УДК 582.28:577.15
HIGHLY STABLE LACCASE FROM REPEATED-BATCH CULTURE
OF Funalia trogii ATCC 200800
© 2014 O. Yesilada*, E. Birhanli*, N. Ozmen**, and S. Ercan*
* Inonu University, Arts and Science Faculty, Department of Biology, 44280, Malatya, Turkey **Inonu University, Education Faculty, Department of Science, 44280, Malatya, Turkey e-mail: ozfer.yesilada@inonu.edu.tr Received March 25, 2013
The effect of temperature, pH, different inhibitors and additives on activity and stability of crude laccase obtained from repeated-batch culture of white rot fungus Funalia trogii ATCC 200800 was studied. The crude enzyme showed high activity at 55—90°C, which was maximal at 80—95°C. It was highly stable within the temperature intervals 20—50°C. The half life of the enzyme was about 2 h and 5 min at 60°C and 70°C, respectively. pH optimum of fungal laccase activity was revealed at pH 2.5. The enzyme from F. trogii ATCC 200800 was very stable between pH values of 3.0—9.0. NaN3 and KCN were detected as the most effective potent enzyme inhibitors among different compounds tested. The fungal enzyme was highly resistant to the various metal ions, inorganic salts, and organic solvents except propanol, at least for 5 min. Because of its high stability and efficient decolorization activity, the use of the crude F. trogii ATCC 200800 laccase instead of pure enzyme form may be a considerably cheaper solution for biotechnological applications.
DOI: 10.7868/S0555109914010139
The copper-containing enzyme, laccase (benzene-diol:oxygen oxidoreductase; EC 1.10.3.2), can oxidize and degrade various compounds. This enzyme could be used in various industrial and environmental applications [1—3]. The production of highly stable laccase and possible use of crude enzyme from microorganisms without any purification are very important for biotechnological applications. Laccase may have different properties and stabilities depending on culture conditions and species/strains. Temperature stability, optimal pH and substrate specifity of this enzyme can vary considerably [4, 5].
Different cultivation methods such as liquid or solid state fermentation could be used to produce laccase [6, 7]. In our previous study, it was shown that laccase from Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200 801 can be produced in high amounts by repeated-batch fermentation, a different method than batch method [8].
Textile effluents contain different dyes. Most dyes may have toxic and genotoxic effects [9, 10]. Decolorization of dyes by physicochemical treatments has several disadvantages, and conventional biological treatment systems have low decolorization efficiency. Therefore, enzyme-based processes can provide an alternative method to decolorize and remove dyes. Lac-case is the main enzyme with decolorization activity towards dyes [11]. Deveci et al. [12] reported the decolorization of remazol brilliant blue R by crude laccase F. trogii ATCC 200800 without mediator. On the other hand, Murugesan et al. [13] found the same ob-
servation for crude laccase obtained from solid state culture of Ganoderma lucidum, though they stated that this source could decolorize remazol black 5 only in presence of mediator 1-hydroxybenzotriazole.
The aim of this study was to explore the effect of pH and temperature on activity and stability of crude laccase obtained from repeated-batch culture of Funalia trogii ATCC 200800. The action of various inhibitors and additives on enzyme activity was also tested. Textile dye decolorization activity of the F. trogii ATCC 200800 crude enzyme was also determined by a single step detection method on native PAGE gels and in aqueous textile dye solution without any mediator.
MATERIALS AND METHODS
Organism. The stock culture of the white rot fungus F. trogii ATCC 200800 from Inonu University, Arts and Science Faculty, Department of Biology (Malatya, Turkey) was used in the study. It was maintained at +4°C, after subculturing at 30°C every 2— 3 weeks on Sabouraud's dextrose agar (SDA) plates.
Laccase production under repeated-batch cultivation. Firstly, the F. trogii ATCC 200800 pellets were produced to use for laccase production under repeated-batch condition. The fungus was precultured in 250 mL flasks containing 100 mL Sabouraud's dextrose broth (SDB) at 30°C and 150 rpm for 5 days and then, 7 mL of gently homogenized preculture (myce-lial suspension) was transferred into 600 mL fresh SDB media in 1000 mL flasks and incubated as stated
above. After incubation, fungal pellets were harvested by filtration with sterile plastic filters and used for lac-case production. During repeated-batch studies, 50 mL of stock basal medium (SBM) [8] was inoculated with pellets and these pellets were incubated repeatedly in fresh SBM medium [8]. After 5 cycles of incubation, the culture was filtrated from sterile filter paper and the filtrate was used as crude laccase source.
Laccase activity assay. Laccase activity was determined spectrophotometrically (Shimadzu-UV-1601, Japan) by monitoring the oxidation of the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) to its cation radical (ABTS'+) at 420 nm for 1 min at 30°C. The assay mixture contained 100 mM sodium acetate buffer (pH 5.0), 0.5 mM ABTS and a suitable amount of crude enzyme. One unit of laccase activity was defined as the amount of enzyme that oxidized 1 ^mol of substrate (ABTS) per min at 30°C. All values were the means of at least 3 replicates, with standard deviation of the mean shown as ± values.
Effect of pH and temperature on crude laccase activity and stability. To determine pH optimum of F. trogii ATCC 200800 crude laccase, it was assayed within a pH range of 2.0—7.0 at 30°C for 1 min. pH stability of the enzyme was detected within a pH range of 3.0-9.0 for different times (0-24 h) and then the activity was determined as described in laccase activity assay section. For testing the effect of temperature on crude fungal laccase activity, the enzyme was assayed within a temperature range of 5-95°C for 1 min. To determine the enzyme stability, the crude laccase was incubated at 20-80°C for different times (0-24 h). After cooling at ice bath, the activity was measured at 30°C.
Effect of different inhibitors and other additives on laccase activity. In order to determine the effect ofvar-ious inhibitors such as NaN3, KCN, EDTA and SDS on F. trogii ATCC 200 800 crude laccase activity, different amounts of inhibitors were added to reaction mixtures separately at final concentrations of 0.01-1 mM for NaN3, KCN, EDTA and 0.01-1% for SDS. The enzyme activity was assayed for 1 min using ABTS as a substrate and expressed as the relative activity (%) of the control. Controls were determined without any inhibitors. The effect of different additives such as heavy metals (Cu2+, Mn2+, Co2+, Zn2+, Ag+ and Cd2+), inorganic salts (NaCl and Na2SO4) and organic solvents (ethanol, methanol, propanol and acetone) on crude fungal laccase stability was also tested. The crude enzyme source was incubated with different additives for 5, 60 and 300 min. Then, remaining residual laccase activities were determined using ABTS as a substrate and expressed as the relative activity (%) of the control. Controls were determined without any additives.
Protein bands, activity and decolorization determination by PAGE. SDS-PAGE and native PAGE were performed on 10% separating and 4% stacking gels. Protein bands on SDS-PAGE gel were visualized by
staining with Coomassie brilliant blue R-250. After electrophoresis, native polyacrylamide gels were incubated with ABTS for activity determination [8]. Dye decolorization activity of this crude laccase was also determined on the native gels. In order to test the dye decolorization activity of F. trogii ATCC 200 800 crude enzyme loaded on native gels was stained with 2 textile dyes, reactive black 5 (RB 5) or reactive blue 171 (RB 171). The dye structures are shown in Fig. 1. After incubation for 15 min at 50°C, the dye solutions were discarded and gels were examined for decolorization activity.
Decolorization of dyes by crude laccase. The dye
decolorization ability of crude laccase in aqueous textile dye solutions was also determined by using RB 5 and RB 171 textile dyes, separately. The crude enzyme samples (20 ^L and 1 ^L for RB 5 and RB 171, respectively) were added into 110 mM citrate phosphate buffer (pH 2.5) containing RB 5 (50 mg/L) or RB 171 (50 mg/L) dyes. They were incubated at 30°C for 1 min. Decolorization was determined by monitoring OD597 and OD620 for RB 5 and RB 171, respectively.
Substrate specificity of crude laccase. ABTS, 2,6-dimethoxyphenol (DMP) and syringaldazine at 0.5 mM concentrations were used to test the substrate specificity of the crude F. trogii ATCC 200800 laccase. Laccase activity was determined spectrophotometri-cally at 420, 469 and 530 nm by using substrates of ABTS, DMP and syringaldazine, respectively. Relative activities for each substrate were calculated from the activity for ABTS used as control.
RESULTS AND DISCUSSION
Enzyme production. Different culture conditions such as batch, repeated-batch or solid substrate fermentations differently affect laccase production of white rot fungi [8]. Therefore, here, pellet forms of F. trogii ATCC 200800 were firstly obtained and used to produce laccase under repeated-batch cultivation. The culture was filtrated after 5 cycles of incubation and the filtrate was used as crude laccase enzyme source. To determine the best substrate for F. trogii ATCC 200800 crude laccase, the most commonly used substrates, ABTS, DMP and syringaldazine, were used. The relative activites were 100%, 55% and 80% for ABTS, DMP and syringaldazine, respectively. Thus, ABTS was detected as the most suitable substrate for this crude enzyme.
Effect of pH on enzyme activity and stability. Reaction pH can affect enzyme activity and stability. Therefore, the effect of reaction pH on F. trogii AT
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