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A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye

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ABSTRACT

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.

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Elution of laccase on DEAE-Cellulose column using 0.2M NaCl.The chromatogram represents concentration of laccase eluted (absorbance A280 at 280 nm) on Y axis with amount of eluted fraction (ml) on X axis. Each fraction were collected and measured for laccase activity (absorbance at A420) using ABTS as substrate
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pone.0175144.g001: Elution of laccase on DEAE-Cellulose column using 0.2M NaCl.The chromatogram represents concentration of laccase eluted (absorbance A280 at 280 nm) on Y axis with amount of eluted fraction (ml) on X axis. Each fraction were collected and measured for laccase activity (absorbance at A420) using ABTS as substrate

Mentions: Spirulina platensis (CFTRI, Mysore) laccase was purified from the culture filtrate (Table 1). In cold acetone precipitation, the specific activity was increased from 293.42 to 983.6 U/mg protein and its yield and purification factor were 88% and 3.3 fold respectively. The precipitate was dialyzed against 50 mM Tris HCl buffer and applied to a DEAE-cellulose column. Linear gradient elution (0.1-1M NaCl) and fractions were analyzed spectrophotometrically with (ABTS as substrate). The peak showing laccase activity was eluted at 0.2 M NaCl in a DEAE column (Fig 1). In DEAE cellulose column chromatography, the specific activity was increased to 1117.18 U/mg protein with 3.8 fold purification factor and 62.8% yield. The fraction containing major laccase activity were pooled and again dialyzed against 50 mM of Tris HCl buffer and loaded on to Sephadex G- 100 column. The protein concentrations and laccase activity were determined and compared at each step of purification (Table 1). The results depicted downhill slope of concentration and total protein with each passing step whereas an increase in specific activity and fold purification. The specific activity of protein increased from 1117.8 to 1721.5 U/mg along with increase in 3.8 to 5.8 fold purification and 51.5% yield at the end of purification. Mostly laccase purification had been done from fungal sources till date which includes, Phellinus igniarius, Pleurotus ostreatus, Trametes gibbosa, Trametes hirsuta and Trametes versicolor etc [6] and only one laccase has been purified and characterized in a green alga [8]. Previously, presence of laccase was reported in Phormidium valderianum and Oscillatoria boryana but there are no reports on the purification of laccase from cyanobacteria [23]. The presence of laccase gene for putative LAC in cyanobacteria was confirmed in Synechococcus sp. RS9917 by sequencing [24]. The most commonly used method for laccase purification is salt elution from an anion-exchange resin due to higher stability at neutral to alkaline pH and its the isoelectric point (pI) of laccase [25]. The extracel1ular cyanobacterial laccase exhibit strong binding with DEAE cellulose column and elution was done at 0.2 M NaCI gradient. Laccases show variable binding and many studies have also reported the elution from 0.1 M to 0.3 M NaCl gradient [26]. Previously laccase purification was reported from fungi Pleurotus sajor-caju MTCC 141 with 10.71 fold and 3.46% yield while in bacteria 28.46 fold purification of laccase from Bacillus tequilensis SN4 with 13.34% yield was reported [27, 28]. However in green algae Tetracystis aeria 120 fold purified having very low yield of 2.5% was obtained [8].


A novel multicopper oxidase (laccase) from cyanobacteria: Purification, characterization with potential in the decolorization of anthraquinonic dye
Elution of laccase on DEAE-Cellulose column using 0.2M NaCl.The chromatogram represents concentration of laccase eluted (absorbance A280 at 280 nm) on Y axis with amount of eluted fraction (ml) on X axis. Each fraction were collected and measured for laccase activity (absorbance at A420) using ABTS as substrate
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Related In: Results  -  Collection

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pone.0175144.g001: Elution of laccase on DEAE-Cellulose column using 0.2M NaCl.The chromatogram represents concentration of laccase eluted (absorbance A280 at 280 nm) on Y axis with amount of eluted fraction (ml) on X axis. Each fraction were collected and measured for laccase activity (absorbance at A420) using ABTS as substrate
Mentions: Spirulina platensis (CFTRI, Mysore) laccase was purified from the culture filtrate (Table 1). In cold acetone precipitation, the specific activity was increased from 293.42 to 983.6 U/mg protein and its yield and purification factor were 88% and 3.3 fold respectively. The precipitate was dialyzed against 50 mM Tris HCl buffer and applied to a DEAE-cellulose column. Linear gradient elution (0.1-1M NaCl) and fractions were analyzed spectrophotometrically with (ABTS as substrate). The peak showing laccase activity was eluted at 0.2 M NaCl in a DEAE column (Fig 1). In DEAE cellulose column chromatography, the specific activity was increased to 1117.18 U/mg protein with 3.8 fold purification factor and 62.8% yield. The fraction containing major laccase activity were pooled and again dialyzed against 50 mM of Tris HCl buffer and loaded on to Sephadex G- 100 column. The protein concentrations and laccase activity were determined and compared at each step of purification (Table 1). The results depicted downhill slope of concentration and total protein with each passing step whereas an increase in specific activity and fold purification. The specific activity of protein increased from 1117.8 to 1721.5 U/mg along with increase in 3.8 to 5.8 fold purification and 51.5% yield at the end of purification. Mostly laccase purification had been done from fungal sources till date which includes, Phellinus igniarius, Pleurotus ostreatus, Trametes gibbosa, Trametes hirsuta and Trametes versicolor etc [6] and only one laccase has been purified and characterized in a green alga [8]. Previously, presence of laccase was reported in Phormidium valderianum and Oscillatoria boryana but there are no reports on the purification of laccase from cyanobacteria [23]. The presence of laccase gene for putative LAC in cyanobacteria was confirmed in Synechococcus sp. RS9917 by sequencing [24]. The most commonly used method for laccase purification is salt elution from an anion-exchange resin due to higher stability at neutral to alkaline pH and its the isoelectric point (pI) of laccase [25]. The extracel1ular cyanobacterial laccase exhibit strong binding with DEAE cellulose column and elution was done at 0.2 M NaCI gradient. Laccases show variable binding and many studies have also reported the elution from 0.1 M to 0.3 M NaCl gradient [26]. Previously laccase purification was reported from fungi Pleurotus sajor-caju MTCC 141 with 10.71 fold and 3.46% yield while in bacteria 28.46 fold purification of laccase from Bacillus tequilensis SN4 with 13.34% yield was reported [27, 28]. However in green algae Tetracystis aeria 120 fold purified having very low yield of 2.5% was obtained [8].

View Article: PubMed Central - PubMed

ABSTRACT

A novel extracellular laccase enzyme produced from Spirulina platensis CFTRI was purified by ultrafiltration, cold acetone precipitation, anion exchange and size exclusion chromatography with 51.5% recovery and 5.8 purification fold. The purified laccase was a monomeric protein with molecular mass of ~66 kDa that was confirmed by zymogram analysis and peptide mass fingerprinting. The optimum pH and temperature of the enzyme activity was found at 3.0 and 30°C using ABTS as substrate but the enzyme was quite stable at high temperature and alkaline pH. The laccase activity was enhanced by Cu+2, Zn+2 and Mn+2. In addition, the dye decolorization potential of purified laccase was much higher in terms of extent as well as time. The purified laccase decolorized (96%) of anthraquinonic dye Reactive blue- 4 within 4 h and its biodegradation studies was monitored by UV visible spectra, FTIR and HPLC which concluded that cyanobacterial laccase can be efficiently used to decolorize synthetic dye and help in waste water treatment.

No MeSH data available.


Related in: MedlinePlus