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.

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.

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

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

2.1 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

- 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, TCA was 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 toµmol 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 (1000µL); v: volume of sample (100µL); 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 50µL 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

Table 1 Laccase activity of 15 strains

Strains Phenol Guaiacol RBBR Strains Phenol Guaiacol RBBR

-(+): 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

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:

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

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

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

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