Carbohydr. Polym. 2004, 56, 279−286. (18) Sakamoto, Y.; Watanabe, H.; Nagai, M.; Nakade, K.; Takahashi, M.; Sato, T. Lentinula edodes tlg1 encodes a thaumatin-like protein that is involved in lentinan degradation and fruiting body senescence. Plant Physiol. 2006, 141, 793−801. (19) Sakamoto, Y.; Nakade, K.; Konno, N. Endo-β-1,3-glucanase GLU1, from the fruiting body of Lentinula edodes, belongs to a new glycoside hydrolase family. Appl. Environ. Microbiol. 2011, 77, 8350− 8354. (20) Konno, N.; Sakamoto, Y. An endo-β-1,6-glucanase involved in Lentinula edodes fruiting body autolysis. Appl. Microbiol. Biotechnol. 2011, 91, 1365−1373. (21) Eastwood, D. C.; Challen, M. P.; Zhang, C.; Jenkins, H.; Henderson, J.; Burton, K. S. Hairpin-mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus. Mycol. Res. 2008, 112, 708−716. (22) Salame, T. M.; Knop, D.; Tal, D.; Levinson, D.; Yarden, O.; Hadar, Y. Predominance of a versatile-peroxidase-encoding gene, mnp4, as demonstrated by gene replacement via a gene targeting system for Pleurotus ostreatus. Appl. Environ. Microbiol. 2012, 78, 5341−5352. (23) Nakade, K.; Watanabe, H.; Sakamoto, Y.; Sato, T. Gene silencing of the Lentinula edodes lcc1 gene by expression of a

ASSOCIATED CONTENT

S Supporting Information *

Construction of a RNAi vector pChG′-ivr-exg2 (Figure S1). Pictures of fruiting body of the EXG2 downregulated transformant (Figure S2). Analysis of the transcription level of the exg2 gene in the fruiting bodies of SR-1 (original strain), pChG′ (control strain), and an EXG2 downregulated strain (ivr-exg2#51) after harvesting. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +81-197-68-2911. Fax: +81-197-68-3881. E-mail: [email protected] (Y. Sakamoto). *E-mail: [email protected] (N. Konno). Funding

This research was supported by a Grant-in-Aid for Scientific Research to N.K. (no. 2510648) from the Japan Society for the Promotion of Science (JSPS), by grants for project research (Development of fundamental technology for analysis and evaluation of functional agricultural products and functional foods), and by the Japan Science and Technology Agency (JST). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Akiko Uchidate, Miyuki Ito, Ayumi Obara, Junko Kawaguchi, and Shiho Sato for their help with experiments. We are grateful to Dr. Arend F. van Peer for his suggestions and comments.

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REFERENCES

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homologous inverted repeat sequence. Microbiol. Res. 2011, 166, 484− 493. (24) Sakamoto, Y.; Irie, T.; Sato, T. Isolation and characterization of a fruiting body-specific exo-β-1,3-glucanase-encoding gene, exg1, from Lentinula edodes. Curr. Genet. 2005, 47, 244−252. (25) Sato, T.; Okawa, K.; Hirano, T. Construction of novel vectors for transformation of Lentinula edodes using a chitin synthase gene promoter. J. Biosci. Bioeng. 2011, 111, 117−120. (26) Sato, T.; Yaegashi, K.; Ishii, S.; Hirano, T.; Kajiwara, S.; Shishido, K.; Enei, H. Transformation of the edible basidiomycete Lentinus edodes by restriction enzyme-mediated integration of plasmid DNA. Biosci. Biotechnol. Biochem. 1998, 62, 2346−2350. (27) Somogyi, M. Notes on sugar determination. J. Biol. Chem. 1952, 195, 19−23. (28) Konno, N.; Takahashi, H.; Nakajima, M.; Takeda, T.; Sakamoto, Y. Characterization of β-N-acetylhexosaminidase (LeHex20A), a member of glycoside hydrolase family 20, from Lentinula edodes (shiitake mushroom). AMB Express 2012, 2, 29. (29) Mizuno, M.; Minato, K.; Tsuchida, H. Preparation and specificity of antibodies to an anti-tumor β-glucan, lentinan. Biochem. Mol. Biol. Int. 1996, 39, 679−685. (30) Ishida, T.; Fushinobu, S.; Kawai, R.; Kitaoka, M.; Igarashi, K.; Samejima, M. Crystal structure of glycoside hydrolase family 55 β-1,3glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 2009, 284, 10100−10109.

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dx.doi.org/10.1021/jf501578w | J. Agric. Food Chem. 2014, 62, 8153−8157