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

Abstract

Laccases are multicopper oxidases that catalyze aromatic and nonaromatic compounds with concomitant reduction of molecular oxygen to water. They are of great interest due to their potential biotechnological applications. In this work we statistically improved culture media for recombinant GILCC1 (rGILCC1) laccase production at low scale from Ganoderma lucidum containing the construct pGAPZαA-GlucPost-Stop in Pichia pastoris. Temperature, pH stability, and kinetic parameter characterizations were determined by monitoring concentrate enzyme oxidation at different ABTS substrate concentrations. Plackett-Burman Design allowed improving enzyme activity from previous work 36.08-fold, with a laccase activity of 4.69 ± 0.39 UL−1 at 168 h of culture in a 500 mL shake-flask. Concentrated rGILCC1 remained stable between 10 and 50°C and retained a residual enzymatic activity greater than 70% at 60°C and 50% at 70°C. In regard to pH stability, concentrated enzyme was more stable at pH 4.0 ± 0.2 with a residual activity greater than 90%. The lowest residual activity greater than 55% was obtained at pH 10.0 ± 0.2. Furthermore, calculated apparent enzyme kinetic parameters were a Vmax of 6.87 × 10−5 mM s−1, with an apparent Km of 5.36 × 10−2 mM. Collectively, these important stability findings open possibilities for applications involving a wide pH and temperature ranges.

A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases

“…Concerning to the pH they detected that it varied considerably, preserving 3%, 7%, and 28% of its relative activity at pH 3, 4 and 5, respectively, after 4 h of exposure [208]. For Ganoderma lucidum rGlCC1 expressed in P. pastoris, thermodynamic stability analyses performed during 1 h of exposure at different temperatures it was found that preserve its relative activity between 75 and 100%, in pH and temperature ranges from 2 • C to 11 • C and 10 • C to 60 • C, respectively [209]. Bao et al, (2013) found that laccase lac1 from Coprinus comatus maintained a residual activity of 51% after one hour at 60 • C. In terms of best pH for enzyme reaction, it varied depending on the substrate; resulting in pH of 3.0, 6.0, 5.0 and 6.0 for ABTS (2,2'azino bis (3-ethylbenzothiazolin-6-sulfonate)), guaiacol, DMP (2,6 dimethoxyphenol), and syringaldazine (SZ), respectively [210].…”

Recombinant laccase rPOXA 1B real-time, accelerated and molecular dynamics stability study

“…For the enzyme kinetic assay ABTS dissolved in 0.1 M citrate buffer, was used as a substrate (concentration between 0.1–3 mM), pH 3.0 ± 0.2. An enzyme solution with an activity of 10 UL −1 at 25 °C [ 46 ]. All kinetic tests performed in triplicate.…”

Process simulation-integrated optimization of lignocellulolytic enzyme production

“…Laccase. Lac activity was measured as described elsewhere [30] with following modifications: 15 mM ABTS (2,2′-Azino-bis(3ethylbenzothiazoline-6-sulfonic acid) diammonium salt was used as substrate. 0.950 ml citrate buffer, 0.05 ml enzyme solution (if dilution was needed, final enzyme volume was kept as 0.050 ml), 0.2 ml ABTS (dissolved in 0.1 M sodium citrate buffer (pH 3.0)) solution were mixed in spectrophotometric cuvette at room temperature for 3 min and absorbance was read at 420 nm right after timeout.…”