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%.
Highlights
IntroductionLaccases (EC 1.10.3.2, benzenediol:oxygen oxidoreductases) are multicopper oxidases which catalyze the oxidation of a broad range of substrates, e.g., phenolic and polyphenolic compounds, polyamines, aryl diamines, aminophenols, and lignins
The results described in this study, observed for the KbLcc1 laccase, are the first to demonstrate the ability of the laccaselike enzyme to produce vanillin from ferulic acid
Range, and organic solvent tolerance) which predetermine its application in organic synthesis reactions
Summary
Laccases (EC 1.10.3.2, benzenediol:oxygen oxidoreductases) are multicopper oxidases which catalyze the oxidation of a broad range of substrates, e.g., phenolic and polyphenolic compounds, polyamines, aryl diamines, aminophenols, and lignins. Depending on the value of redox potential (RP), two groups of laccases are distinguished, namely low RP (0.4–0.6 V) and high RP (0.6–0.8 V) [1]. When the redox potential of laccase is lower than the RP of the substrate, the enzyme cannot oxidize it. It is necessary to use small molecules capable to act as electron transfer mediators, such as 2,20 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybanzotriazole (HBT). Mediators added to reaction mixtures enhance the use of these enzymes for industrial processes, including the detoxification of industrial dyes, delignification of lignocellulosic biomass, and bioremediation of xenobiotic compounds, polycyclic aromatic hydrocarbons, and pesticides [2]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
