Selection of High Laccase-Producing Coriolopsis gallica Strain T906: Mutation Breeding, Strain Characterization, and Features of the Extracellular Laccases

Abstract

Commercial application of laccase is often hampered by insufficient enzyme stocks, with very low yields obtained from natural sources. This study aimed to improve laccase production by mutation of a Coriolopsis gallica strain and to determine the biological properties of the mutant. The high-yield laccase strain C. gallica TCK was treated with N-methyl-N-nitro-N-nitrosoguanidine and ultraviolet light. Among the mutants isolated, T906 was found to be a high-production strain of laccases. The mutant strain T906 was stabilized via dozens of passages, and the selected ones were further processed for optimization of metallic ion, inducers, and nutritional requirements, which resulted in the optimized liquid fermentation medium MF9. The incubation temperature and pH were optimized to be 30℃ and 4.5, respectively. The mutant strain T906 showed 3-times higher laccase activity than the original strain TCK under optimized conditions, and the maximum laccase production (303 U/ml) was accomplished after 13 days. The extracellular laccase isoenzyme 1 was purified and characterized from the two strains, respectively, and their cDNA sequence was determined. Of note, the laccase isoenzyme 1 transcription levels were overtly increased in T906 mycelia compared with values obtained for strain TCK. These findings provide a basis for C. gallica modification for the production of high laccase amounts.

Introduction

Laccase (p-diphenol oxidase, E.C. 1.10.3.2) is part of a group of copper-containing polyphenol oxidases of the blue oxidase family [27]. Laccases can modify organic and inorganic substrates via four-electron reduction of O2 to H2O with the concomitant oxidation of phenolic compounds [20]. Therefore, these enzymes have broad application prospects and potential value, such as in pulp bio-bleaching, dye decolorization, wastewater treatment, food processing, medical care, and biomass energy. In previous reports, the immobilization of laccase on several supports has proved to be promising for its biotechnological applications [10,23,24]. However, commercial application of laccases is often hampered by insufficient enzyme stocks, with very low yields obtained from natural sources [11]. Laccases are widely spread among fungi, bacteria, and plants. Fungal laccases usually show higher redox potential and yield than their bacterial counterparts [15], and their secretion patterns are affected by the fungal species, substrates, and eco-physiological factors [4]. The most important fungal laccase sources are white-rot fungi (WRF), which are involved in lignin degradation [16]. The potential applications of lignocellulolytic enzymes in industrial and environmental technologies require large amounts of biodegradation enzymes at low cost. Therefore, it is urgent to select new organisms with high synthesis of enzymes such as laccase, and develop strategies for their overproduction. There are many reports about using conventional mutants, such as UV irradiation or chemical mutagenesis, in order to improve enzyme production, including laccase, cellulase, and hemicellulase [1]. Recently, an attempt was made to develop a UV mutant for enhanced production of cellulase with reduced sensitivity to catabolite repression: the mutant strain Trichoderma asperellum SR1-7 was shown to produce filter paper enzyme, carboxymethyl cellulose, and β-glucosidase under optimized conditions, reaching 1.4-, 1.3-, and 1.5-fold increases compared with the wild type [26].

Although constitutive blue oxidases, such as fungal laccase, are usually produced in small amounts, the yield can be significantly enhanced by a wide variety of substances, including phenolic compounds (e.g., 2,5-xylidine) and metal ions (e.g., copper or cadmium). Therefore, the effects of different inducers, such as aromatic compounds and lignin-containing plant extracts, on laccase production were assessed. Indeed, copper was shown to increase laccase gene expression levels in Podospora anserina [12], Trametes versicolor [14], and Pleurotus ostreatus [22]. Hence, in the regulatory sequence upstream of the laccase genes, metal regulatory element consensus sequences were found in the promoter regions in Podospora anserina [12] and Phanerochaete chrysosporium [5].

In this study, using the high laccase-producing Coriolopsis gallica strain TCK as parental strain, a mutant strain named T906 was obtained by a combination of chemical and UV treatments. Based on the optimization of temperature, pH, carbon source, nitrogen source, and metallic ion, the optimized liquid fermentation medium MF9 was developed. Comparing with wild-type C. gallica strain TCK, the laccase activity in mutant strain T906 was 303 U/ml under optimized conditions, which reached about 7.5-times to the initiate MF liquid culture, and showed 3-times higher laccase activity than the parental TCK wild type. These findings demonstrated that C. gallica can be modified to improve its laccase production.

 

Materials and Methods

Chemicals and Microorganisms

All chemicals were of analytical grade and purchased from Invitrogen (USA), except 2,2’-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS), syringaldazine, and vanillic acid, which were from Sigma-Aldrich (Germany). C. gallica strain TCK was isolated from poplar in the north of China (CCTCC number: M2011317). It was identified as C. gallica by comparison of internal transcribed spacer region sequences of the 18S rRNA and 5.8S rRNA genes in GenBank (GenBank Accession No. EF458487) as well as morphology observations. The strain was maintained on potato medium (PDA), and used as the parent for mutation breeding of a higher laccase-producing mutant in this study.

Laccase Enzyme Assays

Culture of the WRF C. gallica strains, and laccase production were carried out as described previously [19]. Protein concentration was determined using a Bradford Protein Assay Kit (Tiangen, China) with bovine serum albumin as the standard. Laccase activity was assayed by measuring oxidation of ABTS at 30℃ in 0.1 M NaAc-HAc (pH 4.0). The reaction solution consisted of 1.95 ml of 0.1 M NaAc-HAc, 2 ml of 0.5 mM ABTS, and 50 μl of enzyme solutions. After 3 min, substrate oxidation by laccase was monitored at 420 nm. One unit of activity was defined as the amount of enzyme needed to oxidize 1 μM of ABTS per minute [18]. All samples were measured in triplicates.

Mutant Screening

The conidia of C. gallica TCK were collected after culture on PDA medium for 21 days at 30℃, dispersed with sterilization glass beads by vortexing, and cultured in PDA liquid medium at 30℃ for 10 h. Ten milliliters of log-phase culture was transferred onto PDA-remazol brilliant blue R (PDA-RB) medium plates. TCK spore suspensions were treated with 20 μg/ml NTG for 30 min, followed by UV irradiation under magnetic stirring as following: a 15 W or 30 W UV lamp was used at a distance of 30 cm with irradiation times of 10, 20, 30, 40, 50, 60, 80, 100, and 150 sec, respectively. The PDA-RB plates were incubated at 30℃. Colonies with large color rings were inoculated into MF medium and cultured for 13 days at 30℃. Laccase activities of the isolated strain were determined as described above.

Optimization of Submerged Fermentation Conditions for Laccase Production

Culture of the WRF C. gallica strains were carried out as described previously. To enhance the laccase production, the fermentation conditions were optimized. First, the fermentation temperature and initial pH values were screened ranging from 25℃ to 40℃ and pH values 3 to 7, respectively. Then, based on the liquid fermentation culture MF (millfeed 25 g/l, glucose 10 g/l, KH2PO4 0.2% (w/v), MgSO4·7H2O 2 g/l, CaCl2·2H2O 0.5 g/l, ammonium tartrate 0.1 g/l, VB1 1.84 g/l, Tween-80 10 mg/l, MnSO4·H2O 0.05% (w/v), NaCl 7 mg/l, FeSO4·7H2O 7 mg/l, CaCl2 7 mg/l, ZnSO4·7H2O 1.0 mg/l, CuSO4·7H2O 0.5 mg/l, and H3BO3 0.5 mg/l (pH 4.0)), the effects of carbon source (maltose, sucrose, glycerol, glucose, carboxymethylcellulose sodium, starch, or millfeed), nitrogen source (casein, yeast powder, peptone, ammonium nitrate, ammonium sulfate, or ammonium dihydrogen phosphate), metal elements (Cu2+, Fe2+, Mn2+, Zn2+, Ca2+, Co2+, and Mg2+), the initial reaction pH, and optimal temperature of the fermentation cultures were assessed. Meanwhile, several laccase inducers (veratryl alcohol, ABTS, gallic acid, tannic acid, and guaiacol) were added at 0.5 mM after 96 h of incubation.

Biochemical Characterization of the Purified Laccase from TCK and T906 Strains

Culture supernatants of TCK and T906 at 11 days were collected by centrifugation at 12,000 rpm for 15 min at 4℃. Laccase isolation and purification were carried out as described by Martin et al. [19]. Mass spectrometry was utilized to identify laccase protein sequences in the TCK and T906 strains, respectively, which were compared with known sequences in the National Center for Biotechnology Information (NCBI; http://blast.ncbi.nlm.nih.gov/) nr database, using the Mascot search tool of Matrix Science Company (http://www.matrixscience.com). The temperature optimum and stability of purified laccases from the two strains were assessed by using 0.1 mM ABTS as substrate in 100 mM sodium tartrate buffer (pH 4.0) at 30℃ [6]. The temperature stability of laccases was tested by incubating the purified enzymes for 1 h. To determine the pH optima of purified laccase, a series of assessments were performed by using 100 mM sodium tartrate buffer (pH 2.0–7.0) and 100 mM sodium phosphate buffer (6.0–8.0). The pH stability of laccase was tested by incubating the purified enzymes for 24 h at pH 2.0 to 7.0 in 30℃. Metal ions were added to a final concentration of 2 mM in 0.1 mM ABTS reaction buffer, and laccase activity was determined after 1 h at 30℃ and pH 4.0, respectively.

Sequence Analysis of Laccase Genes from C. gallica TCK and T906 Strains

Mycelia of C. gallica strains TCK and T906 grown for 8 days in MF9 fermentation culture were collected by centrifugation. Genomic DNA and total RNA were isolated from mycelia of the TCK and T906 strains using the DNA Extraction Kit (Qiagen, Germany) and RNA easy Mini kit (Qiagen), respectively. Primers (Laccase-gF, 5’-TCT TCG GCA GTC CTC CCC AAT C-3’ and Laccase-gR, 5’-AAC GAC GCC ATA AGC CCA AAC C-3’) were designed according to nucleotide sequences deduced from the homolog proteins obtained by mass spectrometry analysis. Reverse transcription was performed using the First Stand cDNA Synthesis Kit (ToYoBo, Japan) according to the manufacturer’s instructions. Polymerase chain reaction (PCR) products amplified from laccase genomic DNA and cDNA were cloned into PCR2.1 TA Vector (Invitrogen, USA) and sequenced. Sequences were compared using the BLAST search tool of the NCBI database.

Expression Analysis of the Laccase Gene in C. gallica TCK and T906 Strains

To analyze the expression profile of the laccase gene, mycelia of the C. gallica TCK and T906 strains at 3, 5, 7, 9, 11, 13, and 15 days were collected. Total RNA from each sample was obtained as described above. qRT-PCR was conducted on cDNA samples with three independent replicates (Primers: lac-F1, 5’-CCT ACT GCC GAT GCG ACT CTC A-3’; lac-R1, 5’-TGA CAC CAG GCG GAA GCG ATA-3’). The 18S-RNA of C. gallica was used as an internal control (Primers: 18s-F1, 5’-TTG CTG GTT GCC GTC TTC TTA GA-3’; 18s-R1, 5’-CTC GCT GGC TCT GTC AGT GTA G-3’). qRT-PCR was carried out on an Eppendorf Mastercycler Ep Realplex, with SYBR Premix Ex Taq (Takara Bio, China). The PCR conditions were as follows: 95℃ for 30 sec; 40 cycles at 95℃ for 15 sec and 63℃ for 30 sec; a denaturation step to check the absence of unspecific products or primer dimers. Data analysis was carried out by the relative quantification method described by Muller et al. [21]. An assay was carried out by adding 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mM Cu2+ in the fermentation culture, respectively. Mycelial culture flasks were then incubated at 30℃ and 150 rpm. At 11 days, mycelial extracts were separated by centrifugation, respectively, and qRT-PCR was performed as above.

Statistical Analysis

The data variance (three replicates) were analyzed by one-way ANOVA, and paired comparison between the groups was performed using the S-N-K method. Statistical significance differences were set at a level of p < 0.05. The values were expressed as the mean ± SD.

 

Results

Identification of the C. gallica Mutant Strain T906 with Higher Laccase Yield

The laccase activity of the C. gallica wild-type strain TCK was 15.40 U/ml after 13 days of culture in the MF medium. To achieve higher laccase activity and satisfy the industry need, mutagenesis by both NTG and UV irradiation of the C. gallica strain TCK was conducted, and more than 120 mutants were obtained. The mutants showing large color rings on RBBR-containing PDA plates were screened, and six high laccase output mutant strains (T901, T902, T915, T918, T920, and T906) were selected for further analysis. Finally, the mutant strain T906, which stably showed the highest laccase activity (49.08 U/ml at 13 days in MF liquid fermentation culture), was further assessed.

Growth Characteristics of C. gallica TCK and T906 Strains

To evaluate the growth traits of the two strains, their morphological properties were compared after 3 and 8 days of culture on PDA. The colonies of the mutant strain T906 (Fig. 1B) were overtly smaller, but thicker than those of the TCK strain (Fig. 1A). In addition, T906’s mycelia (Fig. 1D) were shorter, broader, and arranged more regularly compared with those of the wild-type strain TCK (Fig. 1C) at 3 days, as assessed by scanning electron microscopy (SEM). At day 8, mycelium aging was more pronounced in strain TCK (Fig. 1E) compared with T906 (Fig. 1F). These findings indicate great differences in morphology between the mutant T906 and the original strain TCK. Previous studies have revealed that the morphology of filamentous fungi is mainly influenced by medium constituents, physical culturing conditions, genetic material, and phase of cultivation [31]. In this case, the mycelia of mutant strain T906 were smaller compared with the wild strain TCK, which leads to the volumetric power input in strain T906 increase in submerged culture [4]. Meanwhile, the higher division speed resulted in an increased proportion of mycelia in the logarithmic growth phase of strain T906, which may be one of the possible explanations for the morphology differences in SEM images between T906 and TCK. However, the changes of enzyme activities or metabolic pathways are other possible explanations.

Fig. 1.Growth characteristics of C. gallica wild type and its mutant strain T906. Wild strain TCK colonies (A) were larger, but thinner compared with those of T906 (B). In addition, mycelia of strain T906 (D, F) were shorter, broader, and arranged more regularly than those of strain TCK (C, E) as assessed by SEM at 3 days (C, D) and 8 days (E, F).

Culture Conditions Affect Laccase Yields in C. gallica Strain T906

The results showed that C. gallica strain T906 had maximum laccase production at 30℃. Mycelial growth and laccase production decreased markedly below 25℃ or over 35℃. The maximum laccase production (93.44 U/ml) in the MF medium was achieved at an initial pH of 4.5. Therefore, further experiments for fermentation condition optimization were designated at 30℃ and initial pH 4.5 with 200 rpm. Laccase activity levels of strain T906 under different carbon and nitrogen sources were evaluated: the highest values of 83.07 and 96.66 U/ml were obtained with glycerol and ammonium dihydrogen phosphate as carbon and nitrogen sources, respectively. Additionally, millfeed, ammonium sulfate, and casein were also good sources of carbon and nitrogen. The best combination of carbon and nitrogen sources was also analyzed: an optimum culture medium consisted of 1.5% casein, 2.5% wheat bran, 1% glycerol, and 0.5% di-ammonium hydrogen phosphate, instead of the original carbon and nitrogen sources in the MF medium. Indeed, laccase activity of T906 reached 153 U/ml with carbon and nitrogen sources supplied in the medium. Optimal metal ion concentrations were as follows: MgSO4·7H2O, 5g/l; CuSO4·5H2O, 0.6g/l; MnSO4·H2O, 0.05 g/l; FeSO4·7H2O, 0.04 g/l; CaCl2, 0.08 g/l; ZnSO4·7H2O, 0.15 g/l; and CoCl2, 0.01 g/l. The inducer types and concentrations were also analyzed (Fig. 2A). Interestingly, induction with resveratrol (0.5 mM) of C. gallica showed the best outcome, followed by tannic acid and guaiacol. Meanwhile, ABTS and gallic acid did not affect laccase production. Finally, we established the optimized liquid culture, named MF9 liquid medium. At 13 days of incubation in the optimal mycelium liquid fermentation, the highest laccase activity of T906 reached 303 U/ml, corresponding to a laccase activity of 27.67 U/mg of crude protein, whereas 100.1 U/ml corresponding to a laccase activity of 9.40 U/mg of crude protein was obtained for the TCK strain (Fig. 2B).

Fig. 2.Optimization of fermentation conditions. (A) Laccase activity of C. gallica strain T906 using different inducers in MF medium. (B) Laccase activity of TCK and T906 in MF medium and the optimized MF9 medium. The horizontal axes represent time of culture (in days), and the vertical ones laccase activity (U/ml). Data are the mean ± SD from at least three independent experiments performed in duplicates.

Purification and Characterization of Laccase from the T906 and TCK Strains

Extracellular laccase levels in culture supernatants of the two strains were analyzed by native PAGE. Laccase isoenzymes 1 (LAC1) was isolated and purified as described previously. After separation of T906 and TCK samples on a Mono Q column, respectively, laccase was eluted as a single peak. Combined laccase-containing fractions were concentrated and loaded onto a Superdex 200 GL gel filtration column, and distinct peaks of laccase activity were eluted, corresponding to final laccase activities per milligram protein of 15.37-fold and 11.43-fold compared with values obtained for the original supernatants, respectively. Both C. gallica strains T906 and TCK showed homogeneous purified laccase characterized by a single protein as assessed by SDS-PAGE (Fig. 3), and the molecular mass of laccase was 53.7 kDa as analysis by high-performance gel filtration chromatography. IEF indicated that LAC1 is a monomeric protein with an acidic isoelectric point of 4.1.

Fig. 3.SDS-PAGE of extracellular fluid (A) and purified laccase (B) from C. gallica strains TCK and T906. (A) 1, TCK on the 5th day; 2, T906 on the 5th day; 3, TCK on the 7th day; 4, T906 on the 7th day; 5, TCK on the 9th day; 6, T906 on the 9th day; 7, TCK on the 11th day; 8, T906 on the 11th day. (B) 1, purified laccase from TCK; 2, purified laccase from T906; M, Protein molecular weight marker of 99.2, 66.4, 44.3, 29.0, and 20.1 kDa.

Effects of Temperature, pH, and Metallic Ions on LAC1 Activity and Stability

The temperature optima for laccases from the two strains were all 60℃ (Fig. 4A), whereas the enzyme stability temperature of 40℃ was obtained for both strains (Fig. 4B). The residual activities of laccase samples from TCK and T906 at 60℃ after 1 h were 82% and 86%, respectively, but almost undetectable at 70℃ after 1 h, suggesting that the thermal stability of laccase in the two strains was between 20℃ and 60℃ (Fig. 4B). The optimal pH value was 2.5, which yielded 251% and 245% of laccase activity obtained for T906 at pH 4.0, in T906 and TCK samples, respectively (Fig. 4C). Interestingly, laccase activity was stable for 24 h at pH 2.0-4.0, which peaked at pH 4.0 but decreased significantly beyond pH 5.0, with only 7.9% and 9.2% obtained for T906 and TCK, respectively, relative to values at pH 4.0 (Fig. 4D). The supply of Cu2+ obviously promoted laccase activity, which was 196% higher compared with the control group (without metallic ion additive) in strain T906, and 188% in TCK. Supply of Mg2+, Zn2+, and Ba2+ positively affected the enzyme activity, whereas Pb2+ instead greatly decreased laccase activity in T906 (Fig. 4E). As shown in Fig. 4E, SDS and EDTA inhibited the activity of laccase to a certain extent.

Fig. 4.Effects of temperature, pH, and metal ions on the activities of purified LAC1 from C. gallica strains TCK and T906. (A) Temperature optima for pure laccases from TCK and T906; (B) thermostability for pure laccases from TCK and T906; (C) pH optima for pure laccases from TCK and T906; (D) pH stability of pure laccases from TCK and T906. The laccase activity of strain T906 at 30℃ and pH 4.0 was used as the reference (set to 100%). (E) Effects of metallic ions and inducers on laccase activity. The LAC1 activity without metallic ion additive was used as the reference (set to 100%). Data are the mean ± SD from at least three independent experiments performed in duplicates.

Cloning and Sequence Analysis of Laccase Isoenzyme 1 Gene from T906 and TCK

A single extracellular laccase isoenzyme, named LAC1, with relative molecular mass of 61.5 kDa and theoretical isoelectric point of 4.1, was purified from the fermentation suspension of both wild-type TCK and mutant T906 strains. Mass spectrometry analysis suggested that the LAC1 protein was similar to C. gallica laccase. The ORF of the laccase isoenzyme 1 gene (LAC1) in both strains was 1,554 bp, encoding a protein of 517 amino acids (GenBank No. KP135562 for T906 and KP135563 for TCK). The LAC1 protein was predicted to contain a 22-residue N-terminal signal peptide and two copper-binding sites. There were 14 nucleotide differences, but only four amino acid variations between the two strains (positions 145, 273, 450, and 453). The deduced amino acid sequence of T906 LAC1 showed 99%, 96%, 91%, and 81% identity with laccase homologs from C. gallica (GenBank No. ABD93940.1), Coriolopsis trogii (GenBank No. CAC13040.1), Coriolopsis caperata (GenBank No. AGE13770.1), and Trametes sp. C30 (GenBank No. ACO53432.1), respectively.

Expression Profiles of LAC1 in Mutant T906 and Wild-Type Strains

LAC1 transcriptions levels in TCK and T906 were analyzed using qRT-PCR at different time points (Figs. 5A and 5B), as well as under different copper concentrations (Figs. 5C and 5D). Data were normalized to LAC1 transcription levels in wild-type strain TCK at 3 days. The highest LAC1 transcription level in T906 detected at 11 days was 34.13 times higher than TCK values (Fig. 5A). In agreement with the laccase transcription levels, the extracellular laccase activities of both strains peaked at 11 days, with the mutant strain T906 showing 289.6 U/ml and the wild-type strain TCK showing 99.6 U/ml (Fig. 5B). These results indicated that at the peak of laccase production in the mutant T906, gene transcription levels were greatly increased, suggesting that significantly increased transcription of LAC1 leads to higher laccase activity in the mutant T906 compared with the TCK amounts. Subsequently, after 13 days, LAC1 transcriptional levels fell relatively sharply to 4.86-fold the TCK values; all LAC1 transcription levels of the mutant strain T906 were significantly higher than those obtained for the original strain (Fig. 5A). According to qRT-PCR data, LAC1 transcription levels increased in both strains with copper ion concentrations. When T906 was grown for 11 days with copper ions at 2.5 mM in the culture medium, LAC1 transcription levels were more than 5-times higher compared with values obtained with 0.5 mM of copper ions; meanwhile, TCK grown for 11 days with copper ions at 2.5 mM showed LAC1 mRNA levels of about 2 times that obtained with medium containing copper ions at 0.5 mM. Therefore, copper ions greatly enhance LAC1 transcription levels, especially in the mutant T906 (Figs. 5C and 5D).

Fig. 5.LAC1 gene transcription levels and laccase activity in T906 and TCK strains under various conditions. (A) Fold transcription of LAC1 in inductive-mutant T906 and wild-type TCK strains at 3 to 15 days. The LAC1 mRNA level at 3 days in MF9 culture medium of TCK was used as the reference (set as 1). (B) Activity of LAC1 in inductive-mutant T906 and wild-type TCK strains at 3 to 15 days. (C) Fold transcription of LAC1 in strains T906 and TCK at different copper ion concentrations. LAC1 mRNA levels at 11 day in MF9 culture medium of TCK was used as the reference (set as 1). (D) Effect of Cu2+ concentration on laccase activity in both wild-type strain TCK and mutant strain T906. Bars represent the mean ± SD of three replicate samples.

 

Discussion

Laccase attracts a lot of attention and can be potentially used in industry for broad applications, including biodegradation of environmental pollutants as well as pulping and bleaching of paper [27]. Nevertheless, the laccase yield from natural sources is usually very low, while affected remarkably by the fungal strain [4], so it is urgently necessary to select sufficient enzyme stocks for optimal performance. It has been reported that strains exposed to UV display increased reactive oxygen species production, with activation of some small molecules such as riboflavin, tryptophan, and porphyrin [25]. Besides this, traditional methods such as chemical mutagenesis have been used to improve the production of enzymes such as laccase, cellulase, and hemicellulase to a certain extent. In this study, the mutant T906 was identified with 3-fold higher laccase production and stable yield after a successful combination of chemical and UV mutagenesis, and was further assessed.

It is known that laccase production in fungi can be significantly enhanced by several factors, including medium composition, substances, pH, temperature, and aeration rate [17]. Assessment of LAC1 transcriptional regulation in C. gallica strains TCK and T906 was important to further unveil the mechanism of laccase production, as well as the physiological roles played by different laccases. In this study, the highest laccase activity levels of 83.07 U/ml and 96.66 U/ml were obtained with glycerol and ammonium dihydrogen phosphate as carbon and nitrogen sources, respectively. As described above, copper ions starkly enhanced the laccase activity, especially in T906. Copper is involved in laccase regulation at the transcriptional level in a number of basidiomycetes such as Trametes versicolor [9]. How copper activates the transcription of laccase genes and binds response elements is not completely understood. A possible reason is that an ACE1-like transcription factor is essential for laccase induction with copper addition. Another possible reason is that laccases might act against oxidative stress to chelate Cu2+ ions, given that they contain copper-binding sites [2]. Previous studies have also demonstrated differential transcription in response to copper in Volvariella volvacea [7] and Pleurotus ostreatus [22], which was consistent with the results in this study.

Fungi may synthesize a number of laccase isoenzymes, with some in response to regulatory signals and others constitutively expressed, depending on differences in their localization and physicochemical kinetic properties, especially in regulatory mechanisms. Indeed, different laccases are produced at distinct growth periods of filamentous fungi. Of note, inducers such as veratryl alcohol, veratraldehyde, vanillic acid, and ferulic acid can promote laccase production, and although they have no significant effect on total laccase activity, they yield different laccase isoenzyme patterns [13]. In this study, only one laccase isoenzyme band was found, albeit this result does not necessarily mean that TCK and T906 have only one laccase isoenzyme. Indeed, it is plausible that besides the extracellular LAC1 found in this study for the two s tains, more isoenzymes may be found intracellularly, at fruiting and/or spore stages, or under other culture conditions. Further research is needed for clarification.

Laccase isoenzymes, named LAC1, with purification folds (15.37 for T906 and 11.43 for TCK) were successfully purified. The physicochemical and catalytic properties of the LAC1 from the two strains were similar. Both of them showed a temperature-dependent oxidation of ABTS, which peaked at pH 2.5. Temperature optima for the LAC1s reported in this study were slightly higher (60℃) than that in other fungi previously reported, which show maximum catalytic activity at 50℃ [8]. The activities of purified the LAC1s were stable in the temperature range from 20℃ to 60℃, with more than 82% residual activity being maintained after 1 h. Hence, the LAC1 reported in this study can be applied in a broad temperature range, particularly under high temperatures. The pH is also critical for laccase stability and activity, as it impacts the enzyme’s active site, thus affecting the binding to amino acids. In this study, the activities of the LAC1s were stable in the pH values 2.0-4.0, and peaked at pH 4.0. Those were consistent with previous studies, where fungal laccases typically exhibit pH optima lower than 4.0 [3]. Metallic ions can also affect the production and stability of extracellular laccases. A previous report assessing a purified polyphenol oxidase from tobacco leaf suggested that in the presence of Cu2+, the highest oxidase activity levels were achieved. Zn2+, Na+, Ca2+, Ba2+, Mg2+, and syringic acid caused a slight increase in extracellular laccase activity, whereas addition of Cd2+, Fe3+, Mn2+, Cr2+, and caffeine did not alter laccase activity at all [13]. In this study, the supply of Cu2+ overtly promoted laccase activity in both T906 and TCK strains. In addition, Mg2+, Zn2+, and Ba2+ positively affected the enzyme activity, whereas Pb2+ greatly decreased the activity. These findings provide a basis for the treatment of metal-contaminated laccase with these chemicals.

Overall, the mutant C. gallica strain T906 obtained in this study showed increased laccase activity of more than 3-fold compared with the original strain TCK. An optimized liquid fermentation medium was developed and named MF9. The extracellular laccase isoenzyme 1 was purified and characterized from both strains. Our findings provide a basis for fungus engineering in order to increase laccase production.