Optimization, purification and characterization of laccase from Ganoderma leucocontextum along with its phylogenetic relationship

The aim of this work to study an efficient laccase producing fungus Ganoderma leucocontextum, which was identified by ITS regions of DNA and phylogenetic tree was constructed. This study showed the laccase first-time from G. leucocontextum by using medium containing guaiacol. The growth cultural (pH, temperature, incubation days, rpm) and nutritional (carbon and nitrogen sources) conditions were optimized, which enhanced the enzyme production up to 4.5-folds. Laccase production increased 855 U/L at 40 °C. The pH 5.0 was suitable for laccase secretion (2517 U/L) on the 7th day of incubation at 100 rpm (698.3 U/L). Glucose and sucrose were good carbon source to enhance the laccase synthesis. The 10 g/L beef (4671 U/L) and yeast extract (5776 U/L) were the best nitrogen source for laccase secretion from G. leucocontextum. The laccase was purified from the 80% ammonium sulphate precipitations of protein identified by nucleotides sequence. The molecular weight (65.0 kDa) of purified laccase was identified through SDS and native PAGE entitled as Glacc110. The Glacc110 was characterized under different parameters. It retained > 90% of its activity for 16 min incubation at 60 °C in acidic medium (pH 4.0). This enzyme exerted its optimal activity at pH 3.0 and temperature 70 °C with guaiacol substrate. The catalytic parameters Km and Vmax was 1.658 (mM) and 2.452 (mM/min), respectively. The thermo stability of the laccase produced by submerged fermentation of G. leucocontextum has potential for industrial and biotechnology applications. The results remarked the G. leucocontextum is a good source for laccase production.

www.nature.com/scientificreports/ The consensus sequence was generated in BioEdit version 7.2.5 26 . The homology searches were performed at the NCBI using BLASTn. The sequences of this study were deposited in GenBank. The alignment was manually edited at 593 positions. The Amauroderma rude (Berk.) Torrend was used as an outgroup to support the tree. The MEGA 10.0 was used for phylogenetic analysis with 1000 replicates of bootstrap 27 (Fig. 2A). The sequences under MK713839 and MN134012 accession numbers were deposited in the GenBank.
Ganoderma leucocontextum laccase genes were identified by degenerate primers accordingly D'Souza et al. 28 . The laccase genes of exoenzyme were confirmed by a guaiacol test. The primers Cu1F (5′-CAT(C) TGG CAT(C) GGN TTT(C)TTT(C) CA-3′) and Cu2R (5′-G G(A)CT GTG GTA CCA GAA NGT NCC-3′) exhibited the better results during amplification. The template of genomic DNA was isolated from fungal material for single PCR. The amplifications steps were comprised DNA extracts (3 µL) added to a reaction mixture (50 µL) containing of 10X Taq buffer (5 µL) with MgCl 2 , 10 mM dNTPs (4 µL), Taq DNA polymerase (0.2 µL) and 60 µM of both primers (1 µL). The nucleotide sequences of our specimens were deposited in NCBI under the mentioned accession numbers (GenBank MK713839; MN134012). The coding regions and protein sequences after alignment were carried out with identified laccase of different Ganoderma species. The deduced coding regions of sequence released and the additional laccase sequences were retrieved from GenBank, which manually aligned by BioEdit program. The phylogenetic tree was constructed by MEGA10 software using the Maximum Likelihood (ML) method with 1000 replications. The Gaeumannomyces graminis was used as outgroup to root the laccase tree (Fig. 2B).
The phylogenetic identification was confirmed by the author Aisha Umar. These specimens were submitted to "Lahore Herbarium (LAH)" of University of the Punjab.
Qualitative analysis. Malt Extract Agar media was prepared in g/L by adding Malt Extract 7, Agar 10, MgSO 4 ·7H 2 O 0.5, K 2 HPO 4 0.5, KH 2 PO 4 0.5, ZnSO 4 0.005, MnSO 4 0.05, Peptone 2.5 and Glucose 15 29 at pH 5.0 (Fig. 1B). The streptomycin (200 mg/L) was added as an antibacterial agent. The above mentioned nutrient agar media was sterilized in an autoclave for 20 min at 121 °C. After autoclaving 0.02% guaiacol was added to MEA media to screen the best laccase producing specimens. All the plates were incubated at 30 °C for 5 days. The formation of reddish brown oxidation zone on agar plate media was used to screen the laccase from G. leucocontextum.
Quantitative analysis. Kirk's medium was designed for quantitative analysis of laccase activity with a little modification in the shake flasks. The macronutrients and tracer elements (g/L) of Kirk's medium were taken in shake flasks for the growth of mycelium. The macronutrients with little modifications were mixed in g/L (glucose 10 g, yeast extract 5 g, starch 1 g, while tracers MgSO 4 ⋅7H 2 O 0.5 g, NaCl 0.5 g, FeSO 4 ⋅7H 2 O 0.5 g, KH 2 PO 4 0.046 g, K 2 HPO 4 0.1 g, CaCl 2 ⋅2H 2 O 0.5 g, ZnSO 4 0.02 g, CuSO 4 ⋅5H 2 O 0.5 g, H 4 PO 4 1.0 g, Na 4 HPO 4 0.05 g, MnSO 4 0.001 g, ZnSO 4 0.001 g 30 . The pH of liquid medium was adjusted to 5.0. The complete medium of 1 L shake flask was autoclaved and cool down before inoculum with mycelium discs. Three mycelia plugs (5 mm diameter inoculum) of pure culture were added in the above mentioned autoclaved liquid medium (100 mL) of each flask and incubated at 27 ± 2 °C in static condition for 3 days. After 3rd day, culture medium was moved dynamically via shaker to optimize the growth and nutritional conditions. Add the ingredients one by one in continued culture till the optimization of each parameter achieved. The liquid samples in shake flask or submerged conditions were used for the analysis of laccase activity 31 .
The enzyme activity was determined by 100 mM guaiacol substrate dissolved in 100 mM sodium acetate buffer (pH 5.0). This reaction mixture contained 1.5 mL acetate buffer, 1.5 mL guaiacol and 1.0 mL of crude enzyme source. The laccase activity was measured at 27 ± 2 °C or room temperature after 15-30 min 32 . The changed in the absorbance of reaction mixture comprised guaiacol was monitored for 3 min at 470 nm by UV Spectrophotometer 33 . This activity was determined in triplicate by monitoring the absorbance for 3-5 min and expressed in U/L 34 .
Optimization of cultural and nutritional growth conditions for laccase production. Each culture flasks (100 mL) with few mycelia discs were incubated for 7 days at different temperature (20 °C, 40 °C, 60 °C) and pH (3.0, 5.0, 6.0). Each fermentation broth (100 mL) with a few mycelia discs was incubated for 7, 10 and 15 days to maximize the laccase production at different rpm (50,100,150) to maximize the laccase production.
Liquid medium of flask was modified by changing the concentration and nature of nutritional sources. Three actively grown discs from mycelium of G. leucocontextum were taken via cork borer and inoculated in 250 mL Erlenmeyer flasks contained liquid broth (pH 5.0). These cultural flasks were incubated on a rotary shaker (40 °C) at 100 rpm. After 10 days of incubation, the laccase activity was measured 35 . The liquid culture was decanted on Whatman No.1 paper, and the filtrate collected to optimize the following factors: Different carbon sources (20 g and 25 g) like maltose, glucose and sucrose were evaluated for laccase production. The suitable inorganic and organic nitrogen sources were selected to check the maximum laccase production. The organic nitrogen sources included peptone, beef extract and yeast extract (5 g, 10 g), and inorganic ammonium sulphate, sodium nitrate and potassium nitrate were amended in the concentrations of 5 g/L and 10 g/L. The flasks were incubated for 10 days at 40 °C.  www.nature.com/scientificreports/ Purification of laccase isozymes. A complete set up of culture broth (1000 mL) was designed separately under best optimized nutritional and growth condition. The broth was filtered through Whatman filter no. 1 and filtrate centrifuged at 13,000×g for 15 min at 10 °C. The supernatant was collected for partial purification of laccase. The finely grounded powder of (NH 4 ) 2 SO 4 was mixed thoroughly in cold supernatant till the saturation level was achieved (60% and 80%) for protein precipitation 36 . This saturated enzyme assay was incubated overnight at 4 °C and these precipitates were collected by centrifuging at 12,000×g for 35 min. After that the protein pellets were dissolved in 20 mM citrate-phosphate buffer (pH 5.0). The same buffer was used in dialysis at 4 °C for 1 day 37 .
Laccase molecular weight. The yield of expressed protein was evaluated by SDS-PAGE, using a Criterion XT gel system (Bio-Rad, CA, USA). Estimated protein molecular weight (MW) of laccase was made against the standard protein markers (14.3-97.0 KDa). In order to assign ∼ 65 kDa laccase, a native PAGE was performed and stained with guaiacol. The separated protein was visualized by incubating the gel in 50 mM sodium acetate buffer (pH 5.0) containing 100 mM guaiacol.
Biochemical characterization of laccase. The pH (2.0-8.0) was maintained to examine the laccase activity and stability at 40 °C in 50 mM citrate phosphate buffer. The relative enzyme activity was taken after every 15 min. Temperature effected the laccase activity, which measured by incubating the protein at optimal pH. The temperature (10 °C to 80 °C) adjusted to determine the thermo-stability and readings were taken after every 10 min by increasing 10 °C temperature on each round. The effect of metal ions on laccase was determined to check the stability and relative activity. The metal ions (Cu 2+ , Ca 2+ , Zn 2+ ) with sulfate donor in concentration of 1, 3, 6, and 9 mM was used in this study. The aliquot of enzyme, 50 mM citrate-phosphate buffer (pH 3.0), particular metal ion concentration was mixed throughly, and then incubated for 30 min at 40 °C. A few inhibitors were examined by incubating the purified laccase for 30 min at room temperature. A control was run parallel without inhibitors to estimate the % inhibition on laccase activity and its performance. The concentration of SDS and EDTA was 1, 3, 6 and 9 mM, whereas 0.01, 0.05 and 0.1 mM of NaN 3 investigated in this study.
Kinetic studies. The kinetic parameters K m and V max of purified laccase isozyme was determined by using substrate at different concentration of 1 mM, 2 mM, 3 mM, 5 mM and 10 mM guaiacol in 100 mM citrate-phosphate buffer (pH 3.0).

Statistical analysis.
The data collected from various parameters during presented study was subjected to statistical analysis in computer software, Co-Stat version 3.01. Assays were carried out in triplicate and the values were presented as mean ± standard deviation.

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Results
Ganoderma species identification. Ganoderma leucocontextum was identified by molecular method. The ITS-5.8S rDNA sequence of 570 bp was amplified from the genomic DNA. After NCBI blasting, the sequences with great identity were selected and used to generate the phylogenetic tree. It can be seen from the topology that specimens showed the maximum identity with G. leucocontextum (Fig. 2). The closely related species in the phylogenetic tree were G. lucidum (Curtis) P. Karst., G. oregonense Murrill, G. tsugae Murrill., G. ahmadii Steyaert. and G. valesiacum Boud. The bootstrap 96 (Fig. 2), where specimens (ANP1, AY2B) were nested in a well-supported clade of G. lucidum complex by forming a discrete lineage.
The phylogenetic identification was confirmed by the author Aisha Umar. These specimens were submitted to "Lahore Herbarium (LAH)" of University of the Punjab under the proper voucher/LAH number (LAH36345, GenBank MK713839; LAH36346, GenBank MN134012).
Qualitative analysis. The laccase producing G. leucocontextum was preliminarily screened to produce the reddish brown halos on MEA plates (Fig. 1B,C) contained guaiacol indicator. This exhibited a biggest reddish brown colored zone around the colony after 7 days of incubation at 27 °C (Fig. 1B,C).
Optimization of culture growth conditions. Individual culture flasks (100 mL) with few mycelia discs of G. leucocontextum were incubated for 7 days at different temperature (30 °C, 35 °C, 40 °C). The best optimized 40 °C exhibited maximum laccase production (855 U/L). At 20 °C, laccase production reduced as the temperature was increased from 40 to 60 °C (Fig. 3A).
The fermentation broth was inoculated with mycelia discs and incubated for 7 days at different pH (3.0, 5.0, 6.0). The suitable pH was 5.0 for guaiacol assay. The maximum production of laccase was 2517 U/L, whereas very low and higher acidity reduced the secretion level of laccase. So, the best suitable pH was 5.0, which enhanced the synthesis of laccase (Fig. 3B).  www.nature.com/scientificreports/ Optimum time of incubation required for the production of maximum laccase in this study. The incubations days were 14 in total. The harvested cultures were evaluated after 3 days interval. After 7 days, the secretion was enhanced (522.2 U/L) but decreased as the medium exhausted due to shortage of nutrients for mycelium after 10 days (Fig. 3C).
The 50, 100 and 150 rpm of orbital shaker was set to optimize the maximum laccase secretion and mycelium growth. The maximum production rate of laccase from G. leucocontextum was achieved at 50 and 100 rpm. The laccase activity 5551.9 U/L at 50 rpm and 698.3 U/L at 100 rpm was achieved in this work (Fig. 3D).
Organic carbon sources were selected to enhance the production of laccase including glucose, sucrose and maltose (20 g/L and 25 g/L) in fermentation broth contained mycelium of G. leucocontextum. The 20 and 25 g/L glucose has been exhibited the greater production of laccase than sucrose. The both concentrations of maltose showed less than 1500 U/L of laccse. So, glucose and sucrose were better to enhance the yield than maltose (Fig. 3E).
The extracellular enzyme from G. leucocontextum was studied in the presence of different organic and inorganic nitrogen sources. The organic nitrogen sources stimulated the more laccase production. Peptone, yeast and beef extracts were also evaluated (Fig. 3F). The optimal enzyme production was attained by using peptone nitrogen source. The 10 g/L beef and yeast extract was the best defined organic nitrogen source for laccase production i.e., 4671 U/L and 5776 U/L, respectively from G. leucocontextum (Fig. 3F).
The effect of inorganic nitrogen sources (KNO 3 , NaNO 3 and (NH 4 ) 2 SO 4 ) were determined by using 5 g/L and 10 g/L concentrations (Fig. 3G). The influence of inorganic nitrogen on laccase activity was dependent on the nature and concentrations. Opposite to this; KNO 3 maximized the enzyme production (6540 U/L) at low concentration (5 g/L) rather than higher. It was apparent that 10 g/L KNO 3 inhibited the enzyme formation. On Purification and identification of laccase isozyme. A complete set up of culture broth (1000 mL) was designed separately under the best optimized conditions. Filtrate was centrifuged at 13,000×g for 15 min at 10 °C and finely grounded powder of (NH 4 ) 2 SO was mixed thoroughly in supernatant (60% and 80%) of filtrate. The best suitable concentration was 80% ammonium sulphate yielded 65% laccase. The laccase was purified 4.5-folds from its initial culture broth with a final yield (82%). The total activity of the purified enzyme was 15,228.5 ± 22.0 U/L by guaiacol (100 mM) assay. The protein (Glacc 110) molecular weight of ∼ 65.0 kDa was estimated by SDS-PAGE (Fig. S1A) and Native PAGE (Fig. S1B). A single brown band of ∼ 65.0 kDa in a lane was stained by guaiacol, which indicated the presence of an active laccase of G. leucocontextum extract.
Characterization of purified laccase. Various buffers were tested to evaluate the laccase stability at different pH. The optimum pH to achieve the maximum activity of purified laccase was 3.0, while this enzyme also stable at pH 4.0. The relative activity was > 90% evaluated for 16 min. Readings were taken after 2 min time interval, whereas the activity dropped as the pH increased (Fig. 4A).
The temperature-dependent activity and thermal stability of purified laccase from G. leucocontextum showed optimum activity in sodium acetate buffer (100 mM, pH 3) at 70 °C. The higher temperature reduced the enzyme activity. The thermal stability of the purified laccase from G. leucocontextum was maximal at 40 °C to 60 °C, while decreased abruptly beyond 70 °C. The relative activity was 77% and 96% at 40 °C and 70 °C, respectively (Fig. 4B).
The effects of various inhibitors on purified Glacc110 activity was investigated by using SDS, EDTA and NaN 3 . The laccase activity was measured by pre incubating the purified Glacc110 in the presence of each inhibitor at 70 °C for 15 min. The 1 mM and 3 mM was effective to retain the maximum relative activity than 6 mM and 9 mM of EDTA. The relative activity was 88% and 87% at 1 mM and 3 mM of EDTA, respectively.
SDS was a bad inhibitor for the laccase activity than EDTA at all concentrations. The NaN 3 was also a bad inhibitor for laccase activity at all the concentrations used in this work rather EDTA and SDS (Fig. 4D1,D2) and negligible relative activity exhibited by Glacc110.
Kinetic studies. The time course of the oxidation of guaiacol in the presence of purified laccase is shown in this study (Fig. 5). The kinetic constants of purified G. leucocontextum laccase were determined in this study. The K m and V max of G. leucocontextum laccase was 1.658 (mM) and 2.452 (mM/ min), respectively.

Discussion
The factors leading to taxonomic confusions are morphological features, inter hybridization, geography, abiotic factors, and genetic heterogeneity 38,39 . Phylogenetic reconstructions of DNA sequences increased the reliability of evolutionary framework. This powerful data answered the ambiguous questions related to species identification 40 . Our new Ganoderma record (ANP1, AY2B) was grouped well within the sequences of Chinese G. leucocontextum 4,22 .
Guaiacol is a reliable and easily detectable screening step of laccase activity. The halo formed due to oxidation of indicator via lignolytic enzymes 41 . The production of intense brown, brown, and reddish brown color under and around the fungal colony was considered the positive result of guaiacol oxidation 42 . The obtained results had good agreement with Kiiskinen et al 41 . Temperature is a significant environmental factor to regulate the secretion of laccase isozymes 43 . This is fungal dependent parameters that stimulate the enzyme production 44 . Industrial applications demand not only the optimal temperature, but need to measure the robustness of laccase at higher temperature. Laccase in fungi act as phenol oxidases prefer the temperature range of 30-55 °C to get catalytic activity 45 , which varies under the optimal temperature (30 °C and 55 °C) 46 . The fungal activity of enzyme was condensed or minimized, when cultivated above 30 °C 47 . The maximum enzyme production under high temperature indicated an adaptive step of white rotting basidiomycetes to grow and survive in hot nature of environment.
The pH variation is an important factor for the improvement of laccase quantity 48 . Fungal laccase exhibited higher stability in acidic pH (pH 4-6/3.6-5.2) to promote the catalytic efficiency 45,49 . The mobility of polypeptide chain increased electrostatic interactions at pH 3.0. Low pH causes the unfolding of protein due to accumulation of hydrophobic surfaces. This was a logical hint to loss the laccase activity 50 . The maximum laccase quantity in a shorter period is advantageous in industrial applications, while fungal species require longer period for laccase production 51 . In the same way, researchers prefer 14 to 20 days for maximum laccase secretion from wood rotting fungi 52 .
The fungal mycelium biomass covered the liquid medium on 3rd day of incubation to secrete the maximum laccase. But this view about Gaonderma lucidum was contradicted to our results 48 . In this study, mycelium biomass fully covered the broth on day 6th and 7th. The reported laccase activity was 27 U L −1 of Ganoderma sp. 53 , 80 U L −1 of G. australe 54 , and 120 U L −1 from Ganoderma sp. En3 55 . Rodrigues et al. 10 used 3 fungal discs (5 mm) of G. lucidum on PDA medium and incubated at 27 °C for 8 days at 150 rpm for maximum production of laccase. In this work, G. leucocontextum formed filamentous mats due to restriction of oxygen between fungal mycelium and medium. The similar views were represented by Madhavi and Lele 55 . The earlier studies showed the highest laccase production in agitated cultures 56 .
The carbon sources are significant inducers in production of laccase 57 . These sources symbolized the first sign of mycelial growth within 24 h, while completely colonized within 6 days 58 . The excessive glucose concentration www.nature.com/scientificreports/ was inhibitory to extracellular laccase production from several Ganoderma species 59 . Li et al. 60 given two opinions to the scientist: (1) glucose is a strong inhibitor of laccase expression from Ganoderma isolates or (2) glucose is an important nutrient to convince the G. lucidum to secrete the laccase 60 . The activity of laccase is dependent on concentration, nature of carbon sources and mushroom species 61 . Presence of sugar reduced the enzyme yield by repress the catabolites 62 . This repression is associated with laccase expression 63 . Several authors reported low carbon-nitrogen ratio 64 , while a few prefer the high carbon-nitrogen ratio 65 . In basidiomycete, high concentrations of glucose inhibit the laccase transcription 66 . The excess of any nutrient cause the blockage in induction, and permit the constitutive enzyme production. Sugar supported the sufficient mycelium biomass formation but not the guarantor of maximum enzyme yields 67,68 . The laccase activity is dependent on concentration and nature of nitrogen sources in wood rotting fungi [67][68][69] . The organic nitrogen sources are more efficient than inorganic 10 . The nitrogen was not affected the enzyme activity and yield of some fungal species 9 . The concentrations of nitrogen suppressed as well as stimulate the ligninolytic enzyme activity in several species e.g., Trametes trogii 69  Zou et al. 83 explained that many fungal laccases are functional under acidic or neutral pH and lost their functionality under alkaline conditions. They found the laccase activity at pH 5.0 after 24 h incubation. Laccase of G. lucidum-CDBT1 was most stable at pH 5.0 and 30 °C 84 . The preferable stable acidic pH region was 5.0 in Hericium erinaceum 85 and Lentinula edodes 86 . Purified laccase of Ganoderma sp. was active under acidic pH (3.0-5.5), stable at pH 3.0 to 5.0 and maximum activity was found at pH 4.5 33 . Purified laccase retained 95% residual activity at pH 5 and 50% at pH 6.0 33 . Rate of laccase inactivation increased with increase in temperature from 10 to 60 °C, while stable at 10 to 30 °C 87 . Thermostabilty depend upon time and substrate e.g., laccase at 60 °C was stable for 24 h extracted from C. gallica 88 , 5-9 h of Peniophora sp., and 10 min in T. gallica 89 . The residual laccase activity of G. lucidum was maintained and examined every 10 min for 80 min at pH 3.0, when incubated under 60 °C 90 . The laccase was very stable at pH 6.0 and 7.0, whereas moderately stable at pH 5.0 and 8.0 (25 °C) of T. versicolor 91 . Temperature greater than 60 °C dropped rapidly the activity of fungal laccase 92 . The laccase activity increased smoothly from 30 to 60 °C (sharply increased at 50 °C and 60 °C), while decreased over 80 °C in Echinodontium taxodii 93 . In this study, favorable highest temperature range was 50 °C to 60 °C. Similarly more than 70% laccase activity of Glac15 was maintained at 50 °C by guaiacol substrate 94 , whereas 25-35 °C significantly reduced this activity 79 .
Fonseca et al. 43 produced the highest laccase activity at 0.5 mM Cu 2+ in culture medium of G. applanatum and Peniophora sp. The enzyme production and fungal growth was inhibited under the higher concentration of Cu 2+ (3 mM) 95 . The optimal concentrations of Cu 2+ were 0.1 and 0.5 mM enhanced the laccase from P. ostreatus and Streptomyces lavendulae 96 , respectively. In fungal organisms, copper ions are essential to form the intact and active structures of laccase, whereas many reports showed that copper ions badly affect the laccase production ability 95 . Copper induction also influences the genetic transcription level of laccase 96,97 .
Ions (K + , Ca 2+ , Fe 2+ , Ba 2+ , Fe 3+ Zn 2+ or Al 2+ ) closely bind the T1 site of laccase. These ions perform a function like competitive inhibitor for edonors by hindering the access of substrates to the T1 site or stop the emovement to T1 active site. This action leads to inhibition of laccase activity 98 . The NaN 3 is a typical laccase inhibitor, which bind to the type II and III Cu sites. The NaN 3 binding affects the internal electron transfer, thereby inhibit the laccase activity 99 . Contrary, SDS enhanced the enzyme activity, which also changes the structure of enzyme 100 . The purified laccase was sturdily inhibited by SDS at 0.5 mM (13%) and 1 mm (6%) of Trametes sp. LS-10C 64 . Vantamuri and Kaliwal 101 supplemented 20 mM EDTA in the purified enzyme of Marasmius species BBKAV79, while Das and associates 36 determined the laccase activity from Pleurotus florida. This was interesting to know that 1 mM EDTA represented 116% inhibition rate of recombinant laccase from T. versicolor 20 .
The V max is dependent on enzyme concentration 102 . Except slow oxidation, wood rotters laccase exhibited low affinity and catalytic constants (K m ) with guaiacol. These are higher than other substrates found in this study. The K m (mM) value of purified laccase was 2.50 of Pleurotus sajor-caju and 0.107 of G. lucidum GaLc3 103 . The higher K m (0.107 mM) value of laccase from G. lucidum was indicated the low enzyme affinity to the substrate 104 . The published value of K m from wood rotters were 1.2 mM for P. ostreatus 105 , 0.550 for P. pulmunarius Lcc2 (mM) 106

Conclusion
Ganoderma leucocontextum was identified by ITS markers. In this work, first time laccase was purified and characterized at the industrial level from G. leucocontextum. In conclusion, this study reported that laccase produced from G. leucocontextum cultures shown the isozymes by SDS and Native-PAGE with interesting properties like stability at higher temperature and acidic pH. There are still many wood rotting mushrooms/fungi, which not described till yet. The new researchers have to explore the maximum new diversity of genus Ganoderma with their biochemical characterizations and laccase production. This laccase is suitable for industrial and biotechnological applications. The few challenges for future researchers are the use of this laccase as a biocatalyst offer economically feasible domino processes for the preparation of bioactive compounds, immobilization of laccase, immobilization methods, evaluation methods for laccase activity, factors affecting the laccase expression, and heterologous expression.

Data availability
The data set generated and analyzed during the current study are available at institute of plant sciences, University of the Punjab.