ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 6, с. 592-599
UDC 577.112
PURIFICATION AND CHARACTERIZATION OF LACCASE SECRETED BY Phellinus linteus MTCC-1175 AND ITS ROLE IN THE SELECTIVE OXIDATION OF AROMATIC METHYL GROUP
© 2013 P. K. Chaurasia, A. Yadav, R. S. S. Yadav and S. Yadava
Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur 273009, Uttar- Pradesh, India
e-mail: rssy_chemistry@rediffmail.com Received Novembar 26, 2012
A laccase from the culture filtrate of Phellinus linteus MTCC-1175 has been purified to homogeneity. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion exchange chromatography on DEAE-cellulose. The SDS-PAGE and native-PAGE gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 70 kDa. Using 2,6-dimethoxyphenol, 2,2'[azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 4-hydroxy-3,5-dimethoxybenzaldehyde azine as the substrates, the Km, kcat and kcat/Km values of the laccase were found to be 160 ^M, 6.85 s-1, 4.28 x 104 M-1 s-1, 42 ^M, 6.85 s-1, 16.3 x 104 M-1 s-1 and 92 p.M, 6.85 s-1, 7.44 x 104 M-1 s-1, respectively. The pH and the temperature optima of the P. linteus MTCC-1175 laccase were 5.0 and 45°C, respectively. The activation energy for thermal denaturation of the enzyme was 38.20 kJ/mole/K. The enzyme was the most stable at pH 5.0 after 1 h reaction. In the presence of ABTS as the mediator, the enzyme transformed toluene, 3-nitrotoluene and 4-chlorotoluene to benzaldehyde, 3-nitroben-zaldehyde and 4-chlorobenzaldehyde, respectively.
DOI: 10.7868/S0555109913060068
Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a polyphenol oxidase, which belongs to the superfamily of multicopper oxidases [1, 2] and catalyzes [3-5] the four-electron reduction of molecular oxygen to water. Laccases are dimeric or tetrameric glycoproteins. To perform their catalytic functions, laccases use Cu atoms that are distributed at the 3 different copper centres. They are called blue (type-1), normal (type-2) and coupled binuclear (type-3) copper centres and differ in their EPR characteristics [6, 7]. The organic substrate is oxidized by one electron at the active site of the laccase generating a reaction radical which further reacts non-enzy-matically. The electron is received at type-1 Cu centre and shuttled to the trinuclear cluster where oxygen is reduced to water.
Ortho- and para-diphenols, aminophenols, polyphenols, polyamines, lignins, and arylamines and some of the inorganic ions are the substrates for laccases. The ability of laccases to catalyze the oxidation of various phenolic compounds, coupled to the reduction of molecular oxygen to water makes them valuable from the point of view of commercial applications [4, 8-10]. The biotechnological importance of laccases has increased after the discovery that substrate range for oxidizable reaction could be further extended in the presence of small readily oxidizable molecules called mediators [11, 12]. During the last two decades, laccases have turned out to be the most promising enzymes for industrial uses [9, 10]
having applications in food, pulps, paper, textile, and cosmetics industries and in synthetic organic chemistry [13-16].
Laccases purified from different sources exhibit different properties and are suitable for many applications [3-6]. Thus, there is a scientific need to study laccases from new sources so that laccases with novel properties could be found.
Moreover, there are reports in the literature that the fungal strains which secrete blue laccases in liquid growth media, secrete yellow laccases when grow in the media containing solid lignocellulosic substrates like wheat straw [17, 18]. The yellow laccases lack the absorbance band around 610 nm found in case of blue laccases. Moreover, yellow laccases do not require the presence of mediator molecules for the oxidation of non-phenolic compounds [18].
Phellinus linteus MTCC-1175 is a white rot fungus growing on logs and its laccase has not been purified so far. It is not known which type of laccase (blue or yellow) is secreted when fungus grows in medium containing solid lignin-containing substrates. The aim of the research was to purify laccase from P. linteus MTCC-1175, to determine its type and to study phys-icochemical properties. Results reported have shown that P. linteus MTCC-1175 secretes blue laccase when grows in the presence of solid lignin-containing natural substrate, wheat straw, and this enzyme is suitable
for the conversion of aromatic methyl group to aldehyde group in the presence of mediator ABTS.
MATERIALS AND METHODS
Materials. 4-Hydroxy-3,5-dimethoxybenzalde-hyde azine (syringaldazine), 4-chlorotoluene and DEAE-cellulose were obtained from Sigma (USA). 2,2'-Azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) diammonium salt (ABTS) and 2,6-dimethoxy phenol (DMP) were purchased in Fluka Chemi (Switzerland). All other chemicals used in these investigations were obtained either from Himedia laboratory Ltd. (India) or from Merck (Germany) and used without further purifications. The chemicals used in the gel electrophoresis of the protein samples and the protein molecular weight markers were purchased from Bangalore Geni Pvt. Ltd., (India).
The fungal strain and its growth. Phellinus linteus MTCC-1175 was procured from the Microbial Type Culture Collection Center and Gene Bank, Institute of Microbial Technology, Chandigarh, (India) and maintained on agar slants as reported in MTCC Catalogue of strains-2000 [19]. The growth medium for the fungus contained (g/L): malt extract — 20.0 and agar — 20.0; the pH was adjusted to 6.5.
In order to detect the extracellular secretion of the laccase by P. linteus MTCC-1175, the liquid growth medium reported by Coll et al. [20] was used. This medium consisted of (g/L): glucose — 10.0, asparagine — 1.0, yeast extract - 0.5, MgSO4 • 7H2O - 0.01 and FeSO4 • 7H2O — 0.01. The liquid growth medium was supplied with natural lignin substrates like coir dust, corn cob, wheat straw, saw dust or bagasse particles. Each of these substrates was prepared by adding 0.5 g to 25 mL of growth medium in 100 mL culture flasks which were sterilized. The sterilized growth media were inoculated with small pieces of the P. linteus MTCC-1175 mycelia (0.5 x 0.5 cm) under aseptic conditions and the fungal cultures were grown under stationary culture conditions at 25°C in a biological oxygen demand (BOD) incubator. In order to monitor the production of the laccase in the liquid culture medium, 0.5 mL aliquots of the growth medium were withdrawn at the regular intervals of 24 h and filtered through sterilized Millipore filter 0.22 ^m (USA). The filtered extract was analyzed for the activity of the lac-case using DMP as the substrate by the method [21] given below in assay section. The optimal time for extracellular secretion of the P. linteus MTCC-1175 lac-case in the liquid medium was determined by plotting the enzyme activity (unit/mL) in the growth medium against the number of days after inoculation of the fungal mycelia. Each point on the curve was an average of 3 measurements. The growth medium for the control experiment did not contain natural lignolytic substrate. In order to optimize the conditions for maximum production of the laccase by P linteus MTCC-1175, the
amount of the best enzyme inducer, wheat-straw particles, was varied from 100 to 1000 mg in 25 mL of the growth medium. The amount of the inducer in the growth medium which gave the maximum of the enzyme activity was taken as the optimal.
Enzyme assay. The assay solution using DMP as the substrate [20] contained 1.0 mM DMP in 50 mM sodium malonate buffer (pH 4.5) at 37°C, using ABTS as the substrate [22] contained 0.5 mM ABTS in 0.1 M sodium acetate buffer (pH 5.0) at 25°C and using syringaldazine as the substrate [23] contained 0.1 mM syringaldazine in 50 mM sodium phosphate buffer (pH 6.0) at 50°C. In case of DMP, the reaction was monitored by measuring OD468 and using the molar extinction coefficient [20] value of 49.6 mM-1 cm-1. In case of ABTS, the reaction was assayed by measuring OD420 and using the molar extinction coefficient [22] value of 36.0 mM-1 cm-1. In case of syringaldazine, the reaction was performed by measuring OD530 and using molar extinction coefficient [23] value 64.0 mM-1 cm-1. The UV/Vis spectrophotometer Hitachi (Japan) model U-2900 fitted with electronic temperature control unit was used for absorbance measurement. One enzyme unit produced 1 ^M of the product per min under the specified assay conditions.
Purification of laccase. For the purification of the laccase, P. linteus MTCC-1175 was grown in ten 100 mL culture flasks each containing 25 mL of growth medium with 400 mg of the inducer, wheat-straw particles, under stationary culture conditions in a BOD incubator at 30°C. The maximum activity of the laccase was revealed on 9 day after the inoculation of the fungal mycelia. On the 9 day, mycelia from all flasks were removed by filtration through 4 layers of cheese cloth. The culture filtrate was saturated up to 30% with ammonium sulphate and centrifuged at 5480 x g and 4°C for 20 min. The precipitate was discarded and the supernatant was saturated up to 85% by further addition of ammonium sulphate. The resulting suspension was centrifuged at the same conditions and the supernatant was discarded. The precipitate was dissolved in 100 mM Na-acetate buffer (pH 4.5) and dialyzed against 10 mM Na-ace-tate buffer (pH 4.5) with 3 changes of buffer during 8 h. Thirty mL of dialyzate containing 2.7 mg/mL protein was concentrated using Amicon concentrator cell model 8200 (USA) and loaded on the DEAE-cel-lulose column (size 0.75 x 18.0 cm) which was pre-equilibrated with 10 mM Na-acetate/acetic acid buffer (pH 4.0) with flow rate 15 mL/h. The column was washed w
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