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An alkaline fungal laccase overexpressed in Coprinopsis cinerea shows application potential in treating high-salt dye wastewater
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Xianhua WANG1, 2, 3, Xueling ZHU1, 2, 3, Jiayi LIN1, Xianfu GU1, Qixia PENG1, 2, 3, Yazhong XIAO1, 2, 3, Kües Ursula4, Zemin FANG1, 2, 3, *
Acta Microbiologica Sinica | 2024, 64(8) : 3014 - 3029
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Acta Microbiologica Sinica | 2024, 64(8): 3014-3029
• Research Articles •
An alkaline fungal laccase overexpressed in Coprinopsis cinerea shows application potential in treating high-salt dye wastewater
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Xianhua WANG1, 2, 3, Xueling ZHU1, 2, 3, Jiayi LIN1, Xianfu GU1, Qixia PENG1, 2, 3, Yazhong XIAO1, 2, 3, Kües Ursula4, Zemin FANG1, 2, 3, *
Affiliations
  • 1 School of Life Sciences, Anhui University, Hefei 230601, Anhui, China
  • 2 Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China
  • 3 Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui, China
  • 4 University of Goettingen, Goettingen 237077, Germany
Published: 2024-05-17 doi: 10.13343/j.cnki.wsxb.20240085
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[Objective] To homologously overexpress an alkaline fungal laccase PIE5 (CcPIE5) in Coprinopsis cinerea FA2222 under the control of the Agaricus bisporus gpdII promoter. [Methods] The laccase activity reached (24.2±1.1) U/mL in the supernatant after 7 days of cultivation at 37 ℃ in the mKjalke medium. The purified CcPIE5 showcased the best performance at pH 8.0 and 60 ℃. [Results] Unlike other characterized fungal laccases, CcPIE5 was tolerant to high concentrations of NaCl. Particularly, both kcat and Km decreased when the concentration of NaCl was increased from 0 to 1.5 mol/L, which indicated that CcPIE5 demonstrated application potential in the dye decoloring of texile finishing. In dye decolorization, CcPIE5 efficiently degraded (92.9±2.3)% indigo carmine at pH 8.5 and 60 ℃, with syringic acid as the mediator. Isatin 5-sulfonic acid (ISA) was identified by LC-MS as the primary byproduct of indigo carmine degradation. [Conclusion] CcPIE5 is best-suited in decolorizing dyes under high temperatures and alkaline and salty conditions. It serves as a good candidate for specific applications in the environment and industry.

fungal laccase  /  overexpression  /  Coprinopsis cinerea  /  high-salt  /  indigo carmine
Xianhua WANG, Xueling ZHU, Jiayi LIN, Xianfu GU, Qixia PENG, Yazhong XIAO, Kües Ursula, Zemin FANG. An alkaline fungal laccase overexpressed in Coprinopsis cinerea shows application potential in treating high-salt dye wastewater[J]. Acta Microbiologica Sinica, 2024 , 64 (8) : 3014 -3029 . DOI: 10.13343/j.cnki.wsxb.20240085
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Synthetic dyes are widely used in food, cosmetics, paper, and textile industries. It is reported that more than 700 000 tons of synthetic dyes are produced annually worldwide, with more than 15% of them emitted into the environment as effluents. The complexity of wastewater generated from modern textile industries' dyeing processes is steadily increasing[1]. For example, the dye wastewater resulting from contemporary textile finishing procedures exhibits characteristics such as high chroma (ranging from 10 to 250 mg/L), fluctuating alkaline pH levels (between 6.0 and 11.8), and elevated temperatures (ranging from 50 ℃ to 70 ℃). Additionally, high concentrations of salts (ranging from 40 g/L to 100 g/L) are commonly utilized in dye baths to ensure maximal dye fixation onto fibers (with the mass fraction of NaCl and Na2SO4 exceeding 6% and 5%, respectively)[2]. Typically, seawater salinity is characterized by the presence of 3.5% W/W salts[3]. Wastewater exhibiting salinity levels surpassing those found in oceans is classified as hypersaline (3.5%), while wastewater with salinity ranging from 1.0% to 3.5% W/W NaCl is termed highly saline[4]. Moreover, the presence of salinity in wastewater significantly hampers the efficacy of various wastewater treatment methodologies. The persistence of highly stable textile finishing dyes has emerged as a leading contributor to water pollution, exerting detrimental effects on the environment and posing a grave threat to ecological and environmental integrity.
With society's increasing concerns for the ecological environment and health, the treatment of dye wastewater has become an essential part of social development. Many physical and chemical approaches have been developed to treat dye wastewater, including biosorbents, coagulants, electrochemical oxidation, ultrasonic chemical oxidation, ozonation, and irradiation[5]. However, they are costly and susceptible to secondary pollution, restricting their wide application. In contrast, biological processes have advantages of high decolorization rate, low cost, simple operation, low toxicity or non-toxicity of the end product, and have gradually become the primary way for dye wastewater treatment[6-7]. Specifically, the utilization of heme-containing oxidoreductases, including manganese, lignin, and versatile peroxidases, as well as oxidative laccases, is recognized as an effective catalysis for dye biodegradation[8]. Although peroxidases have a higher redox potential (up to 1.40 V vs. a standard electrode), they depend on hydrogen peroxide, leading to heme bleaching and enzyme deactivation even in moderate hydrogen peroxide concentrations[9-10]. By comparison, laccases are four-copper-containing O2-dependent oxidoreductases that oxidize a wide variety of substrates by electron transfer across the copper atoms at the T1, T2, and T3-T4 sites, with water as the only other end product, making them more promising for the biodegradation of dyes[8].
Several fungal and bacterial laccases have been evaluated in dye wastewater treatment with or without a low-molecular-weight mediator (laccase mediator system, LMS)[11-13]. Usually, fungal laccases catalyze the dyes at acidic conditions, showing the characteristics of a high redox potential at the substrate-binding T1 copper, high specific activity and superior affinity for a broader range of substrates[14]. In comparison, most bacterial laccases show optimum activity at neutral and alkaline conditions and the advantages of thermostability and high chloride tolerance. However, bacterial laccases possess a relatively low redox potential (430−460 mV vs. >730 mV of fungi) and specific activity (<10 U/mg vs. >100 U/mg with ABTS (2,2′-azino- bis(3-ethylbenzothazoline-6-sulfonate)) as the substrate)[15]. Thus, it is still challenging to degrade modern-process-produced dye wastewater using any of the characterized natural fungal and bacterial laccases, due to their inherent technical deficiencies. More efforts remain to find specific fungal laccases that show high activity at alkaline pH and high chloride tolerance for wastewater treatment processes.
Because of their low natural production yields, fungal laccases secreted from native sources are typically unsuitable for large-scale industrial applications[16]. Thus, large-scale production of laccases at lower costs is another bottleneck for applying laccases in dye wastewater treatment. Various strategies, including optimizing culture conditions and adding inducers for overexpression, have been tried to scale up the production of laccases but have resulted in only limited improvements[17]. Expressing a laccase gene in heterologous organisms has sometimes been shown to be an effective strategy. For example, more than 30 fungal laccases have been heterologously expressed in Pichia pastoris with various productivities[16, 18]. However, the laccase obtained often changed in biochemical properties because protein modification by glycosylation differed depending on the heterologous host cells[17]. To this end, the over-expression of laccase in the homologous host cells should enable the production of a well-glycosylated laccase at low cost[19].
Basidiomycete fungal laccases have been instrumental in integrating biotechnological processes across diverse sectors such as paper and pulp, food and feed, textile, and pharmaceutical industries. The prevailing milieu for most basidiomycete laccases is within mildly acidic parameters (pH 4.0−6.0) and a temperature range of 30−50 ℃, which may preclude their utilization in contexts necessitating alkaline conditions and elevated temperatures. Coprinopsis cinerea, a prominent model basidiomycetous fungus, harbors 17 distinct laccase genes within its genome and boasts well-established protocols for genetic manipulation[20]. Various inquiries have underscored the promise of heterologous expression of laccases from C. cinerea in efficacious dye wastewater treatment. Notably, CcLcc2 and CcLcc6, when expressed heterologously in P. pastoris, exhibit potential in decolorizing dye-contaminated wastewater[21-22]. Additionally, a newly identified laccase, Lac1, from the basidiomycete Coprinus comatus, sharing 66% genetic similarity with the laccase gene of C. cinerea, has shown remarkable prowess in decolorizing three different azo dyes[23]. Recently, the basidiomycete laccase, C. cinerea Lcc9, has emerged as exceptionally thermophilic and alkaliphilic, demonstrating promise in decolorizing dye-contaminated wastewater[24-25]. Fungal laccase PIE5, a mutant derived from the native laccase Lcc9 of C. cinerea, represents the foremost thermo- and alkali-philic fungal laccase engineered with an optimal catalytic pH in alkaline realms (pH range of 8.0−8.5), particularly towards guaiacol, offering potential for the treatment of dye wastewater. Nevertheless, the expression level of PIE5 reached only 1.2 U/mL when expressed in P. pastoris, presenting challenges for its industrial-scale application. In this current investigation, PIE5 was successfully overexpressed in C. cinerea, yielding a final output of (24.2±1.1) U/mL. This alkali-philic enzyme demonstrated exceptional chloride tolerance, presenting a novel advantage for the biotreatment of high-salt dye wastewater, as illustrated in this study through the degradation of indigo carmine under highly saline alkaline conditions.
1 Materials and Methods
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1.1 Strains, plasmids and chemicals
C. cinerea FA2222 (A5, B6, acu1, trp1-1, 1-6), used as a trp1-auxotrophic monokaryon in transformation, is available at the DSMZ in Braunschweig. C. cinerea expression vector pYSK7 with laccase gene lcc1 with its native terminator sequences subcloned behind the Agaricus bisporus gpdII promoter[19] was used in vector construction, marker-plasmid pBD5 with the C. cinerea trp1+ gene[26] for selection for prototrphy in C. cinerea co-transformations. P. pastoris GS115 and the expression vector pPIC9K were from Invitrogen. Plasmid pPIC9K-pie5 from a lcc9 mutant library contains the developed allele pie5[27]. 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt (ABTS), guaiacol and indigo carmine were from Sigma-Aldrich. All other chemicals and reagents were of analytical grade.
1.2 Expression of PIE5 and wild-type Lcc9
The expression of PIE5 in P. pastoris GS115 using pPIC9K-pie5 was carried out under the control of the inducible AOX1 promoter, along with the α-factor signal coding sequences from the pPIC9K vector for secretion. The laccase expressed in P. pastoris was subsequently purified from the culture supernatant, as detailed by Xu et al.[25], and hereafter referred to as PpPIE5. The wild-type Lcc9, originating from the coculture of C. cinerea and Gongronella sp. w5, was synthesized and purified following the procedures outlined by Pan et al.[24].
1.3 Construction of a PIE5 overexpression C. cinerea FA2222 strain
Plasmid pPIC9K-pie5 was used as the template to amplify PCR of the lcc9 mutant allele pie5[27]. A primer pair was used with homologous overlaps to the DNA regions of the gpdII promotor and the lcc1-terminator present in plasmid pYSK7[19, 28], pYSK7-pie5F 5′-AATCCACCATCTCCGTTTTCTCCCATCTACACACAACAAGCTTATCGCCATGCAAATCCTCGGCCCC-3′ and pYSK7-pie5R: 5′-TGACTATAGCAGCCTCCTACCACTGGCCCTCTGGTCAACTATAATATTATTTAAGGGGTAGGGACGATTTGG-3′ (underlined fragments are homologous overlaps sites). PCR was conducted using the PrimeSTAR® Max DNA Polymerase Kit (TaKaRa). Plasmid pYSK7-lcc1 was linearized using Hpa Ⅰ and BamH Ⅰ (TaKaRa) to double-digest. Saccharomyces cerevisiae strain Y1H (TaKaRa) was transformed with the pie5 PCR amplicon and the linearized pYSK7 to generate the final plasmid pYSK7-pie5 based on in vivo homologous recombination[19]. The Saccharomyces cerevisiae plasmid was transformed into Escherichia coli JM109 to increase its copy number.
C. cinerea FA2222 was cultivated on solid YMG/T medium (per liter 4 g yeast extract, 10 g malt extract, 4 g glucose, 100 mg tryptophan, and 1% agar) at 37 ℃. Protoplast transformation was performed using oidia collected from fully grown plates and harvested in sterile water, filtered through sterile glass wool, washed, protoplasted, and transformed as described before by Dörnte and Kües et al.[28]. For each co-transformation, 1 μg plasmid pYSK7-pie5 and 1 μg pBD5 harboring the trp1+ gene were used. Transformed protoplasts were plated onto regeneration agar according to Dörnte et al.[28]. When required to test laccase activities of obtained transformants in further growth, 0.5 mmol/L ABTS (1st round of selection) and 1 mmol/L guaiacol (2nd round of selection) were added for further testing into regeneration agar, respectively. Finally, prototrophic transformants were collected onto minimal medium[26] and cultivated for further use at 37 ℃.
To further screen transformants with high laccase activity, clones positive in the two rounds of screening on regeneration agar were then cultivated at 37 ℃ and 120 r/min in 100 mL mKjalke liquid medium (per liter: 10 g yeast extract, 20 g glucose, 2 g KH2PO4, 0.5 g CaCl2·2H2O, 50 mg MgSO4·7H2O) supplemented with 0.1 mmol/L CuSO4 in 500 mL Erlenmeyer flasks[17]. Laccase activity in supernatants was tested every 24 h.
1.4 Purification of PIE5 expressed in C. cinerea FA2222
To purify overexpressed PIE5 from C. cinerea FA2222, the C. cinerea FA2222-pie5 transformant with highest laccase activity (clone 14) was cultured in 0.1 mmol/L Cu-supplemented mKjalke medium at 37 ℃, as described above. The culture broth was collected on the 6th day of incubation, centrifuged at 4 ℃ and 8 000×g for 10 min, and further concentrated using an ultrafiltration device (Millipore) to get the crude enzyme solution. After dialysis using citrate-phosphate buffer (20 mmol/L, pH 6.5), the crude enzyme solution was applied onto a DEAE-Sepharose FF column (10 mm×200 mm, Amersham Pharmacia, Uppsala) and eluted according to protocols reported previously[25]. The purified protein was designated as CcPIE5.
The homogeneity of the purified protein was determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) with a 12% polyacrylamide gel. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue R-250. The protein concentration was assayed using the Bradford method at 595 nm, with bovine serum albumin as the standard.
1.5 Laccase assays
Laccase activity was determined at 30 ℃ for 3 min in 100 mmol/L sodium tartrate buffer at pH 4.0 with 1 mmol/L ABTS as the substrate (ε420=36 000 L/(mmol·cm)). The reaction was initiated by adding the enzyme into the solution. The absorbance was measured at 420 nm[29]. Alternative substrates for measuring laccase activity were 1 mmol/L guaiacol (ε465=12 000 L/(mol·cm)), 0.5 mmol/L 2,6-dimethylphenol (2,6-DMP, ε468=49 600 L/(mol·cm)), 100 μmol/L syringaldazine (SGZ) (ε525=65 000 L/(mol·cm)). One unit of activity was defined as the amount of enzyme needed to oxidize 1 μmol substrate per minute. All measurements were performed in triplicates from three biological repeats of culture.
1.6 Additional biochemical characterization of laccase
The effect of pH on laccase activity was assayed in 50 mmol/L citrate-phosphate buffer (4.0−8.0) and 50 mmol/L Tris-HCl buffer (7.5−9.5) at 25 ℃ using 1 mmol/L guaiacol as the substrate. The effect of temperature was determined by incubating protein in a temperature range of 35−75 ℃ at optimum pH. The enzyme stabilities against pH and temperature were determined by incubating proteins at various temperatures and pH 6.5 and 8.5. Then, the residual activities were determined with 1 mmol/L guaiacol as mentioned in section 2.4. The effect of ions (NaCl, NaBr, Na2SO4, KCl, NH4Cl, and CaCl2) on PIE5 activity was tested at 60 ℃. The salt concentration was always 1.0 mol/L but for NaCl that ranged from 0.0 mmol/L to 2.0 mol/L. All experiments were performed at least three independent times from three different purified protein batches, with at least three technical repeats of measurement per batch.
1.7 Kinetic properties
The kinetic parameters of the enzyme were tested under optimum conditions, and Km and Vmax were calculated by nonlinear regression of the Michaelis-Menten equation (Eq. 1) with GraphPad Prism 8.0 (GraphPad Software).
1.8 The dye removal properties of CcPIE5
CcPIE5 was used to decolorize indigo carmine in the presence of 1.0 mol/L NaCl. The initial testing system contained Tris-HCl buffer (50 mmol/L, pH 8.5), purified enzyme (0.2 U/mL), and indigo carmine (100 mg/L), and mediators (vanillin, citronellal, syringic acid (SA), ferulic acid, vanillic acid, and SGZ) when required (final concentration, 200 μmol/L) in a total volume of 1 mL. The reaction mixtures were incubated at 60 ℃ for 2 h.
The effects of concentrations of enzymes, mediators and their concentrations, pH, temperature, and incubation time on dye decolorization were evaluated one by one, based on a single-factor optimization strategy. The decolorization rate of indigo carmine by laccases was monitored according to the decrease in absorbance at 610 nm. All experiments were performed at least three independent times with at least three technical repeats of measurement. The heat-treated enzyme was used as the negative control.
The decolorization ratio was calculated using the following equation (Eq. 2).
A0 and A represent the initial and final absorbance of indigo carmine, respectively.
1.9 End product determination
The reaction mixture was separated using centrifugal filters (3 kDa cut-off value) to discard CcPIE5 to analyze the degradation products. Electrospray ionization mass spectrometry (ESI-MS) analysis was performed with a mass spectrometer (ThermoFisher Scientific) operating in the negative ion mode. The conditions of the MS were: auxiliary gas flow rate of 15 bar; sheath gas flow rate of 70 bar; capillary voltage of 15 V; capillary voltage of 15 V; and capillary temperature of 350 ℃[30]. The expected product structures were drawn with the aid of the mass fragmentation tool of KingDraw and these were then verified with the m/z values obtained from the ESI-MS study[31].
2 Results and Discussion
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2.1 Overexpression of PIE5 in C. cinerea FA2222 and purification
A total of 56 positive transformants were obtained after the two rounds of regeneration medium plate screening, using ABTS and guaiacol as the substrates (Figure 1A). The same positive clone is shown in reaction with ABTS (middle) and guaiacol (right). Reactions with guaiacol in the regeneration agar were, however, less intense, with visually smaller orange halos generated around the orange-brown stained colonies. Randomly selected transformants were then cultured in mKjalke liquid medium (Figure 1B). Because a target gene is randomly inserted at ectopic sites into the C. cinerea genome with different copy numbers during transformation[32], transformants showed different laccase activities of (2.1±0.5) U/mL to (18.1±1.1) U/mL in liquid cultures, measured with ABTS (Figure 1B), which in its large variability is similar to previous observations from culture supernatants (YMG/T plus 0.1 mmol/L CuSO4) of transformants carrying lcc1 under the control of different basidiomycetous promoters[19]. The transformant with the highest laccase activity of about 18.0 U/mL (clone 14, Figure 1B, from now on called CcPIE5-14) was chosen for further research in this work.
Laccase activity in the culture supernatant of clone 14 reached (24.2±1.1) U/mL when using ABTS as the testing substrate after 7 days of cultivation in shaking flasks (Figure 1C). The laccase production was stable even after the overexpression transformant CcPIE5-14 was subcultured for up to 20 generations (Figure 1C). The enzymatic activity of CcPIE5 recombinantly expressed by C. cinerea FA2222 CcPIE5-14 was 20-fold higher than that of PpPIE5 expressed in P. pastoris GS115[27] and was about 13-fold higher than that expressed by C. cinerea monokaryon Okayama 7 from the endogenous lcc9 gene during fungal coculture in competition with Gongronella sp. w5 in our former studies[33] (Figure 1D). Furthermore, the yield of recombinant laccase activity in this study was 2.6-fold higher than in the best case observed so far. Previously, five laccases (Lcc1, Lcc2, Lcc6, Lcc8, and Lcc9) from C. cinerea have been heterologously or homologously recombinantly expressed[17, 21-22, 25, 34], where the laccase yield of the lcc1 transformant pYSK7-26 reached 9.2 U/mL in Cu-supplemented mKjalke medium at 25 ℃ and represented the highest recombinant laccase activity reported before in C. cinerea[17, 24, 35-36].
CcPIE5 was purified 12.71-fold from the culture supernatant (Figure 1E), with a final yield of 40.41% (Table 1). It showed a specific activity of 640.12 U/mg. By comparison, when expressed in P. pastoris, PpPIE5 showed a specific activity of 318.00 U/mg[27]. Varied glycosylation may cause the changed specific activity of PIE5. As shown on a SDS-PAGE gel, although PIE5 had the predicted molecular weight of 55.6 kDa, PpPIE5 and CcPIE5 showed heavier molecular weights than 55.6 kDa, indicating that they were glycosylated proteins[37]. Furthermore, CcPIE5 shifted faster in gels than PpPIE5, suggesting it had lighter glycosylation than the latter. Similar observations with distinct degrees in glycosylation were reported for the wild-type Lcc9 when expressed in the native host and in P. pastoris. Glycosylation were shown to contribute to protein stability. N313 and N454 were determined in mutagenesis approaches to be the specific amino acid that are glycosylated in Lcc9[25]. Generally, the effect of glycosylation on laccase activity is still understudied[38]. However, the studies on a fungal laccase in seven different Steccherinum ochraceum strains suggested a substantial role of N-linked glycosylation in the moderation of enzymatic properties of laccases[39].
2.2 Biochemical characterization of CcPIE5
The effects of pH and temperature on purified CcPIE5 activity were analyzed using the phenolic compound guaiacol as the substrate. CcPIE5 showed the optimum pH and temperature at 7.5−8.0 and 55−60 ℃, respectively, with more than 60% of activity retained at pH of 7.0 and 8.5 and temperatures of 50 ℃ and 65 ℃ (Figure 2A, 2B). In addition, CcPIE5 showed a half-life time of 45 min at pH 6.5 and 8.5 at 60 ℃ (Figure 2C). A comparison of the biochemical properties between CcPIE5 and PpPIE5 showed that they shared nearly identical properties, including optimum pH, temperature, and thermo- and pH-stabilities (Figure 2A−2C), despite their apparent difference in glycosylation.
Dye wastewater usually contains high salt concentrations of more than 1.0 mol/L NaCl or Na2SO4[2]. Thus, the biochemical properties of the two PIE5 versions were tested at 1.0 mol/L NaCl. The optimum pH and temperature changed to 8.0−8.5 and 65 ℃ (Figure 2D−2F). The half-life time of the two PIE5 versions under these conditions at 65 ℃ was 40 min. These results suggested that PIE5 is a thermostable fungal laccase showing high activity at alkaline conditions in the presence of high concentrations of salt. Beyond high salt concentration, wastewater from denim processing is in addition characterized by high pH (>6.0) and high temperature (above 50 ℃)[40]. Thus, the ability of laccases to operate at higher temperatures is particularly important because elevated temperatures often favor high reaction rates in many processes, which means higher decolorization velocity. Furthermore, utilization of thermostable laccases with properties such as the developed PIE5 here avoids the additional cooling process, reduces the operation cost, and fully uses the high temperature of dyeing wastewater to achieve the maximum decolorization rate in a short time[15].
2.3 Chloride tolerance of PIE5
The salt tolerance ability of PpPIE5 and CcPIE5 was further evaluated by employing NaCl and Na2SO4 as salts that are commonly used in dyeing processes[40]. These salts did not negatively affect the PIE5 laccase activity. Laccase activity was even stimulated by 1.0 mol/L Cl− when tested, using guaiacol as the substrate (Figure 3A). Further experiments showed that halide ions, including Cl− and Br−, stimulated the laccase activity, by up to 300% (Figure 3A). Most other fungal laccases are sensitive to and inhibited by halide ions, with an IC50 of 0.02−1 600 mmol/L[35, 41]. For basidiomycete laccases, this value does not usually exceed 100 mmol/L; for example, laccase from Trametes villosa shows an IC50 (Cl−) of 40 mmol/L[42]. Thus, fungal laccases with halide tolerance have recently attracted much attention[43]. Like PIE5, a novel bacterial laccase-like enzyme, Lac1326, showed excellent tolerance to salt and maintained 204.6% and 167.2% of its original activity in the presence of 100 mmol/L and 500 mmol/L of NaCl, respectively. However, this enzyme showed the best catalytic activity in acidic conditions[44]. Enzymatic activity for another bacterial laccase, LacHa of Halomonas alkaliantartica, with an optimum pH and temperature at 7.5 and 45 ℃, respectively, was 1.5-fold increased by NaCl concentrations <500 mmol/L. The enzyme had comparably good stability in higher NaCl concentrations (ca. 60% in NaCl concentrations up to 1 000 mmol/L) but showed only around half of its potential activity at pH 8.0[45]. As far as we know, PIE5 is the first fungal laccase with halide tolerance ability and it is even stimulated by halide at alkaline conditions.
What is the molecular base of the excellent halide tolerance of CcPIE5? Fungal laccases contain four catalytic Cu atoms in the protein (T1 Cu and the tri-nuclear Cu cluster, including T2 Cu, T3α Cu, and T3β Cu). Substrates are assumed to bind near the T1 Cu, which is known as the substrate binding and oxidation site[43]. It is widely accepted that chloride also binds near the T1 site and could act as a competitive inhibitor of electron donors by blocking the access of reducing substrates to the T1 Cu site of laccase or inhibiting the electron transfer at the T1 active site, thus inhibiting laccase activity, while halides with larger molecular radii are less efficient inhibitors (fluoride>chloride>bromide)[35]. In addition, in acidic conditions, chloride may bind to the T2/T3 copper cluster of laccase and affect electron transfer. However, the mechanism of laccase activity stimulation by chloride at alkaline conditions is poorly understood. Therefore, NaCl was used as the chloride donator to investigate the effect of chloride on CcPIE5 activity at pH 8.0. Results showed that the activity stimulation effect of chloride on CcPIE5 was substrate-dependent. For example, NaCl at a concentration of 2.0 mol/L stimulated laccase activity to 237%. By comparison, salt had no significant effect on CcPIE5 activity with 2,6-DMP as the substrate. However, it inhibited the activity when using SGZ as a substrate, with an IC50 of 1 000 mmol/L (Figure 3B). Further experiments showed that the kinetic constants of CcPIE5 changed in various chloride concentrations based on the Michaelis- Menten equation (Table 2). NaCl at low concentrations enhanced the substrate affinity and the catalytic efficiency of the enzyme when using guaiacol as a substrate because the Km and kcat showed a downward trend as NaCl concentration increased to 1.5 mol/L.
Further experiments showed that the kinetic constants of CcPIE5 changed in various chloride concentrations based on the Michaelis-Menten equation (Table 2). gradual increases in kcat/Km values of PIE5 toward guaiacol were observed as chloride increased from 0.0 mol/L to 1.5 mol/L, suggesting that chloride at relatively low concentrations can enhance the affinity of PIE5 to substrate, thereby stimulating the peroxidase activity. Based on the electron transfer theory, a proper substrate orientation and a proper distance between the substrate donor orbitals and the T1-His acceptor orbitals are essential to enhance the electron transfer rate[46]. Accordingly, the relevant active substrate conformations should be kept at a proper distance from T1 Cu[47]. Based on this theory, we hypothesized that NaCl might influence CcPIE5 properties by binding to specific protein sites neat the T1 Cu site and causing subtle changes in local areas, thus enhancing the affinity of the substrate guaiacol to the T1 Cu site by providing a better conformation, shorting the distance between electron donor and acceptor, and finally boosting electron-proton transfer. On the other hand, substrates are different in molar volume (mv). For example, SGZ has an mv of 291.0 cm3/mol, 2,6-DMP has an mv of 135.0 cm3/mol, and guaiacol has an mv of 111.8 cm3/mol. Thus, chloride could inhibit the activity of CcPIE5 on substrates with a relatively large volume by disturbing their proper conformations at the substrate binding site. which showed the salt activation of PIE5 was substrate dependent, and therefore, indicated that different substrates interact with the enzyme at different regions. Thus, the interactions would be affected by the Cl− ion bound to different sites. However, there were decreases in kcat/Km values of PIE5 toward guaiacol as chloride increased from 1.5 mol/L to 2.0 mol/L, indicating that chloride at high concentrations binds to the laccase domain and acts as a competitor of electron donor by blocking the access of substrate to the active site of PIE5 or reducing the electron transfer between them, therefore leading to inhibition of laccase activity[48].
2.4 Indigo carmine degradation by CcPIE5 in salt environment
The predominant environmental concern associated with azo dyes arises from their inherent resistance to degradation, attributable to the presence of one or more azo groups (−N=N−)[49]. Azo dyes pose substantial environmental hazards, capable of modifying both the physical and chemical characteristics of soil, deteriorating aquatic ecosystems, and posing threats to surrounding flora and fauna. Even minute quantities of azo dyes in water are perceptible, adversely affecting the aesthetics, clarity, and gas solubility of the aquatic environment. This results in reduced light penetration, subsequently diminishing photosynthetic activity and inducing hypoxia, thereby disrupting the ecological balance of aquatic organisms. Indigo carmine is one of the most commonly used dyeing agents for food, denim, and polyester fibers[50]. Several laccase-based treatments of excessive concentrations of indigo carmine have been developed under acidic conditions and without high salt concentrations because of typical enzyme salt inactivation[51]. By comparisons, CcPIE5 decolorized 43% of 100 mg/L indigo carmine within 2 h at pH 8.5 in the presence of 1.0 mol/L NaCl. With the addition of mediators vanillin, citronellal, SA, or ABTS, the decolorization rate of CcPIE5 increased significantly. SA was the optimum mediator for CcPIE5. The decolorization rate reached over 90% under the initial reaction conditions (Figure 4A). The order of mediators that promoted the decolorization rate was as follows: SA>citronellal>ABTS>vanillin. By comparison, adding ferulic acid, vanillic acid, and SGZ repressed the decolorization rate (Figure 4A).
The conditions for indigo carmine decolorization were further optimized using a single-factor optimization strategy using SA as the mediator. CcPIE5 showed high decolorization rates in a pH range of 8.0−9.5 and 50−80 ℃, with more than 70% of indigo carmine decolorized (Figure 4B, 4C), following the fact that the enzyme showed the optimum pH at alkaline conditions. CcPIE5 could decolorize indigo carmine at low concentrations of mediator and enzyme. In the presence of 0.1 U/mL CcPIE5 and 10 μmol/L SA, a decolorization rate of more than 90% was obtained after incubation for 2 h. A high concentration of mediator (30 μmol/L) slightly inhibited the decolorization rate. Further optimization of the incubation time showed that CcPIE5 could decolorize (92.9±2.3)% of indigo carmine within 40 min (Figure 4D−4F).
Liquid chromatograph mass spectrometry (LC-MS) was employed to analyze the decolorization products of indigo carmine. Figure 5A depicts the electrospray ionization (ESI) mass spectrum of the untreated indigo carmine solution. The mass spectrum of the original dye solution displayed a primary peak at m/z 443 (Figure 5A), representing the singly charged anion of indigo carmine. Furthermore, an intense anion at m/z 210 was detected, corresponding to indigo carmine in its doubly charged ionic form (3-oxoindoline-5- sulfonate). Upon treatment with CcPIE5, isatin 5-sulfonic acid (ISA) with a nominal mass of 227 (Figure 5B) was identified in its deprotonated form ([2-H]− of m/z 226). Another peak at m/z 244 was observed, corresponding to 4-amino-3 (carboxycarbonyl) benzenesulfonate, a derivative of ISA formed through straightforward chemical reactions[52]. As a result of the disruption of a conjugated system akin to indigo carmine, the solution rapidly decolorized following the addition of CcPIE5. Examination of the indigo carmine dye structure in Figure 5C reveals two distinct degradation pathways. In path (a), (−N=N−) undergoes natural oxidative cleavage to yield 3-oxoindoline-5-sulfonate. In path (b), PIE5 expedites this process by catalyzing the insertion of oxygen atoms at the C2 and C2′ positions, resulting in the formation of a substantial quantity of ISA. ISA can undergo further oxidation to produce 4-amino-3(carboxycarbonyl) benzenesulfonate. Additionally, ISA naturally degrades to yield benzoic acid and aniline, which pose significantly lesser environmental harm[53].
3 Conclusions
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An optimized fungal laccase Lcc9 mutant, namely PIE5, was successfully expressed in C. cinerea. The laccase activity in culture supernatant was (24.2±1.1) U/mL, and compared to PcPIE5 produced in P. pastoris, CcPIE5 did not change its biochemical properties after expression in C. cinerea. NaCl stimulated laccase activity at high concentrations by increasing the affinity of the substrate to the enzyme. CcPIE5 decolorized more than 92.9% indigo carmine within 40 min at pH 8.5 in the presence of high salt, with ISA as the main product. Therefore, CcPIE5 is best-suited in decolorizing dyes under high temperatures and alkaline and salty conditions, and is a good candidate for specific applications in the environment and industry.
References
Less
[1]
DAUD NK, AKPAN UG, HAMEED BH.Decolorization of sunzol black DN conc. in aqueous solution by Fenton oxidation process, effect of system parameters and kinetic study[J].Desalination and Water Treatment,2012: 1-7.
[2]
IMRAN M, CROWLEY DE, KHALID A, HUSSAIN S, MUMTAZ MW, ARSHAD M.Microbial biotechnology for decolorization of textile wastewaters[J].Reviews in Environmental Science and Bio/Technology,2015,14(1):73-92.
[3]
VO HNP, NGO HH, GUO WS, CHANG SW, NGUYEN DD, CHEN Z, WANG XC, CHEN R, ZHANG XB.Microalgae for saline wastewater treatment: a critical review[J].Critical Reviews in Environmental Science and Technology,2020,50(12):1224-1265.
[4]
SRIVASTAVA A, PARIDA VK, MAJUMDER A, GUPTA B, GUPTA AK.Treatment of saline wastewater using physicochemical, biological, and hybrid processes: insights into inhibition mechanisms, treatment efficiencies and performance enhancement[J].Journal of Environmental Chemical Engineering,2021,9(4):105775.
[5]
HOLKAR CR, JADHAV AJ, PINJARI DV, MAHAMUNI NM, PANDIT AB.A critical review on textile wastewater treatments: possible approaches[J].Journal of Environmental Management,2016,182: 351-366.
[6]
KAUSHIK P, MALIK A.Fungal dye decolourization: recent advances and future potential[J].Environment International,2009,35(1):127-141.
[7]
HAYAT H, MAHMOOD Q, PERVEZ A, AHMAD BHATTI Z, ALI BAIG S.Comparative decolorization of dyes in textile wastewater using biological and chemical treatment[J].Separation and Purification Technology,2015,154: 149-153.
[8]
BARBER-ZUCKER S, MATELJAK I, GOLDSMITH M, KUPERVASER M, ALCALDE M, FLEISHMAN SJ.Designed high-redox potential laccases exhibit high functional diversity[J].ACS Catalysis,2022,12(21):13164-13173.
[9]
RUIZ-DUEÑAS FJ, MORALES M, GARCÍA E, MIKI Y, MARTÍNEZ MJ, MARTÍNEZ AT.Substrate oxidation sites in versatile peroxidase and other basidiomycete peroxidases[J].Journal of Experimental Botany,2009,60(2):441-452.
[10]
VALDERRAMA B, AYALA M, VAZQUEZ- DUHALT R.Suicide inactivation of peroxidases and the challenge of engineering more robust enzymes[J].Chemistry & Biology,2002,9(5):555-565.
[11]
AYODEJI FD, SHAVA B, IQBAL HMN, ASHRAF SS, CUI JD, FRANCO M, BILAL M.Biocatalytic versatilities and biotechnological prospects of laccase for a sustainable industry[J].Catalysis Letters,2023,153(7):1932-1956.
[12]
ZHOU WT, ZHANG WX, CAI YP.Laccase immobilization for water purification: a comprehensive review[J].Chemical Engineering Journal,2021,403: 126272.
[13]
BILAL M, ASGHER M, PARRA-SALDIVAR R, HU HB, WANG W, ZHANG XH, IQBAL HMN.Immobilized ligninolytic enzymes: an innovative and environmental responsive technology to tackle dye-based industrial pollutants: a review[J].The Science of the Total Environment,2017,576: 646-659.
[14]
KHATAMI SH, VAKILI O, MOVAHEDPOUR A, GHESMATI Z, GHASEMI H, TAHERI-ANGANEH M.Laccase: various types and applications[J].Biotechnology and Applied Biochemistry,2022,69(6):2658-2672.
[15]
AINIWAER A, LIANG Y, YE X, GAO RJ.Characterization of a novel Fe2+ activated non-blue laccase from Methylobacterium extorquens[J].International Journal of Molecular Sciences,2022,23(17):9804.
[16]
ANTOŠOVÁ Z, SYCHROVÁ H.Yeast hosts for the production of recombinant laccases: a review[J].Molecular Biotechnology,2016,58(2):93-116.
[17]
RÜHL M, LANGE K, KÜES U.Laccase production and pellet morphology of Coprinopsis cinerea transformants in liquid shake flask cultures[J].Applied Microbiology and Biotechnology,2018,102(18):7849-7863.
[18]
RIVERA-HOYOS CM, MORALES-ÁLVAREZ ED, POUTOU-PIÑALES RA, PEDROZA-RODRÍGUEZ AM, RODRÍGUEZ-VÁZQUEZ R, DELGADO-BOADA JM.Fungal laccases[J].Fungal Biology Reviews,2013,27(3/4):67-82.
[19]
KILARU S, HOEGGER PJ, MAJCHERCZYK A, BURNS C, SHISHIDO K, BAILEY A, FOSTER GD, KÜES U.Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters[J].Applied Microbiology and Biotechnology,2006,71(2):200-210.
[20]
KILARU S, HOEGGER PJ, KÜES U.The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies[J].Current Genetics,2006,50(1):45-60.
[21]
TIAN YS, XU H, PENG RH, YAO QH, WANG RT.Heterologous expression and characterization of laccase 2 from Coprinopsis cinerea capable of decolourizing different recalcitrant dyes[J].Biotechnology, Biotechnological Equipment,2014,28(2):248-258.
[22]
WANG B, WANG LJ, LIN YQ, HAN Q, HAN J, GAO JJ, TIAN YS, ZHAO W, PENG RH, YAO QH.Purification and characterization of a laccase from Coprinopsis cinerea in Pichia pastoris[J].World Journal of Microbiology and Biotechnology,2014,30(4):1199-1206.
[23]
BAO SY, TENG Z, DING SJ.Heterologous expression and characterization of a novel laccase isoenzyme with dyes decolorization potential from Coprinus comatus[J].Molecular Biology Reports,2013,40(2):1927-1936.
[24]
PAN K, ZHAO NN, YIN Q, ZHANG TW, XU XL, FANG W, HONG YZ, FANG ZM, XIAO YZ.Induction of a laccase Lcc9 from Coprinopsis cinerea by fungal coculture and its application on indigo dye decolorization[J].Bioresource Technology,2014,162: 45-52.
[25]
XU GF, WANG JJ, YIN Q, FANG W, XIAO YZ, FANG ZM.Expression of a thermo- and alkali-philic fungal laccase in Pichia pastoris and its application[J].Protein Expression and Purification,2019,154: 16-24.
[26]
DORNTE B, KUES U.Split trp1+ gene markers for selection in sequential transformations of the agaricomycete Coprinopsis cinerea[J].Current Biotechnology,2017,6(2):139-148.
[27]
YIN Q, ZHOU G, PENG C, ZHANG YL, KÜES U, LIU JJ, XIAO YZ, FANG ZM.The first fungal laccase with an alkaline pH optimum obtained by directed evolution and its application in indigo dye decolorization[J].AMB Express,2019,9(1):151.
[28]
DÖRNTE B, KÜES U.Paradoxical performance of tryptophan synthase gene trp1+ in transformations of the basidiomycete Coprinopsis cinerea[J].Applied Microbiology and Biotechnology,2016,100(20):8789-8807.
[29]
XIAO Y, TU X, WANG J, ZHANG M, CHENG Q, ZENG W, SHI Y.Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2[J].Applied Microbiology and Biotechnology,2003,60(6):700-707.
[30]
WANG JY, LU L, FENG FJ.Improving the indigo carmine decolorization ability of a Bacillus amyloliquefaciens laccase by site-directed mutagenesis[J].Catalysts,2017,7(9):275.
[31]
AHLAWAT A, JASWAL AS, MISHRA S.Proposed pathway of degradation of indigo carmine and its co-metabolism by white-rot fungus Cyathus bulleri[J].International Biodeterioration & Biodegradation,2022,172: 105424.
[32]
BINNINGER DM, SKRZYNIA C, PUKKILA PJ, CASSELTON LA.DNA-mediated transformation of the basidiomycete Coprinus cinereus[J].The EMBO Journal,1987,6(4):835-840.
[33]
HU J, ZHANG YL, XU Y, SUN QY, LIU JJ, FANG W, XIAO YZ, KÜES U, FANG ZM.Gongronella sp. w5 elevates Coprinopsis cinerea laccase production by carbon source syntrophism and secondary metabolite induction[J].Applied Microbiology and Biotechnology,2019,103(1):411-425.
[34]
SCHNEIDER P, CASPERSEN MB, MONDORF K, HALKIER T, SKOV LK, ØSTERGAARD PR, BROWN KM, BROWN SH, XU F.Characterization of a Coprinus cinereus laccase[J].Enzyme and Microbial Technology,1999,25(6):502-508.
[35]
KITTL R, MUEANGTOOM K, GONAUS C, KHAZANEH ST, SYGMUND C, HALTRICH D, LUDWIG R.A chloride tolerant laccase from the plant pathogen ascomycete Botrytis aclada expressed at high levels in Pichia pastoris[J].Journal of Biotechnology,2012,157(2):304-314.
[36]
RÜHL M, MAJCHERCZYK A, KÜES U.Lcc1 and Lcc5 are the main laccases secreted in liquid cultures of Coprinopsis cinerea strains[J].Antonie Van Leeuwenhoek,2013,103(5):1029-1039.
[37]
XU GF, WU YW, ZHANG YL, FANG W, XIAO YZ, FANG ZM.Role of N-glycosylation on the specific activity of a Coprinopsis cinerea laccase Lcc9 expressed in Pichia pastoris[J].Journal of Bioscience and Bioengineering,2019,128(5):518-524.
[38]
AZA P, de SALAS F, MOLPECERES G, RODRÍGUEZ- ESCRIBANO D, deLa FUENTE I, CAMARERO S.Protein engineering approaches to enhance fungal laccase production in S. cerevisiae[J].International Journal of Molecular Sciences,2021,22(3):1157.
[39]
GLAZUNOVA OA, MOISEENKO KV, KAMENIHINA IA, ISAYKINA TU, YAROPOLOV AI, FEDOROVA TV.Laccases with variable properties from different strains of Steccherinum ochraceum: does glycosylation matter?[J].International Journal of Molecular Sciences,2019,20(8):2008.
[40]
LIU WJ, LIU C, LIU L, YOU YT, JIANG JH, ZHOU ZK, DONG Z.Simultaneous decolorization of sulfonated azo dyes and reduction of hexavalent chromium under high salt condition by a newly isolated salt-tolerant strain Bacillus circulans BWL1061[J].Ecotoxicology and Environmental Safety,2017,141: 9-16.
[41]
MUEANGTOOM K, KITTL R, MANN O, HALTRICH D, LUDWIG R.Low pH dye decolorization with ascomycete Lamprospora wrightii laccase[J].Biotechnology Journal,2010,5(8):857-870.
[42]
XU F.Oxidation of phenols, anilines, and benzenethiols by fungal laccases: correlation between activity and redox potentials as well as halide inhibition[J].Biochemistry,1996,35(23):7608-7614.
[43]
POLYAKOV KM, GAVRYUSHOV S, FEDOROVA TV, GLAZUNOVA OA, POPOV AN.The subatomic resolution study of laccase inhibition by chloride and fluoride anions using single-crystal serial crystallography: insights into the enzymatic reaction mechanism[J].Acta Crystallographica Section D, Structural Biology,2019,75(Pt 9):804-816.
[44]
YANG QH, ZHANG ML, ZHANG MM, WANG CQ, LIU YY, FAN XJ, LI H.Characterization of a novel, cold-adapted, and thermostable laccase-like enzyme with high tolerance for organic solvents and salt and potent dye decolorization ability, derived from a marine metagenomic library[J].Frontiers in Microbiology,2018,9: 2998.
[45]
WANG JJ, ZHANG R, ZHU GL, WANG LL, BAI H, QIAN Y, ZHOU XZ, YIN Q, ZHANG Y.Expression of a deep-sea bacterial laccase from Halomonas alkaliantartica and its application in dyes decolorization[J].Annals of Microbiology,2023,73(1):19.
[46]
LI J, XIE YN, WANG R, FANG ZM, FANG W, ZHANG XC, XIAO YZ.Mechanism of salt-induced activity enhancement of a marine-derived laccase, Lac15[J].European Biophysics Journal,2018,47(3):225-236.
[47]
LI Z, JIANG S, XIE YN, FANG ZM, XIAO YZ, FANG W, ZHANG XC.Mechanism of the salt activation of laccase Lac15[J].Biochemical and Biophysical Research Communications,2020,521(4):997-1002.
[48]
SI J, CUI BK.A new fungal peroxidase with alkaline-tolerant, chloride-enhancing activity and dye decolorization capacity[J].Journal of Molecular Catalysis B: Enzymatic,2013,89: 6-14.
[49]
AYED L, MAHDHI A, CHEREF A, BAKHROUF A.Decolorization and degradation of azo dye methyl red by an isolated Sphingomonas paucimobilis: biotoxicity and metabolites characterization[J].Desalination,2011,274(1/2/3):272-277.
[50]
PAGNACCO MC, MAKSIMOVIĆ JP, NIKOLIĆ NT, BAJUK BOGDANOVIĆ DV, KRAGOVIĆ MM, STOJMENOVIĆ MD, BLAGOJEVIĆ SN, SENĆANSKI JV.Indigo carmine in a food dye: spectroscopic characterization and determining its micro-concentration through the clock reaction[J].Molecules,2022,27(15):4853.
[51]
MAYER A M, STAPLES R C.Laccase: new functions for an old enzyme[J].Phytochemistry,2002,60(6):551-65.
[52]
DALMÁZIO I, de URZEDO APFM, ALVES TMA, CATHARINO RR, EBERLIN MN, NASCENTES CC, AUGUSTI R.Electrospray ionization mass spectrometry monitoring of indigo carmine degradation by advanced oxidative processes[J].Journal of Mass Spectrometry: JMS,2007,42(10):1273-1278.
[53]
TERRES J, BATTISTI R, ANDREAUS J, de JESUS PC.Decolorization and degradation of indigo carmine dye from aqueous solution catalyzed by horseradish peroxidase[J].Biocatalysis and Biotransformation,2014,32(1):64-73.
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Year 2024 volume 64 Issue 8
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doi: 10.13343/j.cnki.wsxb.20240085
  • Receive Date：2024-02-01
  • Online Date：2026-03-19
  • Published：2024-05-17
Article Data
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History
  • Received：2024-02-01
  • Accepted：2024-05-13
Funding
Affiliations
    1 School of Life Sciences, Anhui University, Hefei 230601, Anhui, China
    2 Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China
    3 Anhui Provincial Engineering Technology Research Center of Microorganisms and Biocatalysis, Hefei 230601, Anhui, China
    4 University of Goettingen, Goettingen 237077, Germany

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FANG Zemin, E-mail:
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表12种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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