Purification and characterization of an extracellular laccase from solid-state culture of Pleurotus ostreatus HP-1

Abstract

A native isolate of Pleurotus ostreatus HP-1 (Genbank Accession No. EU420068) was found to have an excellent laccase producing ability. The extracellular laccase was purified to electrophoretic homogeneity from copper sulphate induced solid-state fermentation medium by ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was determined to be monomeric protein with an apparent molecular mass of 68,420 kDa, and an isoelectric point (pI) of 3.5. The inductively coupled plasma spectroscopy showed a presence of iron, zinc and copper in the purified enzyme. The absorption spectrum in the range of 200–700 nm showed the maximum absorption at 610 nm characteristic of fungal laccase and corresponding to the presence of type I copper atom. The laccase was stable at different temperatures up to 70 °C and retained 61 % activity at 50 °C. The enzyme reaction was inhibited by cysteine; sodium azide and EDTA. The enzyme oxidized various known laccase substrates, its lowest Km value being for ortho-dianisidine and highest Kcat and Kcat/Km for ABTS. The purified laccase exhibited different pH optima for different substrates. The N-terminal sequence did not show any similarity with N-terminal sequence of other species of genera Pleurotus.

Optimization of the production, purification and characterization of a laccase from the native fungus Xylaria sp.

“…The laccase from Xylaria sp. had an optimal reaction temperature between 50 and 60ºC (Fig.3b), which is similar to what was obtained for other reported fungal laccases (Forootanfar et al, 2011;Patel et al, 2014). The stability evaluated in the range from 4-70ºC showed that the laccase was mostly stable at temperatures below 30ºC but rapidly inactivated at temperatures of 40ºC and higher (Fig.3c); consistent results were obtained for the laccase of X. polymorpha (Liers et al, 2007), where the enzyme showed great stability at 20ºC for 4 hours but marked changes in residual activity at 40ºC and higher.…”

“…These behaviors are interesting if is considered that one of the main features of the enzyme is that a metal is found on the active site. However, this behavior has also been found in other laccases (De-Souza and Peralta 2003;Patel et al, 2014), and it might be caused by the influence of the substrate on the inhibitory effect of EDTA. Lorenzo et al (2005) found that EDTA strongly inhibits the activity of enzymes when syringaldazine or 2,6-dimethoxyphenol are used as substrates, and when ABTS is used, EDTA did not behave as an efficient inhibitor.…”

“…However, it is important to underscore that the yield obtained for the purification process in different reports, which apply different strategies, is highly variable and is related to the specific characteristics for each crude extract and enzyme. Among this variability, excellent yields such as 77.6% have been obtained by Patel et al, (2014) working with P. ostreatus, but low yields have been reported by other researchers such as Sahay et al, (2008) with 3,46% yield using Pleorotus sajor-caju MTCC 141; or Diaz et al, (2010) with 2,2 and 2,6% yield for laccase isoforms I and II from Coriolopsis rigida; or Junghans et al, (2009) with 0,95% yield using Phoma sp. In contrast, the specific activity value obtained in this study was superior to the activity value obtained for the purified enzymes from other organisms.…”

Immobilization of Laccase on Magnetic Carriers and Its Use in Decolorization of Dyes

“…No activity of laccase was measured from 65 °C upwards. This is similar as in articles 30,31 . For immobilized laccase on microparticles, activity decreased from 20 °C.…”

Plackett-Burman Design for rGILCC1 Laccase Activity Enhancement in Pichia pastoris: Concentrated Enzyme Kinetic Characterization

“…Nonetheless, a higher affinity to ABTS is reported in comparison to other substrates such as syringaldazine or guaiacol among others, with lower oxidation velocity and higher K m values [4, 5, 35]. …”