Purification and Characterization of Laccase from ...

Research Journal of Biotechnology

Vol. 8 (9) September (2013) Res. J. Biotech

Purification and Characterization of Laccase from newly isolated Thermophilic Brevibacillus sp. (Z1) and its applications in removal of Textile Dyes Ceyda Bozoglu1, Ahmet Adiguzel1*, Hayrunnisa Nadaroglu2*, Derya Yanmis3 and Medine Gulluce3 1. Faculty of Science, Department of Molecular Biology and Genetic, Ataturk University, 25240, Erzurum, TURKEY 2. Department of Food Technology, Erzurum Vocational Training School, Ataturk University, 25240, Erzurum, TURKEY 3. Faculty of Science, Department of Biology, Ataturk University, 25240, Erzurum, TURKEY *[email protected], *[email protected]

Abstract Laccases are multicopper-containing oxidases (EC 1.10.3.2.) and they are widely found in fungi, higher plants and bacteria. Because of their high pH stability and thermostability, laccases from this strain have a potential for the treatment of textile bleaching effluents. In this research, the thermophilic test strain isolated from the Agri-Diyadin thermal springs, in the east of Turkey, were subjected to analysis of 16S rRNA sequence. The obtained sequence data were compared with data from the database in the Genebank and as a result it has been identified that this organism is a member of Brevibacillus genus (GenBank number KC292196). The laccase from Brevibacillus sp. (Z1) was purified by using precipitate of saturation (NH4)2SO4, DEAE-cellulose and Sephacryl S200. The purification degree and the molecular mass of the enzyme (93 and 110 kDa) were determined by SDS-PAGE and gel filtration chromatography.

were major steps in the field of biotechnology. Especially the success of T. aquaticus achieved in the commercial area has directed scientists towards studies on isolation and identification of thermophilic microorganisms in geothermal areas, not identified or designated yet1. In the identification and characterization of unknown microorganisms of different ecosystems, the classical methods (morphological, physiological and biochemical) had been used until the molecular methods become available. Today, however, it has been understood that the identification and characterization of the microorganisms by classical methods is not enough and this paved the way for rapid developments in molecular biology2. After the thermostable properties of the thermophilic microorganisms have been identified, in line with the identification and characterization studies, researches on revealing the potential industrial uses of theses enzymes which were purified from these organisms and having economic importance, were increased significantly 2. The dyes, which are important industrially and used in diverse applications in different industrial fields such as cosmetics, food, paper and textile, have negative impacts on the photosynthetic activity, due to the decrease in luminous transmittance and this has toxic effects on living organisms3,4.

In order to determine the enzyme's resistance against the metal ions, the effect of metal ions such as Ca2+, Ba2+, Mn2+, Ni2+, Co2+, Cu2+, Zn2+, Fe2+, K+ and certain chemical substances such as EDTA and ascorbic acid on the pure laccase enzyme activity have been investigated and no significant change was observed. Vmax and Km values were calculated using Lineweaver Burk plots for ABTS, 2,6-dimetoxyphenol and guaiacol. Purified laccase enzyme was also used to remove some textile dyes which are used in textile industries and known to cause major environmental problems such as Reactive Black 5, Fuchsine, Allura Red and Acid Red 37 from waste water. Consequently, it is concluded that the purified laccase from Brevibacillus sp. (Z1) can successfully be used for the removal of the textile dyes from wastewater.

A large portion of these synthetic dyes are azo dyes which are toxic, mutagenic and carcinogenic5. These dyes have the potential to accumulate in the environment at higher levels since they are resistant to degradation due to their azo bonds. Treatment of these dyes is difficult with the classical treatment methods, since they present a stable structure in acidic and alkaline conditions and resistant to aerobic degradation, heat and light6. Industrially important laccase enzyme (EC 1.1.3.2, pdiphenol, dioxygen oxidoreductase) belongs to a gene family named as the blue-copper proteins of the oxidases7 and is produced by 4 type of living organisms including bacteria, insects, higher plants and fungi 8. So far, very few bacterial origin laccase enzymes have been purified and characterized. The first study on this subject is the prokaryotic laccase which was derived from the Azospirillum lipoferum, a rizospheric bacterium. It was determined in this bacterium that the enzyme was

Keywords: Laccase, Brevibacillus, Purification, Characterization, Removal textile dyes.

Introduction The discovery of life at high temperatures and the isolation of Thermus aquaticus from the Yellowstone National park (56)



composed of a catalytic subunit and is a multimeric enzyme, consisting of 1 or 2 larger chains.9

in this study was isolated from water and sludge samples of Diyadin Hotspring in provinces of Agri in Turkey. The water temperature of this hot spring was around 70 °C. The samples were streaked on Nutrient Agar (NA) plates and then incubated in aerobic and anaerobic incubators at 55-60 °C for 24-48 hours. After incubation, different colony developed in the media were selected and purified by subculturing. The purity of the test isolate was assessed by using colony morphology and microscopy. Isolated and purified bacterial strains were stored in Nutrient Broth containing 15% glycerol at -86 °C for further studies16.

Laccase enzyme was purified from the Marinomonas mediterranea, which is a melanogenic marine bacterium. It has also been determined that these bacteria produce two different polyphenol oxidase, capable of oxidizing the substrates of both laccases and tyrosinases9. In a study conducted recently, Ruijssenaars and Hartmans10 have identified a protein of Bacillus halodurans encoded by ORF bh2082 as a potential bacterial laccase. There are very few studies performed with thermophilic bacteria. Recent studies have been conducted with thermophilic organisms including Laccase derived from Streptomyces lavendula REN-711, laccase-like protein derived from Pyrobaculum aerophilum IM2, which is a thermophilic archaea12, laccase 31-like protein derived from Aquifex aeolicus VF5 which is a hyperthermophilic bacterium13 and laccase derived from Thermus thermophilus9,14 and Bacillus pumilus.

Morphological, physiological and biochemical characterizations of isolate: The temperature range for growth was determined by incubating strain from 35 to 80 °C with 1 °C of intervals. The pH dependence of growth was tested from pH 4.0 to pH 12.0. Cell morphology of isolates was investigated by light microscopy. The effect of NaCl on the thermophilic bacterial growth was studied in NB medium containing 2.0, 3.0, 4.0, 5.0, 8.0, 10.0% (w/v) NaCl. Cell and colony morphology, gram reaction, the presence of catalase, oxidase and amylase were investigated according to the methods described by Harley and Prescott17.

Laccase mediated catalysis occurs with reduction of oxygen to water, accompanied by the oxidation of substrate. Laccases thus oxidize polyphenols, methoxysubstituted phenols, aromatic diamines and a range of other compounds15. Consequently, it is possible to use the laccase enzyme in many industrial areas such as the removal of textile dyes, phenols removal and waste detoxification, since it does not have the substrate specificity. For this reason, purification and characterization of the laccase enzyme produced intracellularly by Brevibacillus sp. (Z1) bacteria (Genbank number KC292196) which were isolated in the Agri-Diyadin thermal springs and investigating the usability of this enzyme in removing textile dyes in wastewater were aimed in this study.

DNA extraction from pure culture: Total genomic DNA was extracted from bacteria samples using a modified method previously described by Adiguzel2. PCR amplification and cloning of 16S rRNA gene fragment: The 16S rRNA gene was selectively amplified from purified genomic DNA by using oligonucleotide primers designed to anneal to conserved positions in the 3' and 5' regions of bacterial 16S rRNA genes. The forward primer, UNI16S-L (5'-ATTCTAGAGTTTGATCATGGCT CA-3'), corresponded to positions 11 to 26 of Escherichia coli 16S rRNA and the reverse primer, UNI16S-R (5'-AT GGTACCGTGTGACGGGCGGTGTGTA-3'), corresponded to the complement of positions 1411 to 1393 of Escherichia coli 16S rRNA18. PCR reaction conditions were carried out according to Beffa et al19 and the PCR product was cloned to a pGEM-T vector system (Promega, UK).

Material and Methods Materials: Bovine serum albumin (BSA), ABTS [2,2’azino-bis(3-ethylbenzylthiazoline-6-sulfonic acid)], DEAEsephedex, Sephacryl S-200, ethylene diamine tetra acetic acid (EDTA), dithioerythritol, ß-mercaptoethanol, tris(hydroxymethyl)aminomethane, hydrogen peroxide (H2O2), 4-methylcatechol, guaiacol, pyrogallol, hydroquinone, Reactive Black 5 (RB5), fuchsine (C20H19N3.HCl), allura red (C18H14N2Na2O8S2), Acid red 37 diammonium salt (C18H16N4O8S 2.2H3N) and agents for SDS-PAGE were purchased from Sigma (USA). Sodium acetate (CH3COONa), ammonium sulphate [(NH4)2SO4], sodium chloride (NaCl), BaCl2, CuCl2, MnCl2, CoCl2, ZnCl2, FeCl2, CaCl2, NiCl2 HgCl2, ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHCO3), sodium acetate (CH3COONa), ascorbic acid (C6H8O6) and sodium hydrogen phosphate monohydrate (Na2HPO 4.H2O) were purchased from Merck (Darmstadt, Germany). All other chemicals were of analytical grade.

Sequencing analysis: Following PCR amplification and cloning of the 16S rRNA gene of the strain, the 16S rRNA gene sequence with an Applied Biosystems model 373A DNA sequence was determined by using the ABI PRISM cycle sequencing kit (Macrogen, Korea). The sequence consisting of about 1425 nucleotides (nt) of the 16S rRNA gene was determined. This sequence was compared with those contained within GenBank 20 by using a BLAST search21. The 16S rRNA gene sequences of the species most closely related to our strain were retrieved from the database16. Purification of Laccase from Brevibacillus sp. (Z1): The crude enzyme extract was filtered and centrifuged for 15

Isolation of strain: The thermophilic bacterial strain used (57)



min at 5000 rpm and then the ammonium sulphate enzyme homogenate was precipitated at 0-20%, 20-40%, 40-60%, 60-80% and 80-100% intervals. Then ammonium sulphate was used to saturate the obtained supernatant at 60-80%. After dissolving the precipitate in 20 mM of phosphate buffer (pH 5.0), it was dialyzed against the same buffer above.

SDS polyacrylamide gel electrophoresis: Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed through the 10-30% discontinued electrophoresis method which was defined by Laemmli28. 20 µg of protein was applied to each of the samples. Electrophoresis in a gelcasting apparatus (BioRAD) execution buffer (0.25 M Tris, 1.92 M Glycine, 1% SDS (pH 8.3) was applied at 4 °C.

Anion exchange chromatography: After the suspension obtained by ammonium sulphate, precipitation was dialyzed, the 20 mM of phosphate buffer (pH 5.0) and previously equilibrated DEAE-cellulose ion exchange column (2.5 x 30) were studied. The column was washed until the eluate protein detection fails with the same buffer. Following this, NaCl gradient from 0 up to 1 M was applied to elute the proteins attached to the column. The flow rates of the collected fractions were 3 ml with a 3 mL/min. The absorbance of protein elution was spectrophotometrically measured at 280 nm using ABTS substrate to measure the activity in the fractions. The active fractions were pooled (combined) and allowed to stand at 4 °C22.

The gel was dyed with 0.1% Coomassie Brilliant Blue R250 in 50% methanol, 10% acetic acid and 40% distilled water for 1.5 h. Then the gel was washed with 50% methanol, 10% acetic acid and 40% distilled water until its surface becomes limpid. The electrophoretic pattern was photographed. Molecular weight determination by gel filtration: Molecular weight of the laccase enzyme was performed by using column (3x70 cm) of Sephadex G100. The column was equilibrated until reaching zero absorbance, by using 0.05 M Na2HPO4, 1 mM dithioerythretol, pH 7.0 buffer. Standard protein solution (bovine serum albumin, 66 kDa, egg ovalbumin, 45 kDa, pepsin, 34 kDa, trypsinogen, 24 kDa, ß-lactoglobulin and lysozyme, 14 kDa) was applied to the column and was eluted from the column with the same buffer. Then, the pure laccase enzyme was applied to the column and was eluted under the same conditions. The flow-rate through the column was 20 mL/h. The elution volume was compared with standard proteins29.

Gel filtration: Using an Amicon membrane concentrator with a 10 kDa cut off, the active fractions which have been obtained from anion exchange column were combined, dialyzed, desalted and concentrated. Obtained enzyme solution was applied to Sephacryl S-200 column (120 cm × 1 cm), which has been pre-equilibrated with 20 mM of phosphate buffer (pH:5.0) having 0.5 M NaCl and later the enzyme has been obtained with the very same buffer. And all of fractions have been analyzed as stated above. And for later use, the active fractions have been combined, concentrated and allowed to stand at 4 °C.

Studies on certain in vitro chemicals and metal ions: The impact of the metal ions of Fe2+, Cu2+, Zn2+, Hg2+, Ba2+, Ca2+, Co2+, Mn2+, Ni2+, EDTA and ascorbic acid in different inhibitory concentrations on laccase enzyme was investigated. All metal ions were tested in three runs for each concentration. Laccase activity was measured in different concentrations of the inhibitor. I50 values were calculated for those that present inhibition30.

Protein concentration: The protein concentration was determined spectrophotometrically with an absorbance at 280 nm in addition to the Bradford's method23 using Bovine Serum Albumin (BSA) as a standard.

Remediation of some textile dyes: In this study, it was investigated that whether the purified laccase enzyme can be used in the removal of some dyes used in industry or not. For this purpose, Reactive black 5, Fuchsine, Allura Red and Acid red 37 diammonium salt were used as 4 different dyes. The dye solutions were prepared at 50 mg/L concentration and by taking 50 mL from these solutions 1 mL (5 µg protein), the purified laccase enzyme was added. The reaction medium was set to pH 4.0 and 55 °C, which is the optimum pH and temperature for the enzyme and samples were taken at 15th, 30th, 60th, 90th, 120th, 150th and 180th minutes from the medium and the absorbance was measured spectrophotometrically for Reactive Black 5, Fuchsine, Allura Red, Acid Red 37 Diammonium Salt, at 597 nm, 547 nm, 513 nm and 504 nm respectively. The results obtained are shown in the chart as Absorbance vs. mg dye plots.

Determination of laccase activity: The laccase activity has been determined using ABTS substrate24,25. The reaction mixture was prepared by adding 0.5 mL of the enzyme solution on top of the ABTS (3 mM) substrate dissolved in 0.5 mL of 0.1 M acetate buffer (pH = 4.5) and then it was incubated at 55 °C. The oxidation of ABTS was determined by monitoring the increase in absorbance at 420 nm. And the one unit of a laccase activity was defined as the required amount of enzyme to oxidize 1 μmol of ABTS/min (ε420 = 36,000 M-1 cm-1)26. In order to find out the Km and Vmax values, the laccase activity was determined by measuring at 420 nm, 470 and 468 nm respectively by making use of 3 mM ABTS, guaiacol and 2,6-dimetoksifenol substrates. The blind was prepared by adding the enzyme-containing buffer, instead of enzyme solution, which was used as the sample27.

Statistical analysis: All of the tests were conducted in (58)



three runs in order to determine the laccase activities of the samples. Data were expressed in terms of mean ± standard errors. Statistical analyses were performed using SPSS version 10.0 software (SPSS Inc., Chicago, IL., USA) and the significant differences were determined with a 95% confidence interval (p