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Decolorization of Synthetic Dyes by Crude Laccase from Lentinus Polychrous Lev.
| Content Provider | Semantic Scholar |
|---|---|
| Author | Ratanapongleka, Karnika Phetsom, Jirapa |
| Copyright Year | 2014 |
| Abstract | In the present study, the addition of copper (2mM) to the fungus L. polychrous cultivated in solid media influenced the laccase activity. Laccase production was increased in the presence of copper and reached a maximum activity of 145 U/ml on 12 th day. Crude laccase was capable to decolorize different dye structures. The enzyme showed great decolorization efficiency toward Acid Blue 80 (85% decolorization in 120 min). Reaction kinetics of the enzyme on Acid Blue 80 followed Michaelis-Menten behavior and the initial rate of decolorization depended on the dye concentration. The kinetic parameters of the enzyme were determined and calculated from Lineweaver-Burk plots. The results indicated that the Km value was 0.36 mM and Vmax value was 0.0017 mM/min. The pH value for maximum decolorization of Acid Blue 80 was 5.0. Manuscript received September 8, 2013; revised November 10, 2013. This work was financial supported by Ubon Ratchathani University, Thailand. K. Ratanapongleka is with the Chemical Engineering Department, Faculty of Engineering, Ubon Ratchathani University, Ubonratchathani 34190, Thailand (e-mail: k_ratanapongleka@ubu.ac.th). J. Phetsom is with the Biology Department, Faculty of Science, Mahasarakham University, Kantharawichai District, Maha Sarakham 44150, Thailand (e-mail: phetsom2000@yahoo.com). B. Crude Laccase Extraction and Laccase Activity Crude laccase enzyme was prepared using water extraction. A 3:1 ratio of water and media was used for extraction. The mixed solution was filtered through miracloth and centrifuged at 4,000g for 10 minutes. The supernatant was defined as crude laccase and kept at 4 C for further study. Laccase activity was determined following the change in optical density at 420 nm using 2, 2′-azino-bis (3-ethylbenzthiazoline-6-sulfonate (ABTS) as a substrate ( = 36,000 M cm). Briefly, the assay mixture consisted of 0.1 M acetate buffer pH 4.5, 10 mM ABTS and enzyme. The mixture was incubated at 32 C for 10 min and stopped the reaction with 50% (w/v) TCA. One unit of enzyme was defined as the amount of enzyme required to oxidize 1 μmol ABTS per minute. All assays were carried out in triplicate. C. Effect of Salts and EDTA on Laccase Activity The effect of salts on laccase activity was tested with NaCl, Na2SO4 and Na2CO3. All salts including EDTA were used at a final concentration of 5, 10 and 50 mM. The reaction mixture consisted of 100 mM acetate buffer pH 4.5, 10 mM ABTS and 0.125 U/ml of crude laccase. D. Decolorization Assay Three structural groups of dyes (azo, indigoid and anthraquinone) were selected in this study. All dyes (Reactive Black 5, Reactive Orange 16, Reactive Green 19, Methyl Orange, Indigo Camine, Acid Blue 80 and Water Blue) were purchased from Sigma-Aldrich. The stock solution of dye was prepared by dissolving in distilled water. The desired dye concentration was prepared from the stock solution by dilution. The reactions were carried out directly in the spectrophotometer cuvette. The reaction mixture consisted of crude laccase and an aqueous solution of dye in phosphate buffer pH 7.0 in a total final volume of 3 ml. The cuvette was then inverted using paraffin as a cover, in order to achieve the homogeneity of the mixture prior to the measurement. Decolorized activity was determined by monitoring the decrease in absorbance on a spectrophotometer at a maximal absorbance of each dye (360-630 nm) and expressed in terms of percentage. All experiments were done in triplicate. Controls contained the individual dyes solution with inactive enzyme. E. Effect of Initial Dye and Laccase Concentration on Acid Blue 80 Decolorization The effect of Acid Blue 80 concentration on the enzymatic decolorization was monitored with different concentrations ranging from 20 to 140 mg/l in acetate buffer pH 5.0. The crude enzyme was used at 0.125 U/ml. To test the effect of enzyme quantity, reaction was started with different quantities of enzyme (0.003, 0.006, 0.012, 0.025, 0.062 and 0.125 U/ml). The dye concentration was fixed at 20 mg/l in buffer pH 5.0. The reaction was carried out at room temperature. F. Effect of pH and Temperature on Acid Blue 80 Decolorization The effect of pH on decolorization was tested at different pHs ranging from 2 to 9. The pH of reaction mixture was adjusted by citrate-phosphate and Tris-HCl buffer. To determine the effect of temperature on Acid Blue 80 decolorization, the reaction mixture (pH 5.0) was incubated under a temperature range between 30 and 60 C. The concentration of crude enzyme was controlled at 0.125 U/ml. III. RESULTS AND DISCUSSION A. Laccase Production The production of extracellular laccase from L. polychrous in the presence of Cu was studied (Fig. 1). L. polychrous cultured in natural medium with and without copper was able to produce laccase. During the first week of cultivation, small amounts of laccase were produced (data not shown). The enzyme activity increased obviously after 7 days of cultivation. The presence of Cu in the medium slightly increased the level of laccase activity. The highest laccase activity (145 U/L) was obtained in medium with copper. Laccase production in the absence of Cu gave maximum on 14 day (103 U/L) while in the presence of Cu was on 12 day. It is found that in the presence of inducers many white rot fungi can produce high amounts of laccase into the medium. This study shows that copper induces laccase production of L. polychrous. Similar inductive effect of Cu on laccase production has been reported earlier in P. ostreatus [10] and T. trogii [2], [11]. Zheng et al. [12] indicated that copper could act as an inducer of enzyme activity by affecting the genetic transcription levels in which along with more copper involved in McoA-laccase synthesis. However, copper requirements are usually satisfied at very low concentrations. The presence of 4 mM CuSO4 inhibited the growth of P. hauseri and the cells were almost killed at 4.5 mM CuSO4[12]. Fig. 1. Production of laccase as a function of time in the (■) absence and (▲) presence of Cu. B. Effect of Salts and EDTA on Laccase Activity In addition to dyes, high concentrations of salts have been found in textile industrial effluents and their presence can affect enzyme activity. In this study, the effect of salts and EDTA on laccase activity was tested at various concentrations, namely 5, 10 and 50 mM. It can be seen in Table 1 that laccase activity was significantly inhibited when high amounts of sodium chloride were added. While in the presences of 50 mM of both Na2SO4 and Na2CO3 enhanced the activity. A similar behavior of NaCl on laccase activity 27 International Journal of Chemical Engineering and Applications, Vol. 5, No. 1, February 2014 |
| Starting Page | 26 |
| Ending Page | 30 |
| Page Count | 5 |
| File Format | PDF HTM / HTML |
| DOI | 10.7763/IJCEA.2014.V5.345 |
| Volume Number | 5 |
| Alternate Webpage(s) | http://www.ijcea.org/papers/345-CA428.pdf |
| Alternate Webpage(s) | https://doi.org/10.7763/IJCEA.2014.V5.345 |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
