Isolation, selection and identification of laccase - Producing fungal strains and its dye decolorization ability

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol. 58, No. 9, pp. 132-138 This paper is available online at ISOLATION, SELECTION AND IDENTIFICATION OF LACCASE-PRODUCING FUNGAL STRAINS AND ITS DYE DECOLORIZATION ABILITY Duong Minh Lam and Truong Thi Chien Faculty of Biology, Hanoi National University of Education Abstract. Fungal studies have thus far received little attention from scientists in Vietnam, especially regarding fungal diversity and their application in industrial processes. In this study, we used traditional methods to isolate and screen for fungal laccases that have potential for use in the textile industry (dye decolorization) and a molecular marker to identify studied samples. Fifteen strains of basidiomycetes were isolated and screened for laccase activities on phenol, guaiacol and RBBR. Five strains showed positive laccase activities on all three substrates. The strain CPB30 presented the largest halo zone diameter and was also fastest to decolorize RBBR in a culture broth (within 64 hours of incubation). Using complete ITS sequence analysis, the CPB30 strain was identified as being Trametes maxima (T. maxima CPB30). This strain expresses high laccase activity (614 -796 U/mL) which warrants further applied research. Keywords: Laccase, Trametes maxima, RBBR, decolorization, Cuc Phuong. 1. Introduction Water pollution is a serious problem in many countries and this is especially true in developing countries where uncontrolled industrial processes are taking place. A type of pollutant that is currently being released into the environment is the dyes used in the fabric industry. Laccases (benzenediol: oxygen oxidoreductases; EC 1.10.3.2) have been efficiently used to decolor and detoxify dyes due to the oxidizing capacity of laccases on a wide variety of organic and inorganic compounds, including diphenols, polyphenols, substituted phenols, diamines and aromatic amines, with a concomitant reduction of molecular oxygen to water [12]. Laccases have been found in bacteria (Azospirillum lipoferum, Bacillus subtilis, Streptomyces lavendulae, S. cyaneus) [2, 4, 7, 9] and plants, but it is predominantly found in fungi [1]. Most of the laccases studied are of fungal origin, especially from white-rot fungi, such as Phlebia radiata, Pleurotus ostreatus and Received September 17, 2013. Accepted December 2, 2013. Contact Duong Minh Lam, e-mail address: duong.minhlam@gmail.com 132 Isolation, selection and identification of laccase-producing fungal strains... Trametes versicolor. Recently, laccases have been used in conjunction with other enzymes (cellulases, xylanases) in industrial applications such as pulp delignification, textile dye bleaching, biopolymer modification and bioremediation [3]. Screening for novel laccases with various characteristics that can be used in different industrial applications is important. Different studies have been done on substrates, such as phenolic compounds (tannic, gallic, guaiacol and syringaldazine) and the polymeric dyes remazol brilliant blue R (RBBR) and Poly R-478, in order to discover microorganisms that produce laccases [10]. These substrates are color indicators as they change color when oxidized by laccase which is produced by microorganisms and makes halozone around positive colonies. However, it is not possible to precisely estimate the extent of laccase activity so normally only a general idea of laccase presence and activity can be given. Vietnam is a hot spot for biodiversity in the world, with 12,000 higher plant species. It is estimated that 72,000 fungal species [5] are found in Vietnam and among them perhaps 15,000 - 20,000 are of the Basidiomycota phylum. This is a huge source of new and interesting compounds that are waiting to be discovered. This study aims to 1) isolate fungi from basidiomycetous samples collected in Cuc Phuong National Park, Vietnam, 2) screen for laccase-producing fungi using different color indicators, 3) estimate decolorization of the laccase of selected strain and 4) identify species level using ITS rDNA sequence analysis. 2. Content 2.1. Materials and methods * Materials of the study: 15 fungal samples were collected in Cuc Phuong National Park in June 2011. Chemicals: guaiacol, remazol brilliant blue R (RBBR), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), diammonium salt, phenol, D-glucose. All chemicals were supplied by Sigma and at a high purity level. Media: PDA (Potato Dextrose Agar); Czapeck_Dox (30 g/L saccharose; 0.5 g/L MgSO4; 3.5 g/L NaNO3; 1.5 g/L K2HPO4; 0.5 g/L KCl; 0.01 g/L FeSO4; pH 5 - 5.5). * Methods of the study: - Isolation: Fresh fungal fruiting bodies collected from sites were taken to the laboratory in 12 hours where dust and litter was removed from them. Surface sterilization was done using 96% ethanol for 2 minutes. The fruiting bodies were cut and torn into 2 halves using a sterile razor blade. Internal tissues were removed and placed on a PDA medium. - Screening for laccase: Fungal cultures were grown on a PDA medium containing phenol, guaiacol and RBBR as substrate for laccases. The presence of laccases is shown as a halo around the fungal colony. Phenol and guaiacol were oxidized and become brown while RBBR changed from blue to light brown in the presence of laccases. 133 Duong Minh Lam and Truong Thi Chien - Laccase activity assay: The fungal culture was inoculated in 100 ml of liquid PDA medium in a flask and incubated at 30 ◦C for 90 hours, shaken at 160 rpm. Then, the culture broth was centrifuged at 6000 rpm for 5 mins. The culture supernatant was used as a crude enzyme for laccase activity estimation. Laccase activity was estimated based on the oxidization of ABTS (2,2’-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid), which absorbs light best at 420 nm. The enzymatic reaction includes 800 µL of 0.5 M acetate buffer, pH 5.0; 100 µL of 5 mM ABTS; and 100 µL of crude enzyme. The reaction mixture was incubated for 10 minutes at 40 ◦C. The reaction was stopped by adding 100 µL of TCA 50% (v/v). In the control test, TCAwas added before adding the crude enzyme. All other factors between the control and experimental tests were consistent and identical. The differences in absorbance at OD420nm value between the control and experimental samples were used to calculate activity using the following formula [13]: U/mL = [NA × (106/e420 × d) × V/v × F]/t where NA: increasing absorbance at 420nm; 106/e420× d: conversion to µmol converted substrate/mL using the molar extinction coefficient (ǫABTS, 420 = 3600 M−1.mm−1); d: length of the light path in mm (10 mm); V: total volume (1000 µL); v: volume of sample (100 µL); V/v: dilution in assay (10); F: dilution factor of stock; t: reaction time (10 mins). - Identification of the isolated strain: DNA extraction was carried out using a CTAB lysis buffer and phenol chloroform as outlined by Jeewon et al. [6]. The complete ITS (including 5.8S) regions were amplified using primer pair ITS4 (5’- TCCTCCGCTTATTGATATGC-3’) and ITS5 (5’-GGAAGTAAAAGTCGTAACAA-GG-3’). The amplification conditions were performed in a 50 µL reaction volume as follows: 5 µ of 10X PCR buffer, 0.2 mM each dNTP, 0.3 M of each primer; 1.5 mM MgCl2, 1 unit of Taq Polymerase and 10 ng DNA. PCR parameters were as follows: Initial denaturation 94 ◦C for 3 min, 30 cycles of 94 ◦C for 1 min, 52 ◦C for 50 s, 72 ◦C for 1 min, the final extension at 72 ◦C for 10 min. A characterization of the PCR products was done using agarose gel electrophoresis on a 1% agarose gel containing ethidium bromide as the staining agent. DNA sequencing was performed by the Bioneer Company in Korea using both primers. A BioEdit program was used to generate the consensus sequence of the sample studied. Sequences used in the analysis were chosen from NCBI using a nucleotide Blast tool. A neighbor-joining method was selected for sequence analysis and phylogenetic tree generation. 2.2. Results and discussions 2.2.1. Fungal isolation and laccase screening The collected basidiomycetous samples were isolated on PDA medium, obtaining 15 pure cultures. All 15 strains were screened for laccase on three different substrates (phenol, guaiacol and RBBR) in Petri dishes. Halozone appearing around a colony indicated the presence of laccase. The results can be seen in Table 1. 134 Isolation, selection and identification of laccase-producing fungal strains... Table 1. Laccase activity of 15 strains Substrates Substrates Strains Phenol Guaiacol RBBR Strains Phenol Guaiacol RBBR CPB1 - - - CPB17 + + + CPB4 - + - CPB18 - - - CPB6 - - - CPB21 + + + CPB7 - + + CPB22 - - - CPB8 - - - CPB26 - - - CPB10 - - - CPB28 + + + CPB12 + + + CPB30 + + + CPB15 - - - (+): positive; (-): negative Five strains (CPB12, CPB17, CPB21, CPB28 and CPB30) showed a high level of laccase activity on all three substrates. The typical colors of the oxidized substrates were brown for phenol and guaiacol, and hyaline to pale brown for RBBR (Figure 1A, B). The CPB30 strain was selected for further study due to its large halozone diameter on all three substrates. Figure 1. Laccase activity of the five strains on different substrates: A - Guaiacol, B - RBBR 2.2.2. RBBR decolorization Besides the tests on solid media, the five strains were also screened for decolorization in a culture broth into which was added RBBR 0.01% (w/v). Incubation was done at 30 ◦C, shaking at 160 rpm for 96 hours. The CPB30 strain was the fastest to decolorize RBBR, after 60 hours of incubation, and the slowest was the CP21 strain, after 84 hours. However, the strains showed different colors of oxidized RBBR (Figure 2). The CPB30 strain was the most effective in decolorizing RBBR and was the strain that showed the most promise for application in industrial treatment. 135 Duong Minh Lam and Truong Thi Chien Figure 2. RBBR decolorization of some selected strains 2.2.3. Laccase activity of the CPB30 strain Laccases from the CPB30 strain were quantitatively estimated using the described method considering incubation time in order to get a proper taking-sample time for other experiments (data not shown). The results are presented in Figure 3. Figure 3. Laccase activity of the CPB30 strain along with incubation time The best time to collect the culture broth for laccase estimation was 84 - 90 hours, and the highest enzyme activity was 614.8 U/mL and 796.8 U/mL, corresponding to the broth without RBBR and with RBBR added. The results show that RBBR played the role of substrate and it was also the enzyme producing inducer. Laccases from fungi, especially from white rot fungi, have widely been studied. However, few have been reported as having laccase activity that is as high as that of the CPB30 strain [8, 11]. The results suggest that this CPB30 strain could potentially be used to produce laccases for many different purposes, particularly decolorization at an industrial scale. 2.2.4. Molecular identification of the CPB30 strain The complete coding sequence of ITS1-5.8S-ITS2 of rRNA includes 568 nucleotides, with single stranded molecular weight of 172087.00 Daltons, G+C content = 48.24% and A+T content = 51.76%. The full sequence is shown below: 136 Isolation, selection and identification of laccase-producing fungal strains... The sequence was used to search for similar or close related sequences in the GenBank using the nBlast tool of the NCBI website. The results show that the CPB30 is most closely related to Trametes maxima FPRI376 (JN164918) and T. maxima 9 (JN164933) with a 99% similarity in sequence. Sequence analysis was done using available sequences of different Trametes species. The neighbor-joining method was run in ClustalX8.3 with 10000 replications and a tree was generated (Figure 4). This analysis confirms that the CPB30 strain was derived from Trametes maxima and is therefore named T. maxima CPB30. Figure 4. The relationship of the CPB30 strain with other Trametes species based on the complete ITS sequence analysis using the neighbor-joining method 3. Conclusion From the 15 isolated basidiomycetous cultures, 5 strains showed laccase activity on all three substrates. The five strains differed in time and capacity to decolorize RBBR 137 Duong Minh Lam and Truong Thi Chien added to culture broth. Of the 5 strains, CPB30 was the strongest as it decolored RBBR within 64 hours of incubation while other strains took much longer (up to 84 hours). Molecular identification was applied to CPB30 and it was shown that this strain was derived from Trametes maxima. Acknowledgement. The work was supported by the National Foundation for Science and Technology (NAFOSTED) of Vietnam under grant number 106.07-2011.57. REFERENCES [1] P. Baldrian, 2006. Fungal laccases - occurrence and properties. FEMS Microbiology Reviews, 30, pp. 215–242. [2] Y.H. Chen, L.Y. Chai, Y.H. Zhu, Z.H. Yang, Y. Zheng, H. Zhang, 2012. Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips. Journal of Applied Microbiology, 112, pp. 900-906. [3] J. Fu, G.S. Nyanhongo, G.M. Gu¨bitz, A.Cavaco-Paulo, S. Kim, 2012. Enzymatic colouration with laccase and peroxidases: Recent progress. Biocatalysis and Biotransformation, 30, pp. 125-140. [4] A. Givaudan, A. Effosse, D. Faure, P. Potier, M.L. Bouillant, R. Bally, 1993. Poly-phenol oxidase in Azospirillum lipoferum isolated from rice rhizosphere: evidence for laccase activity in non-motile strains of Azospirillum lipoferum. FEMS Microbiology Letters, 108, pp. 205-210. [5] D.L. Hawksworth, 2001. The magnitude of fungal diversity: The1.5 million species estimate revisited. Mycological Research, 105, pp. 1422-1432. [6] R. Jeewon, E.C.Y. Liew, H.D. Hyde, 2004. Phylogenetic evaluation of species nomenclature of Pestalotiopsis in relation to host association. Fungal Diversity, 17, pp. 39-55. [7] A. Messerschmidt, 1992. Structural studies on copper-containing plant oxidases. Biochem Soc Trans, 20, pp. 364-372. [8] S.S. More, P.S. Renuka, K. Pruthvi, M. Swetha, S. Malini, S.M. Veena, 2011. Isolation, Purification, and Characterization of Fungal Laccase from Pleurotus sp.. Enzyme Research doi:10.4061/2011/248735. [9] K.N. Niladevi, N. Jacob, P. Prema, 2008. Evidence for a halotolerant-alkaline laccase Streptomyces psammoticus: purification and characterization. Process Biochemistry, 43, pp. 654-660. [10] R. Reiss, J. Ihssen, L. Tho¨ny-Meyer, 2011. Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnology, 11, pp. 9-19. [11] R. Sivakumar, R. Rajendran, C. Balakumar, M. Tamilvendan, 2010. Isolation, screening and optimization of production medium for thermostable laccase production from Ganoderma sp.. International Journal of Engineering Science and Technology, 2(12), pp. 7133-7141. [12] E.I. Solomon, A.J. Augustine, J. Yoon, 2008. O2 reduction to H2O by the multicopper oxidases. Dalton Trans, 30, pp. 3921-3932. [13] R.T. Tussell, D. Pérez-Brito1, R. Rojas-Herrera, A. Cortes-Velazquez1, G. Rivera-Mun˜oz, S. Solis-Pereira, 2011. New laccase-producing fungi isolates with biotechnological potential in dye decolorization. African Journal of Biotechnology, 10(50), pp. 10134-10142. 138