Isolation, selection and identification of Laccase-producing fungal strains and its dye decolorization ability

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.

JOURNAL OF SCIENCE OF HNUE Chemical and Biological Sci., 2013, Vol 58, No 9, pp 132-138 This paper is available online at http://stdb.hnue.edu.vn 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 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 uning 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 134 Isolation, selection and identification of laccase-producing fungal strains Strains CPB1 CPB4 CPB6 CPB7 CPB8 CPB10 CPB12 CPB15 Table Laccase activity of 15 strains Substrates Substrates Phenol Guaiacol RBBR Strains Phenol Guaiacol RBBR CPB17 + + + + CPB18 CPB21 + + + + + CPB22 CPB26 CPB28 + + + + + + CPB30 + + + (+): 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 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 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 Figure 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 (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 The relationship of the CPB30 strain with other Trametes species based on the complete ITS sequence analysis using the neighbor-joining method Conclusion From the 15 isolated basidiomycetous cultures, 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 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] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] 138 P Baldrian, 2006 Fungal laccases - 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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... 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,