Biochemical characterization and kinetic studies on a purified yellow laccase from newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decayed plant matter

Abstract

The study investigated the biochemical characteristics and kinetic parameters of laccase from a newly isolated Aureobasidium pullulans NAC8 obtained from soil containing decay plant litters. This was with a view to identifying the type of laccase and its possible suitability for biotechnological applications. The fungal strain was identified as A. pullulans NAC8 by sequencing of its 5.8S rRNA and adjacent internally transcribed sequences (ITS) 1 and 2. A. pullulans NAC8 laccase was purified 2.0-fold with a yield of 59.3% and specific activity of 9.34 μmol/min/mg protein. The kinetic parameters K_M , V _max, k _cat and k _cat/K_M for laccase with guaiacol as substrate were 1.05 ± 0.12 mM, 12.67 ± 0.55 μmol/ml/min, 25.3 × 10^-1 s^-1 and 2.4 × 10³ M^-1 s^-1 respectively. Laccase exhibited maximum activity at 45 °C and optimum pH of 4.5. The enzyme showed stability at a temperature range of 45-55 °C after a 2 h incubation. The molecular weight determined on SDS-PAGE was 68.4 kDa. The enzyme was stable at 10% of all organic solvents used but displayed a loss of activity at 50%. 2.5 mM thioglycolic acid (TGA) and 0.05 mM sodium azide inactivated the enzyme. The substrate specificity was guaiacol > catechol > tannic acid > gallic acid. There was no peak observed at 610 nm and the ratio of absorbance at 280 nm and 610 was 26. This suggests a yellow laccase. The biochemical properties of A. pullulans NAC8 yellow laccase makes it potentially useful in several biotechnological applications.

Keywords: Aureobasidium pullulans NAC8; Biochemical characterization; Kinetics; Yellow laccase.

Figure 1

Phylogenetic relationship based on homology index for Aureobasidium pullulans NAC8. YY7 represents Aureobasidium pullulans strain YY7, HN2.3 represents Aureobasidium pullulans strain HN2.3, NAC8 represent Aureobasidium pullulans strain NAC8.

Figure 2

Ion-exchange chromatography of laccase from Aureobasidium pullulans NAC8 on DEAE-Sephadex equilibrated with 10 mM Tris–HCl buffer, pH 7.2. 2 ml fractions were collected at a flow rate of 30 ml/h. Bound proteins were eluted stepwise with 1 M NaCl. ---●--- protein concentration at 280 nm. ---■--- laccase activity.

Figure 3

SDS–PAGE of purified laccase from Aureobasidium pullulans.

Figure 4

Wavelengths scan of purified laccase from Aureobasidium pullulans NAC8. The wavelength scan was varied from 250 nm to 800 nm.

Figure 5

Heat stability of laccase from Aureobasidium pullulans. 1 ml of laccase was incubated at different temperatures [45 °C–75 °C] for 2 h. 100 μl was withdrawn at every 30 min interval and assayed for laccase activity and the residual activity was determined under the standard reaction conditions. The activity at zero time was taken as 100%. The residual activity was plotted against the time of incubation.

Figure 6

The pH Stability of A. pullulans laccase.

Figure 7

The organic solvent tolerance of the Aureobasidium pullulans laccase. The effect of organic solvents on laccase activity from A. pullulans was obtained by varying the final concentration in the reaction medium at 10% and 50%. Control experiments contained no organic solvents. The values shown represent the average from triplicate experiments. Error bars represent the standard deviation.