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.
Highlights
Laccases are blue multicopper oxidases (EC 1.10.3.2), catalyzing oxidation reactions for an array of compounds such as diphenols, polyphenols, deamines, aromatic amines, inorganic compounds, and nonphenolic compounds in the presence of redox mediators
The strain was P. pastoris X33 containing pGAPZαA-LacGluc-Stop (Clone 1) expression vector with previously optimized synthetic gene GILCC1 coding for Ganoderma lucidum GILCC1 laccase
Pichia pastoris X33/pGAPZαALaccGluc-Stop clone 1 Master Cell Bank (MCB) [21] was thawed and used for inoculating 5 media volume (mL) screw cap tubes with sterile YPD supplemented with 40 μg mL−1 zeocin (Z)
Summary
Laccases are blue multicopper oxidases (EC 1.10.3.2), catalyzing oxidation reactions for an array of compounds such as diphenols, polyphenols, deamines, aromatic amines, inorganic compounds, and nonphenolic compounds in the presence of redox mediators. During the reaction the substrate is oxidized by donating its electron, where molecular oxygen acts as an electron acceptor and is reduced into water [1,2,3]. Laccases are enzymes mainly produced in white rot fungi; they are widely distributed in plants, insects, fungi, and bacteria [1, 4,5,6]. They have important applications in different industrial settings, as they help to reduce the environmental impact of their waste. Heterologous expression is a promising option for greater scale production, using the potential of hosts that are easy to handle and culture, such as bacteria and yeast [12]
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