White-rot fungi are the most efficiency degrader of wood lignin by having extracellular ligninolyitc enzymes system; lignin peroxidases, manganese peroxidases and laccase. Laccase has an important place in ligninolysis and has been widely applicated in some industries. The fungus CP29, which showed high laccase activity, was indentified and chosen the cultivation condition for biosynthesis of laccase. The fruiting bodies of strain CP29 formed effused, very flat, yellow green hymenium layer. The colony was off-white, blossom, regularly circular. The mycelia were thick and closely bound together. Beside ligninase, this fungus could produce other extracellular enzymes such as protease, cellulase and amylase. The biological characteristics of strain CP29 suggest that it belongs to subdivision Basidiomycota. Based on the result of morphological characteristics and the ITS-rDNA region analysis, the fungus CP29 was classified as Perenniporia tephropora. The sequence of ITS–rDNA gene of P. tephropora CP29 deposited onto GenBank (NCBI) under the accession number KX257798. To enhance laccase production, optimal cultivation conditions were investigated. This strain P. tephropora CP29showed the highest laccase activity reached up 7669.05 U/L on medium MT1 containing 0.0625mM CuSO4 after 7 days of cultivation at 37ºC and pH 5.0.

Laccase (EC 1.10.3.2, p-benzenediol: oxygen oxidoreductase) which belongs to a family of blue multicopper oxidases containing four copper atoms in their catalytic center and catalyzes the four electron reduction of oxygen to water (Patel et al. 2016). Laccases can oxidize both phenolic and non-phenolic compounds as well as highly environmental pollutants, so they have been successfully used in textile dye bleaching, deligninfication in the paper and pulp, detoxification for many industrial effluents, bioremediation of contaminated soil and water, production washing powder, transformation of steroids and antibiotics and  in the design of biosensors and nanotechnology (Upadhyay et al., 2016). Laccases are found in plants, insects and bacteria, but the most important sources of these enzymes are white rot fungi. There are some white rot fungi Basidiomycetes such as Phanerochaete chrysosporium, Theiophora terrestris, Lenzites betulina, Phlebia radiata, Pleurotus ostreatus, and Trametes versicolor canproduce high levels of laccase (Shraddha et al., 2011). The isolation and identification of white rot fungi which biosynthesis laccase, plays a vital role in detecting the potential environmental and biotechnological application of fungal and enzyme. Laccase are biosynthesized in the secondary metabolism of white rot fungi and is influenced by many fermentation parameters such as medium composition, temperature, initial pH of medium and time of cultivation (Viswanath et al., 2014). In the present work, we describe the identification of strain CP29 and choose the cultivation condition for biosynthesis of laccase from this fungus strain.

The fungus was grown on 2% malt MEA at 28C, the form, size and color of fungal colonies were observed and recorded. The mycelia were observed under Olympus inverted microscope IX71. The growth rate of mycelia was determined according to the method described by Schwantes and Salttler (1971). The macroscopic characteristics of the fungus were  studied by the methods in Trinh Tam Kiet (2011).

The genomic DNA of fungus CP9 was extracted using alkaline extraction method (Sambrook and Russell, 2001), and subjected to PCR to amplify the 5.8S rDNA gene using primers BF and BR. The thermal cyling progam was described by Gardes and Bruns (1993). The PCR products were sequenced by Axil Scientific Pte., Singapore. The sequences of fungus were compared with similar sequences from GenBank using BLAST program for identification of fungal species. 

Cultivation conditions for production of laccase from CP29 strain was  cultivated  in  the following five media:  MT 1 (Dhakar et al., 2013), MT 2 (Cupul et al., 2014), MT 3 (Dhouib et al., 2005), MT 4 (Chawachart et al., 2003), MT 5 (Kenkebaschivilli et al., 2012) were used. The effects of cultivation  temperature and pH were studied in the range of 25 to 40ºC and pH of 3 to 8 to find the values most favourable for the CP29 strain. Flasks of 250-ml volume containing 50 ml of medium were inoculated with 3 fungal disks (6 mm diameter) taken from a 4-day culture of the CP29 strain. In all experiments, shaker flask cultures were performed on rotary shaker at 150 rpm for 7 days.

Laccase activity was assayed using 0.216 mM syringaldazine  in  methanol  as  substrate  at 37˚C. The reaction mixture included 100 mM potassium phosphate buffer solution (pH 4.5), substrate solution and enzyme solution. The oxidation of the substrate was monitored at 530nm using Spectro Sc spectrophotometer (Labomed, inc.). One unit of  enzyme  activity corresponds to 0.001 change in OD at the reaction conditions and it is expressed in units per litre of culture medium (Brakova et al., 2016).

All experiments were performed in triplicates. The data presented in this work are mean values, were analyzed by one-way analysis of variance (ANOVA), followed by mean comparisons with Least significant difference (LSD) tests at the p<0.05 significance level, using Excel Data Analysis Statistical Software; the error bars represent the standard deviation.

The fruiting bodies of strain CP29 formed effused, very flat, yellow green hymenium (Fig. 1a). No more than a crust spread on the log. There are some water droplets on surface of fungi. Mycelium were white, grew deep into woody. Fungus CP29 could use many substrates because of having abundant extracellular enzyme system such as ligninase, cellulase, proteinase and amylase. Laccase are remarkably non-specific regarding substrates, therefore, guaiacol is widely used as indicator to evaluate laccase activity. On the agar medium supplemented with this indicator, the oxidation of guaiacol by laccase can be visualized as reddish-brown coloration around the fungal colony (Jebapriya  and Gnanadoss, 2014). As shown in Fig. 1b, fungus CP29 produced high activity of laccase.

  

The suitable temperature and pH for growth of fungus CP29 was at 20-30ºC and pH 3–7. This fungus fully colonized the petri dish in 7 days at 30ºC. The mycelia growth rate was 234.3 µm/h. The colony was off-white, blossom, and regularly circular with thick  mycelia closely bound together (Fig. 2a). The generative hypha were septate, with thick wall and clamp connections (Fig. 2b). The clamp connections are usually formed on during cell division of generative hyphae. All fungi that produce clamp connections are members of the Basidiomycota, but not all Basidiomycota produce clamp connections (Hood, 2006). Hence, the isolate CP29 was placed to subdivision Basidiomycota. 

  

The ITS-rDNA region has been successfully applied for identification of wood decay fungi. The fungal specific primer ITS1 and the fungal general primer ITS4 are the earliest PCR primer sets used to be amplified the fungal ITS regions (White et al., 1990), but now, many studies used other specific primers to identify fungi (Jebapriya  and Gnanadoss, 2014). Genomic DNA of CP29 was isolated for PCR amplification. The amplified PCR product exhibited a band with 816 bp in length. The ITS-rDNA sequence of CP29 was compared with similar sequences. The result of phylogenetic analysis is given in Fig. 3. Base on morphological and phylogenetic analyses suggest that fungus CP29 was closest (99% similarity) to the species Perenniporiatephropora, hence the isolated fungus CP29 was identified as Perenniporiatephroporaand name as Perenniporiatephropora CP29. The sequence of ITS – rDNA gene of P. tephropora CP29 deposited onto GenBank (NCBI) under the accession number KX257798.

Perenniporia isa large genus of Polyporaceae, about 90 species have been accepted in this genus and isolated from many parts of the world: China, Japan, Brazil, U.S.A., Europe, Africa (Gerber et al., 1999). The number of available reports on the production of laccase by white-rot fungi Perenniporia is rather limited. The laccase from the white rot fungus P. tephropora MUCL 43536 was purified and characterized by Younes et al. (2006).

The growth and production of enzymes is strongly affected by medium compositions and cultivation conditions. In this work, five media, specifically M1, M2, M3, M4 and M5, were used to study the growth and laccase production of P. tephropora CP29. Also, the effects of cultivation temperature and pH were studied. There was statistically significant effect of test media on laccase production. The effect of different media on the yield of laccase of P. tephropora CP29 was presented by in Fig. 3. The activity of laccase was determined after 7 days of cultivation, except medium M5 that had quite low activity, other four test media were suitable for production of laccase. The best results was obtained in cultures on medium M1, where the laccase activity was of 7010,95 U/L. 

Laccases productions are reported to be induced in the presence of copper ions (Patel et al., 2016). The tested media in this paper both containing CuSO4 but with different concentrations (1:20 mg/L). Therefore, further research is needed to select the appropriate concentration of CuSO4 and the time point to add on medium MT1 for higher laccase production of P. tephropora CP29.

The pH of the culture medium is critical in enzyme production of the white rot fungi. There are some reports indicated initial pH levels set between pH 4 and 6 prior to inoculation, but pH was not controlled during most cultivations (Viswanath et al., 2014). pH ranges of 3–6 was found suitable for laccase production by strain P. tephropora CP29, and the highest activity (7262.81 U/L) was observed when grown in medium M1 initially adjusted at pH 5.0 (Fig. 5). The other authors reported the optimum pH of laccase production in many white rot fungi, falls between 5.0 and 6.0 (Dhouib et al., 2005; Younes et al., 2006; Kenkebashvilli et al., 2012). The differences between laccase activity at the pH were statistically significant.

Temperature, like any other cultivation parameters, plays an important role in growth and laccase production of the white rot fungi (Viswanath et al., 2014). In many cases, the temperature suitable for growth is not the optimum temperature for enzyme biosynthesis. The favourable temperatures for growth of P. tephropora CP29 were from 25 to 30ºC, however, in order to study the effect of temperature on the laccase activity, P. tephroporaCP29 was cultivated at temperatures of 25, 30, 37 and 40ºC. 

   

The highest laccase activity (7669.05 U/L) was achieved at 37ºC (Fig. 6), was statistically significant higher than that at other cultivation temperatures. Higher cultivation temperatures did not suitable for the laccase production, as the activity at 40ºC was decreased about 53%. 

In this paper, it was found that the most favourable cultivation conditions of the laccase production by strain  P. tephropora CP29 include:  medium MT1 containing 0.0625 mM CuSO4, pH 5.0, temperature 37ºC, rotary shaker speed 150 rpm, and  cultivation time 168 hours.  The established conditions enabled the strain to express highest laccase activity. 

The fungus CP29, which showed high laccase activity. In present paper, this fungus strain was studied with respect to its cultivation, enzyme activity, biological characteristics and identification. The results of morphological and phylogenetic analyses suggest that CP29 can be named as Perenniporiatephropora CP29. The most favourable cultivation conditions of the laccase production by strain  P. tephropora CP29 was established. In shaker flask cultivation using medium M1 at pH 5.0 and temperature 37ºC, Perenniporiatephropora CP29 achieved highest laccase activity after 168 hours. Further study will be focused on production and purification of laccase, its chemical structure and aspects of application.

Acknowledgement.The present research is financially supported by the Ministry of Industry and Trade to the project ĐT.05.18/CNSHCB. The National Key Laboratory of Gene Technology, Institute of Biotechnology, provides facilities for this study.

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