《食品生物化学》课程教学资源（文献资料）第二章酶 Important role of fungal intracellular laccase for melanin synthesis：purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies.pdf_大学文库

Mcro6obgy(2003),149,2455-2462 D0110.1099/mic.026414-0 Important role of fungal intracellular laccase for melanin synthesis:purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies Masaru Nagai,Maki Kawata.Hisayuki Watanabe,Machiko Ogawa. Kumiko Saito,Toshikazu Takesawa,Katsuhiro Kanda and Toshitsugu Sato lwate Biotechnology Research Center,22-174-4 Narita Kitakami,Iwate 024-0003,Japan A laccase (EC 1.1032)was isolated from the fully br ed aills of len and size-exclusion chromatography.SDS-PAGEanalysis showed the purified laccase,Lcc 2,to be a monomeric protein of 58-0 kDa.The enzyme had an isoelectric point of around pH69.The optimum pH for enzyme activity was around 3-0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6 sulfonic acid)dian n salt (ABTS),and it was most active at 40( and stable up to 50 The enzyme com d8-69 ana some copper atoms. and oga bu -3.4-D oxidized by a laccase pre from the culture filtrate ofedodes.wasas oidized ceived 10 Aoril 2005 by Lcc 2,and the oxidative product of L-DOPA was identified as L-DOPA quinone by HPLC Revised 13 June 2003 analysis.Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to Accepted 13 June 2003 brown-coloured products,suggesting a role for laccase in melanin synthesis in this strain INTRODUCTION post-harvest ng of Lentin The mechanisms of mushroom browning hav beer T undesirable since it causes n unpleasant and ).Browning r in this species is mainly due to DOPA and GDHB melanins (Jolivet et al. m05 ant ro cler,1986).In general,fungal me ins are th of I harvest storage and that gill browning increased with increasing Tyr activity (Kanda et al.,1996a). GHB),catecho l melanin d fron cases,these phenolic compounds are oxidized enzymically products via various pathways.Recently,Castro-Sowinski eans to b es of melanin npared with strains without this Lcc da et al. 2002) bis(-thy/o mel anin sy nd i Th from L-DOPA and DHN,and an Lcc gene of Aspergillus GD in th In 0002-64142003 SGM Printed in Grear Britain 2455

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies Masaru Nagai, Maki Kawata, Hisayuki Watanabe, Machiko Ogawa, Kumiko Saito, Toshikazu Takesawa, Katsuhiro Kanda and Toshitsugu Sato Correspondence Masaru Nagai nagai@ibrc.or.jp Iwate Biotechnology Research Center, 22-174-4 Narita, Kitakami, Iwate 024-0003, Japan Received 10 April 2003 Revised 13 June 2003 Accepted 13 June 2003 A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58?0 kDa. The enzyme had an isoelectric point of around pH 6?9. The optimum pH for enzyme activity was around 3?0 against 2,29-azino-bis(3-ethylbenzothiazoline-6- sulfonic acid)diammonium salt (ABTS), and it was most active at 40 6C and stable up to 50 6C. The enzyme contained 8?6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. b-(3,4-Dihydroxyphenyl)alanine (L-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of L-DOPA was identified as L-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain. INTRODUCTION The post-harvest preservation or mishandling during picking of Lentinula edodes fruit bodies causes a brown surface discoloration. This gill browning is commercially undesirable since it causes an unpleasant appearance and the concomitant development of an off-flavour, and it is considered to be due to melanin biosynthesis as a result of a stress response. Melanin is known to protect fungi from environmental stresses, such as UV radiation, elevated temperatures, antimicrobial agents and lytic enzymes (Bell & Wheeler, 1986). In general, fungal melanins are classified into four types: b-(3,4-dihydroxyphenyl)alanine (DOPA) melanin derived from tyrosine, c-glutaminyl-3,4-dihydroxybenzene (GDHB) melanin derived from c-glutaminyl-4- hydroxybenzene (GHB), catechol melanin derived from catechol and dihydroxynaphthalene (DHN) melanin derived from pentaketide (Bell & Wheeler, 1986). In all cases, these phenolic compounds are oxidized enzymically to quinones, which polymerize by non-enzymic means to form the melanin pigments. Oxidation of these phenolic compounds is commonly catalysed by tyrosinase (Tyr; EC 1.14.18.1). The mechanisms of mushroom browning have been investigated extensively in Agaricus bisporus (Burton, 1998; Espı´n et al., 1999). Browning in this species is mainly due to DOPA and GDHB melanins (Jolivet et al., 1998), and Tyr seems to play the most important role in their synthesis (Turner, 1974). Burton (1988) reported that epidermal tissues of A. bisporus had a greater activity of non-latent Tyr and a greater concentration of phenols than did the flesh. Previously, we also reported that Tyr activity of L. edodes fruit bodies increased during postharvest storage and that gill browning increased with increasing Tyr activity (Kanda et al., 1996a). Laccases (Lcc; EC 1.10.3.2), catalyse the single-electron oxidation of phenols or aromatic amines to form different products via various pathways. Recently, Castro-Sowinski et al. (2002) reported that a strain of Sinorhizobium meliloti with an intracellular Lcc synthesized different types of melanin compared with strains without this Lcc. Ikeda et al. (2002) also reported a correlation between melanin synthesis and intracellular Lcc in Cryptococcus neoformans. The melanins of Aspergillus conidia are formed from L-DOPA and DHN, and an Lcc gene of Aspergillus nidulans is specifically expressed in the conidia (Aramayo & Timberlake, 1990). In addition, Clutterbuck (1972) Abbreviations: ABTS, 2,29-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt; DHN, dihydroxynaphthalene; L-DOPA, b-(3,4- dihydroxyphenyl)alanine; GDHB, c-glutaminyl-3,4-dihydroxybenzene; GHB, c-glutaminyl-4-hydroxybenzene; Lcc, laccase; Tyr, tyrosinase; PB, 10 mM sodium phosphate buffer. 0002-6414 G 2003 SGM Printed in Great Britain 2455 Microbiology (2003), 149, 2455–2462 DOI 10.1099/mic.0.26414-0

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 showed that yellow-spored mutants of Aspergillus nidulans are deficient in Lcc. Although there are many reports dealing with extracellular Lccs produced by white-rot basidiomycetes (Leonowicz et al., 2001), there are few studies of the intracellular Lccs produced by these fungi (Burke & Cairney, 2002; Schlosser et al., 1997; Roy-Arcand & Archibald, 1991). In A. bisporus, the biological signifi- cance of intracellular Lccs is considered to be very limited because of their low levels (Turner, 1974), but their significance remains unclear. In this paper, we continue in our attempt to clarify the relationship between gill browning and Lcc activity in L. edodes through the isolation and characterization of an Lcc from the mature gills and through the comparison of some properties of the purified enzyme with those of an extracellular Lcc (Lcc 1) purified previously from L. edodes (Nagai et al., 2002). To our knowledge, this is the first report of the purification of intracellular Lcc from a basidiomycete. METHODS Chemicals. Unless otherwise stated, all chemicals were certified reagent grade purchased from Wako Pure Chemicals. Lcc 1, an extracellular Lcc, was purified from the culture filtrate of L. edodes SR-1 as described by Nagai et al. (2002). Tyr was purified from the gills of L. edodes strain Hokken 600 (H 600) as described by Kanda et al. (1996b). Organisms and culture conditions. A commercial dikaryotic strain of L. edodes, strain H 600, was obtained from Hokken Sangyo and was used throughout this study. Mycelia were maintained on 1?5 % agar plates (diam. 90 mm) with 0?256 MYPG medium containing 0?25 % Bacto malt extract (Difco), 0?1 % Bacto yeast extract (Difco), 0?1 % tryptone peptone (Difco) and 0?5 % glucose. For production of fruit bodies, mycelia were cultivated for 50 days in sawdust medium containing 3?7 kg sawdust, 1?3 kg Baideru (a nutrient supplement for mushroom production; Hokken Sangyo) and 7?6 l water according to the method of Matsumoto (1988). Fruit bodies were harvested immediately after the veil had broken. Preparation of crude extract from fruit bodies. Fruit bodies were separated into caps (pigmented rind and flesh), stipes and gills and frozen by liquid nitrogen. The frozen tissue was suspended in 10 times its volume of 10 mM sodium phosphate buffer (PB), pH 6?0, and homogenized using an Excel Auto Homogenizer (Nihon Seiki) at 10 000 r.p.m. for 1 min. The homogenate was centrifuged at 12 000 g for 20 min, after which the supernatant was collected as the crude extract. Purification of Lcc from the browned gills. All steps were carried out at 4 uC. Column chromatography was operated with a gradient controller (AC-5900 GradiCon III; ATTO) or with an FPLC system (Pharmacia). A crude extract was prepared from 40 g sliced, fully browned gills. Powdered ammonium sulfate was then added to the extract to achieve 30 % saturation and the resulting precipitate was removed by centrifugation at 12 000 g for 20 min. The supernatant was applied to a TOYOPEARL Butyl-650 M (Tosoh) column (25680 mm) equilibrated with PB containing 30 % saturated ammonium sulfate. The column was washed with the same buffer and adsorbed proteins were eluted by a linear concentration gradient of ammonium sulfate (300 ml, 30–0 % saturation) in PB, at a flow rate of 2 ml min21 . The fractions containing Lcc activity were collected, dialysed against 20 mM sodium acetate buffer, pH 4?0, and applied to a TOYOPEARL CM-650 M (Tosoh) column (10650 mm) equilibrated with 20 mM sodium acetate buffer, pH 4?0. After washing the column with the same buffer, the adsorbed proteins were eluted by a linear concentration gradient of NaCl (40 ml, 0–500 mM) at a flow rate of 1 ml min21 . The Lcc active fractions were pooled and concentrated to about 250 ml by ultrafiltration using a Centricon-30 concentrator (30 kDa cut-off; Amicon). The concentrated enzyme solution was applied to a Superdex 75 HR 10/30 column (1630 cm; Pharmacia) equilibrated with PB containing 100 mM NaCl. The enzyme was eluted with the same buffer at a flow rate of 250 ml min21 . Fractions exhibiting Lcc activity were pooled and dialysed against PB, and powdered ammonium sulfate was added to achieve 20 % saturation. The enzyme solution was then applied to a Phenyl Superose HR 5/5 column (5650 mm, Pharmacia) equilibrated with PB containing 20 % saturated ammonium sulfate. After washing the column with the same buffer, the final elution was with a linear concentration gradient of ammonium sulfate (20 ml, 20–0 % saturation) in PB, at a flow rate of 500 ml min21 . Enzyme assay. To determine Lcc activity, 2,29-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) (Sigma) was used as the substrate. The reaction mixture for the standard assay contained 1 mM ABTS, McIlvaine buffer (pH 3?0) and the enzyme solution in a total volume of 100 ml. After incubation at 30 uC for 20 min, the reaction was stopped by adding 100 ml 5 % trichloroacetic acid. The formation of the cation radical was detected by measuring the absorbance increase at 420 nm (e420=36 000 M21 cm21 ). One unit of Lcc activity was defined as the amount of enzyme that catalysed the oxidation of 1 mmol ABTS in 100 ml reaction mixture at 30 uC in 1 min. Tyr activity was measured at pH 6?0 using 1 mM catechol as the substrate. The formation of the cation radical was detected by measuring the absorbance increase at 450 nm (e450=2211 M21 cm21 ). One unit of Tyr activity was defined as the amount of enzyme that catalysed the oxidation of 1 mmol catechol in 100 ml reaction mixture at 30 uC in 1 min. Protein assay. Protein concentration was measured with BCA Protein Assay Reagent (Pierce) using BSA (Sigma) as the standard. During Lcc purification steps, protein concentration was monitored spectrophotometrically by A280. Electrophoresis. Native PAGE was performed according to the method of Davis (1965) using a 5–20 % polyacrylamide gradient gel (NPG-520L PAGEL; ATTO) and Premixed 106Tris/Glycine Buffer (Bio-Rad). SDS-PAGE was performed according to the method of Laemmli (1970) using a 10 % polyacrylamide gel (NPU-10L PAGEL; ATTO) and Premixed 106Tris/Glycine/SDS Buffer (Bio-Rad). The samples were boiled in 2 % SDS and 5 % 2-mercaptoethanol for 10 min and then applied to the gel. The isoelectric point (pI) of the enzyme was measured in an isoelectric focusing gel between pH 3?5 and 9?5 (Ampholine PAG plate; Pharmacia) and Multiphore II system (Pharmacia) using an isoelectric focusing calibration kit, pH 3?5–9?3 (Pharmacia). Proteins were stained with Coomassie brilliant blue R 250 (PAGE Blue 83; Daiichi Chemicals). Activity staining was carried out by incubating the gel after native PAGE at room temperature in McIlvaine buffer (pH 3?0) with 1 mM ABTS. Estimation of molecular mass. The molecular mass of the enzyme was estimated by two methods: (1) gel filtration on a Superdex 75 HR 10/30 column with Gel filtration standards (BioRad); and (2) SDS-PAGE as described above with Precision Protein Standards (Bio-Rad). The molecular mass of the enzyme was calculated from the mobility versus molecular mass plots of the marker proteins. 2456 Microbiology 149 M. Nagai and others

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 Determination of carbohydrates. Carbohydrate molecules in the purified Lcc were determined by endoglycosidase treatment. The purified enzyme (1 mg) was boiled with 5 % 2-mercaptoethanol for 10 min, then incubated with 5 mU Endoglycosidase-H (Roche Diagnostics) in PB at 37 uC for 16 h. After this treatment, the molecular mass of the protein was calculated by SDS-PAGE. Effect of pH and temperature on Lcc activity and stability. The effect of pH on Lcc activity was examined at pH values from 1?0 to 6?0, using 0?1 M KCl/HCl buffer at pH values from 1?0 to 2?0, 0?1 M Glycine/HCl buffer at values from 2?0 to 4?0 and McIlvaine buffer at values from 4?0 to 6?0. The effect of pH on enzyme stability was investigated by measurement of the activity remaining after incubation for 16 h at 30 uC in various buffers with 50 mg BSA ml21 . The buffers were 0?1 M KCl/ HCl (pH 1?0–2?0), 0?1 M Glycine/HCl (2?0–4?0), McIlvaine buffer (4?0–6?0), 0?1 M sodium acetate (5?0–7?0), 0?1 M Tris/HCl (7?0–8?0) and CAPS (8?0–10?0). The effect of temperature on enzyme activity was determined at pH 3?0, with reactions performed by incubating at each temperature and pH 3?0 for 10 min. The thermal stability of Lcc was investigated by incubating preparations in PB with 50 mg BSA ml21 for 30 min at various temperatures. After incubation, the activity remaining was determined. Substrate specificity. Spectrophotometric measurement of substrate oxidation by purified Lcc was carried out in a 100 ml reaction mixture containing the test substrates in McIlvaine buffer (pH 3?0– 6?0). Activity against ABTS, p-phenylenediamine, 2,6-dimethoxyphenol, catechol, guaiacol, ferulic acid and L-DOPA was assayed at concentrations of between 0?1 and 1 mM. Activity against pyrogallol was assayed between 1 and 10 mM. All reactions were conducted at 30 uC for 10 min. The rate of substrate oxidation was determined by measuring the absorbance increase, with the molar extinction coefficient (e) obtained from the literature (Eggert et al., 1996; Shin & Lee, 2000). Michaelis constants (Km) were calculated from Lineweaver–Burk plots at the optimum pH in each case. The oxidative products of L-DOPA and catechol were also analysed by HPLC. The analysis was carried out using a reverse phase HPLC cartridge and a Tsk gel ODS-8TM (150 mm64?6 mm i.d.; Tosoh), radially compressed by a separations module (Waters 2960) at 25 uC. The mobile phases (flow rate 1 ml min21 ) consisted of 5 ml PIC B8 (Waters 84283) in 1 l 0?05 % acetic acid for L-DOPA, and CH3CN and 5 % acetic acid (12 : 88) for catechol. Detection was performed between 220 and 400 nm with a photodiode array detector (Waters 996) connected to a Millennium Chromatography Manager (Waters). In vitro gill browning experiments. Gills (4 g) were cut from the fruit bodies of L. edodes, frozen and homogenized in an Excel Auto Homogenizer at 10 000 r.p.m. for 1 min with 10 ml McIlvaine buffer, pH 4?0. Tyr (0?1 U ml21 ), Lcc 1 (0?4 U ml21 ) or Lcc 2 (0?4 U ml21 ) was added to 80 ml of the supernatant of the homogenate and the reaction mixture was incubated at 30 uC for 60 min. An absorbance increase at 460 nm, showing the synthesis of LDOPA quinone, was measured at the end of the incubation period. RESULTS Purification of Lcc 2 We tested Lcc activity to study the production of Lcc during post-harvest preservation. Fruit bodies were preserved in a desiccator at 25 uC and 80 % humidity. Some brown spots appeared on the gills after 2 days preservation, and after 3 days gills were coloured dark brown (Fig. 1a). Lcc activities of the gill, cap and stipe increased over the preservation period. The highest Lcc activity was obtained in the homogenate of gills preserved for 4 days (Fig. 1b), and Lcc was therefore purified from gills at this stage. Before enzyme purification, the enzyme stability was tested at 4 uC for 16 h. The Lcc was stable at a pH range from 3?0 to 7?0. Thus, the purification was done at this pH range. Enzyme yields during purification steps are summarized in Table 1. The procedure yielded 604 mg of the purified enzyme from 40 g gill tissue, and recovery of total Lcc activity was 23?5 %. The purified Lcc, which we designate as Lcc 2, showed as a single protein band on native PAGE and was identified by having the same location as the band stained for activity in a gel run simultaneously (Fig. 2a). Homogeneity, molecular mass, spectroscopy and pI The purified enzyme appeared as a single band in SDSPAGE (Fig. 2b). The molecular mass of Lcc 2 was estimated as 58 kDa by SDS-PAGE and 53 kDa by gel filtration. These 0 2 3 Preservation (days) (a) (b) Fig. 1. (a) Gill browning of L. edodes and (b) laccase (Lcc) activity during post-harvest preservation. (a) The fruit body was placed in a desiccator at 25 6C and 80 % humidity for 4 days to induce gill browning. Arrows indicate brown spots. (b) Lccs were extracted from caps (open circles), stipes (open triangles) and gills (closed circles). The enzyme activity was measured at pH 3?0. http://mic.sgmjournals.org 2457 Fungal intracellular laccase and melanin synthesis

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 results suggest that the enzyme is a monomeric protein. When the enzyme was treated with Endoglycosidase-H, a clear and smaller (53 kDa) protein band was obtained (data not shown), indicating that Lcc 2 was a glycoprotein with 8?6 % glycosylation. Spectrophotometric analysis of Lcc 2 showed a peak absorption at around 610 nm, typical for a type-I copper signal, with a shoulder at 320 nm, typical for a type-II binuclear copper signal (data not shown; Hanna et al., 1988). Isoelectric focusing indicated that Lcc 2 had a pI of around 6?9. Effect of pH and temperature The pH profile for Lcc 2 activity against ABTS showed a single peak of maximum activity at pH 3?0 (Fig. 3a). When the effect of pH on enzyme stability was tested at 30 uC for 16 h, the enzyme was stable over a pH range of 4?0–7?0 (Fig. 3b). The optimum temperature of Lcc 2, determined at pH 3?0, was 40 uC (Fig. 4a). The thermal stability of Lcc 2 was determined by incubating the enzyme at pH 6?0 (Lcc 2 was stable at 30 uC for 16 h at this pH) for 30 min. No loss of activity was observed after incubation at 50 uC, but incubation at 60 uC resulted in a 33 % loss of Lcc activity (Fig. 4b). Thus, we concluded that Lcc 2 is stable up to 50 uC. Effects of metal ions The effects of metal ions on Lcc activity were tested using ABTS as the substrate (Table 2). The enzyme was strongly inhibited by 1 mM Hg2+ (32?9 % inhibition) and 1 mM Sn2+ (31?1 % inhibition), and slightly by 1 mM Mn2+ (16?7 % inhibition). Neither 1 mM nor 10 mM Cu2+ affected its activity. Table 1. Purification of Lcc 2 Purification step Total protein (mg) Total activity (units) Specific activity (units mg”1 ) Purification (-fold) Yield (%) Culture filtrate 3080 783 0?254 – 100 30 % Ammonium 2770 730 0?264 1?04 93?2 sulfate fraction (sup.) TOYOPEARL Butyl-650M 217 703 3?24 12?8 89?8 TOYOPEARL CM-650M 4?24 246 58?0 228 31?4 Superdex 75 HR 10/30 0?977 210 215 846 26?8 Phenyl Superose HR 5/5 0?604 184 305 1200 23?5 1 2 M 1 kDa 150 100 75 50 37 25 (a) (b) Fig. 2. (a) Native-PAGE and (b) SDS-PAGE of purified laccase (Lcc 2). (a) Lanes: 1, purified Lcc 2 (1 mg) stained with Coomassie brilliant blue; 2, purified Lcc 2 (1 mg) stained for activity with ABTS. (b) Lanes: M, molecular mass markers; 1, purified Lcc 2 (1 mg) stained with Coomassie brilliant blue. Fig. 3. Effect of pH on enzyme (a) activity and (b) stability. (a) The enzyme reaction was performed at 30 6C for 10 min. (b) Activity remaining was measured after incubation with 50 mg BSA ml”1 at various pH values at 30 6C for 16 h. Buffers: open circles, 0?1 M KCl/HCl; closed circles, 0?1 M Glycine/HCl; open triangles, McIlvaine; closed triangles, 0?1 M sodium acetate; open squares, 0?1 M Tris/HCl; closed squares, CAPS. 2458 Microbiology 149 M. Nagai and others

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 Substrate specificities of two Lccs from L. edodes Specificities of purified Lcc 2 for various substrates were determined at the optimum pH for each substrate and compared with those of Lcc 1. As shown in Table 3, the conventional substrates of Lccs, such as ABTS, guaiacol and 2,6-dimethoxyphenol, were oxidized by Lcc 2. Both enzymes showed relatively low Km values for these substrates. Kcat/Km values were also determined. As shown in Table 3, significant differences between Lcc 1 and Lcc 2 were found in the activities against 2,6-dimethoxyphenol, catechol, ferulic acid and L-DOPA. L-DOPA, which is the precursor for DOPA melanin synthesis, was not oxidized by Lcc 1 (Nagai et al., 2002), but was oxidized by Lcc 2. Spectrophotometric analysis of the products of Tyr- or Lcc 2-mediated oxidation of L-DOPA and catechol showed identical patterns of absorbance spectra (data not shown). These products of L-DOPA and catechol were also analysed by HPLC. The oxidative products of L-DOPA had retention times of 2?86 min (by Tyr) and 2?87 min (by Lcc 2, Fig. 5). Fig. 6 shows the HPLC profiles of catechol oxidation products mediated by Tyr and Lcc 2. Oxidation by Tyr resulted in three peaks at 3?24, 5?58 and 6?89 min, and that by Lcc 2 resulted in three peaks at 3?23, 5?57 and 6?86 min. All corresponding peaks obtained by Tyr and Lcc 2 reactions showed identical patterns of absorbance spectra (data not shown). Thus, we concluded that Tyr and Lcc 2 produced the same oxidative products of L-DOPA and catechol. Fig. 4. Effect of temperature on enzyme (a) activity and (b) stability. (a) The enzyme reaction was performed in McIlvaine buffer, pH 3?0, for 10 min. (b) Activity remaining was measured after incubating with 50 mg BSA ml”1 in 10 mM PB, pH 6?0, at various temperatures for 30 min. Table 2. Effect of metal ions on laccase activity Inhibitor Concentration (mM) Relative activity (%) None – 100 BaCl2 1 93?7 CaCl2 1 97?7 CdCl2 1 99?3 CoCl2 1 91?9 HgCl2 1 67?1 KCl 1 105 MgCl2 1 94?0 MnCl2 1 83?3 NaCl 1 96?1 PbCl2 1 99?3 RbCl 1 103 SnCl2 1 68?9 ZnCl2 1 89?9 CuSO4 1 93?2 10 96?1 Table 3. Substrate specificities of two purified Lccs from L. edodes Substrate Optimum pH* Km (mM) KcatD/Km Lcc 1 Lcc 2 Lcc 1 Lcc 2 Lcc 1 Lcc 2 ABTS 4?0 3?0 0?108 0?127 404 173 Guaiacol 4?0 4?0 0?917 0?350 0?168 4?13 2,6-Dimethoxyphenol 4?0 3?0 0?557 0?351 6?46 116 L-DOPA ND 4?0 –1?22 – 1?28 Ferulic acid 5?0 5?0 2?86 1?39 0?00263 0?283 Catechol 4?0 3?0 22?4 1?72 0?0670 2?363 p-Phenylenediamine 5?0 4?0 0?256 1?72 0?201 0?337 Pyrogallol 4?0 3?0 0?417 24?6 8?62 16?9 Veratryl alcohol ND ND –– – – Tyrosine ND ND –– – – *ND, Not detected. DKcat (1023 6mol min21 mol21 ). http://mic.sgmjournals.org 2459 Fungal intracellular laccase and melanin synthesis

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 In vitro gill browning by Lcc 2 The results of the in vitro gill browning experiments by purified enzymes are shown in Fig. 7. When gill extracts were incubated with Lcc 1 or without enzymes, the colour changed only slightly. However, Lcc 2 and Tyr clearly browned the gill extract. During the early stages of incubation, 0?4 U Lcc 2 ml21 browned the extract faster than 0?1 U Tyr ml21 and the absorbance increase in the presence of Lcc 2 reached a maximum (about 0?3) at 30 min and changed little thereafter. Ultimately, Tyr was more effective in causing browning, with a maximum absorbance of about 0?65. When both enzymes were added to the same reaction mixture, an early synergistic effect was observed. DISCUSSION Generally, Lccs of basidiomycetes are secreted extracellularly from the vegetative mycelia, increase during fruiting and decrease when the fruit bodies mature (Wood, 1980; Leonard, 1971; Leonard & Phillips, 1973). Ohga et al. (2001) reported that the level of Lcc gene transcripts in L. edodes was maximal during the mycelial growth stage and then rapidly decreased at the fruiting stage. Thus, it is thought that extracellular Lccs degrade lignin and have a role in nutrient uptake to support the developing fruit bodies. Lcc activity in the fruit bodies of L. edodes was investigated by Leatham & Stahmann (1981) who observed highest activity in the pigmented rind. Zhao & Kwan (1999) studied the expression of two Lcc genes of L. edodes and showed that both of these genes (lac1 and lac2) were highly expressed in the caps and that high Lcc activity was detected from the caps when the blocks of fresh fruit bodies were used as enzyme source. These results suggest that Lcc also has a role in pigment synthesis in the rind of caps. We speculated that the synthesis of melanin in the gill during post-harvest preservation (gill browning) might be catalysed Fig. 5. HPLC traces of reaction mixtures containing (a) LDOPA, (b) L-DOPA plus 0?1 U Tyr ml”1 and (c) L-DOPA plus 0?4 U Lcc 2 ml”1 incubated at 30 6C for 5 min. Fig. 6. HPLC traces of reaction mixtures of (a) catechol, (b) 0?1 U Tyr ml”1 and (c) 0?4 U Lcc 2 ml”1 . Reaction mixtures had been incubated at 30 6C for 5 min. Fig. 7. In vitro gill browning by Tyr and Lccs. Gill homogenates were incubated at 30 6C for 60 min without enzymes (open circles) or with 0?4 U Lcc 1 ml”1 (open triangles), 0?4 U Lcc 2 ml”1 (closed circles), 0?1 U Tyr ml”1 (closed triangles) or 0?4 U Lcc 2 ml”1 plus 0?1 U Tyr ml”1 (closed squares). 2460 Microbiology 149 M. Nagai and others

Fungal intracellular laccase and melanin synthesis Kvaues of both typical Le sutrate riod and was proportional to the intensity of gill were similar.but those of Lec 2 fo browning.This result corresponds to that of Tyr activity reported previously (Kanda et al.,1996a). (-M.) ably lo mM fo The (K/K values)of Lec 2 against catechol and L-DOPA significantly higher than those of Lcc Because ls ar ular other char from analysed the activities of Tyr and Lee2 for oxidation of (Thurston, L-DOPA and catechol.As shown in Figs 5 and 6,both enzymes and catech generat th C 2 (53 kDa)wa (55 kDa). of Lc(pl) highe nd values of most Lccs secreted in culture media are acidi polym T 《2是之 aks obtained by HPLC analysis in the stud probably correspond to these quinones or catechol dimers hus,Lc DOPA and catechol to the I by the method activity for the etracted from the fruit bodics (Fig.7).Th was not detected beca 。the n terminus wa slight browning that occurred during incubatio on of gil extracts witho nzymes may have caused by nativ ylated 10 ofthdnidaiy nt, browning,addition of Lce 2 and Tyr did,suggesting that dihy of metal ior by Lec2 but not by Lec 1.Becau e the maximum absorba Nagal er al at aro Ho peak absorption at around 10nm was observed to indicate Le 2 to the Tr reaction mixture.especially in the carh type-l signa Thu speculated 号 with the signal as well as the The corresponding enzyme ratio in gill homogena ate principle pe -I coppe signa prepared from the fruit bo d to When PH of L bs the lolivet et al (1995)rer rted that the amount of phenols authors (tyrosine,GHB and GDHB)in the epidermal tissue was aatiaProte L19 ill porus timum pHs of Lcc 2 for many tsis of latent-t T than those of Lcc 1. that the ntr but an intracellular Lcc 名P xtracellular Lcc the ed that the s to tu substrate specificity of the Lcc contained in the culture Considering the fact that purified Lcc 2 oxidized both from the Lcc contained in the crude catechol, oxyphe Inves http://mic.sgmjournals.org 2461

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 by Lcc 2 as well as by Tyr. As shown in Fig. 1, Lcc activity of the gill homogenate increased over the preservation period and was proportional to the intensity of gill browning. This result corresponds to that of Tyr activity reported previously (Kanda et al., 1996a). The purified intracellular Lcc, Lcc 2, was characterized and compared with an extracellular Lcc, Lcc 1, previously purified from the culture filtrate of L. edodes. The molecular mass of Lcc 2 (58 kDa) was lower than Lcc 1 (72?2 kDa) and other Lccs so far characterized from basidiomycetes (Thurston, 1994). The molecular masses of both Lccs after treatment with endoglycosidase-H were also determined. The molecular mass of deglycosylated Lcc 2 (53 kDa) was lower than that of Lcc 1 (55 kDa). The isoelectric point (pI) of Lcc 2 (pH 6?9) was higher than that of Lcc 1 (around pH 3?0). To our knowledge, the pI values of most Lccs secreted in culture media are acidic (Eggert et al., 1996; Dedeyan et al., 2000; Galhaup et al., 2002; Saparrat et al., 2002), and the pI of Lcc 2 is the highest of all Lccs reported previously, except for an Lcc from Coriolus hirsta (pH 7?4; Shin & Lee, 2000). Thus, the neutral pI of Lcc 2 might be very specific. Although the N-terminal amino acid sequence of Lcc 2 was analysed by the methods described previously (Nagai et al., 2002), the clear sequence was not detected because the N terminus was probably blocked. The facts that the molecular mass of the deglycosylated Lcc 2 was lower than that of Lcc 1 and that the ion charge of the surface of these proteins was different, suggest that the two Lccs are encoded individually. When the effect of metal ions on enzyme activity was tested, 10 mM Cu2+ activated Lcc 1, but not Lcc 2 (Table 2; Nagai et al., 2002). The absorption spectrum of Lcc 1 showed a peak absorption at around 610 nm, suggesting the existence of type-I copper ions. However, no secondary peak absorption at around 310 nm was observed to indicate a type-II copper signal. Thus, we speculated that the activation of Lcc 1 by Cu2+ may be due to the filling of type-II copper-binding sites with copper ions (Nagai et al., 2002). In this study the absorption spectrum of Lcc 2 showed a peak for a type-II copper signal as well as the principle peak at 610 nm indicating a type-I copper signal. When substrate specificities of Lcc 2 were tested, the optimum pH of Lcc 2 for each substrate was different (Table 3). Such differences have been observed by several authors and have been ascribed to variable degrees of substrate protonation under different pH conditions (D’Annibale et al., 1996; Fukushima & Kirk, 1995). The optimum pHs of Lcc 2 for many substrates were lower than those of Lcc 1. Schlosser et al. (1997) have also reported that the optimum pH of the intracellular Lccs from Trametes versicolor is lower than that of the extracellular Lccs. In addition, they have reported that the substrate specificity of the Lcc contained in the culture filtrate was different from the Lcc contained in the crude extract of the mycelia. The Km values of both enzymes for typical Lcc substrates – ABTS, p-phenylenediamine, 2,6-dimethoxyphenol, ferulic acid or guaiacol – were similar, but those of Lcc 2 for catechol and L-DOPA (1?72 and 1?22 mM, respectively), were noticeably lower than those of Lcc 1 (22?4 mM for catechol and zero oxidation for L-DOPA). Also, the activities (Kcat/Km values) of Lcc 2 against catechol and L-DOPA were significantly higher than those of Lcc 1. Because dihydroxyphenols are known to be oxidized by Tyr for synthesis of melanin in vivo (Bell & Wheeler, 1986), we analysed the activities of Tyr and Lcc 2 for oxidation of L-DOPA and catechol. As shown in Figs 5 and 6, both enzymes oxidized L-DOPA and catechol to generate the same products. Both enzymes generated three products from catechol. It is established that catechol is oxidized by Tyr to o-benzoquinone and three types of semiquinone radical, and that these quinones are polymerized to catechol dimers in vivo (Bell & Wheeler, 1986). The three peaks obtained by HPLC analysis in the present study probably correspond to these quinones or catechol dimers. Thus, Lcc 2 appears to oxidize L-DOPA and catechol to the same products as generated by Tyr. Lcc 2 had oxidative activity for the phenolic compounds extracted from the gill of L. edodes fruit bodies (Fig. 7). The slight browning that occurred during incubation of gill extracts without enzymes may have been caused by native enzyme activity or by non-enzymic oxidation reactions. Although the addition of Lcc 1 to the extract did not effect browning, addition of Lcc 2 and Tyr did, suggesting that the phenolic compounds (perhaps especially dihydroxyphenols) in the gills of L. edodes fruit bodies can be oxidized by Lcc 2 but not by Lcc 1. Because the maximum absorbance change was obtained by the addition of Tyr, we speculated that Tyr can oxidize a wider variety of compounds than Lcc 2. However, increased oxidation following the addition of Lcc 2 to the Tyr reaction mixture, especially in the early stages, suggests either synergistic interactions between Tyr and Lcc 2 or perhaps the existence of phenolic compounds which can be oxidized by Lcc 2 but not Tyr. In this study, the reactions were carried out with a ratio of 4 Lcc 2 : 1 Tyr. The corresponding enzyme ratio in gill homogenates prepared from the fruit bodies of L. edodes preserved for 3 days was 22 : 1 (data not shown). Thus, we conclude that Lcc 2 has an important role in gill browning. Jolivet et al. (1995) reported that the amount of phenols (tyrosine, GHB and GDHB) in the epidermal tissue was related to susceptibility to browning in A. bisporus. Kanda et al. (1996a) showed a dependency of gill browning in L. edodes on de novo synthesis of a latent-type Tyr and increasing Tyr activity. Our results suggested that not only Tyr, but also an intracellular Lcc (Lcc 2) is produced during post-harvesting preservation and functions in the oxidation of phenolic compounds to turn gills brown. Considering the fact that purified Lcc 2 oxidized both L-DOPA and catechol, Lcc 2 might have a strong activity against dihydroxyphenols. Investigations into the phenol http://mic.sgmjournals.org 2461 Fungal intracellular laccase and melanin synthesis

Downloaded from www.microbiologyresearch.org by IP: 202.110.209.177 On: Tue, 05 Jun 2018 06:06:56 content of the gills of L. edodes fruit bodies are currently under way. We are also analysing the Lcc 2 gene with a view to its repression or disruption to construct a strain which can be preserved for longer periods without gill browning. REFERENCES Aramayo, R. & Timberlake, W. E. (1990). Sequence and molecular structure of the Aspergillus nidulans yA (laccase I) gene. Nucleic Acids Res 18, 3415. Bell, A. A. & Wheeler, M. H. (1986). Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol 24, 411–451. Burke, R. M. & Cairney, J. W. G. (2002). Laccases and other polyphenol oxidases in ecto- and ericoid mycorrhizal fungi. Mycorrhiza 12, 105–116. Burton, K. S. (1988). The effects of pre- and post-harvested development on mushroom tyrosinase. J Hortic Sci 63, 255–260. Castro-Sowinski, S., Martinez-Drets, G. & Okon, Y. (2002). 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《食品生物化学》课程教学资源（文献资料）第二章 酶 Important role of fungal intracellular laccase for melanin synthesis：purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies

《食品生物化学》课程教学资源（文献资料）第二章酶 Important role of fungal intracellular laccase for melanin synthesis：purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies