Novel Cold-Adapted Recombinant Laccase KbLcc1 from Kabatiella bupleuri G3 IBMiP as a Green Catalyst in Biotransformation

Abstract

Cold-adapted enzymes are useful tools in the organic syntheses conducted in mixed aqueous-organic or non-aqueous solvents due to their molecular flexibility that stabilizes the proteins in low water activity environments. A novel psychrophilic laccase gene from Kabatiella bupleuri, G3 IBMiP, was spliced by Overlap‑Extension PCR (OE-PCR) and expressed in Pichia pastoris. Purified recombinant KbLcc1 laccase has an optimal temperature of 30 °C and pH of 3.5, 5.5, 6.0, and 7.0 in the reaction with 2,2′‑azino‑bis(3‑ethylbenzothiazoline‑6‑sulfonic acid) (ABTS), guaiacol, sinapic acid, and syringaldazine, respectively. Moreover, laccase KbLcc1 is highly thermolabile, as it loses 40% of activity after 30 min at 40 °C and is inactivated at 50 °C after the same period of incubation. The new enzyme remained active with 1 mM of Ni2+, Cu2+, Mn2+, and Zn2+ and with 2 mM of Co2+, Ca2+, and Mg2+, but Fe2+ greatly inhibited the laccase activity. Moreover, 1% ethanol had no impact on KbLcc1, although acetone and ethyl acetate decreased the laccase activity. The presence of hexane (40%, v/v) caused a 58% increase in activity. Laccase KbLcc1 could be applied in the decolorization of synthetic dyes and in the biotransformation of ferulic acid to vanillin. After 5 days of reaction at 20 °C, pH 3.5, with 1 mM ABTS as a mediator, the vanillin concentration was 21.9 mg/L and the molar yield of transformation reached 14.39%.

Expression of a deep-sea bacterial laccase from Halomonas alkaliantarctica and its application in dyes decolorization

“…In the presence of 5% ethanol, the laccase activity was inhibited, and the enzyme activity decreased to 77.35%. Conversely, 1% ethanol had no effect on KbLcc1, although acetone and ethyl acetate reduced laccase activity ( Wiśniewska KM et al,2021).…”

“…In the presence of K + , laccase activity was the lowest, retaining 43.21% of its activity. Metal ions have different effects on different types of laccases, such as Kabatiella bupleuri G3 IBMi (Wiśniewska KM et al,2021) and A. ayderensis SK3-4 (Fang ZM et al,2011). KbLcc1 remained active at 1 mM Ni 2+ , Cu 2+ , Mn 2+ and Zn 2+ and 2 mM Co 2+ , Ca 2+ and Mg 2 , but Fe 2+ greatly inhibited laccase activity (Wiśniewska KM et al,2021).…”

“…Metal ions have different effects on different types of laccases, such as Kabatiella bupleuri G3 IBMi (Wiśniewska KM et al,2021) and A. ayderensis SK3-4 (Fang ZM et al,2011). KbLcc1 remained active at 1 mM Ni 2+ , Cu 2+ , Mn 2+ and Zn 2+ and 2 mM Co 2+ , Ca 2+ and Mg 2 , but Fe 2+ greatly inhibited laccase activity (Wiśniewska KM et al,2021). The inhibitory effect of Fe 2+ may be due to its interaction with the laccase electron transport system.…”

An Engineered Thermostable Laccase with Great Ability to Decolorize and Detoxify Malachite Green

“…During the subsequent electron transferring, oxygen is reduced to water at the trinuclear center formed by Type 2 and 3 copper [2]. Thanks to their broad substrate specificity and the green reaction only requiring oxygen and releasing water as the sole by-product, laccases have been applied for industrial use such as delignification to improve biomass saccharification [3], biobleaching [4], degradation of environmental pollutants [5], and decolorization and detoxification of dyes [6,7].…”