Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation
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 <i>Kabatiella bupleuri</i>...
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MDPI AG
2021-09-01
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| author | Katarzyna M. Wiśniewska Aleksandra Twarda-Clapa Aneta M. Białkowska |
| author_facet | Katarzyna M. Wiśniewska Aleksandra Twarda-Clapa Aneta M. Białkowska |
| author_sort | Katarzyna M. Wiśniewska |
| collection | DOAJ |
| description | 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 <i>Kabatiella bupleuri</i>, G3 IBMiP, was spliced by Overlap-Extension PCR (OE-PCR) and expressed in <i>Pichia pastoris</i>. 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 Ni<sup>2+</sup>, Cu<sup>2+</sup>, Mn<sup>2+</sup>, and Zn<sup>2+</sup> and with 2 mM of Co<sup>2+</sup>, Ca<sup>2+</sup>, and Mg<sup>2+</sup>, but Fe<sup>2+</sup> 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%, <i>v</i>/<i>v</i>) 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%. |
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| language | English |
| last_indexed | 2024-03-10T08:10:12Z |
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| spelling | doaj.art-de769f321f2144f283f2e2314a6a4d7f2023-11-22T10:45:58ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-09-012217959310.3390/ijms22179593Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in BiotransformationKatarzyna M. Wiśniewska0Aleksandra Twarda-Clapa1Aneta M. Białkowska2Institute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego 2/22, 90-537 Łódź, PolandInstitute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego 2/22, 90-537 Łódź, PolandInstitute of Molecular and Industrial Biotechnology, Lodz University of Technology, Stefanowskiego 2/22, 90-537 Łódź, PolandCold-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 <i>Kabatiella bupleuri</i>, G3 IBMiP, was spliced by Overlap-Extension PCR (OE-PCR) and expressed in <i>Pichia pastoris</i>. 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 Ni<sup>2+</sup>, Cu<sup>2+</sup>, Mn<sup>2+</sup>, and Zn<sup>2+</sup> and with 2 mM of Co<sup>2+</sup>, Ca<sup>2+</sup>, and Mg<sup>2+</sup>, but Fe<sup>2+</sup> 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%, <i>v</i>/<i>v</i>) 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%.https://www.mdpi.com/1422-0067/22/17/9593psychrophilic laccase<i>Kabatiella bupleuri</i>recombinant enzyme<i>Pichia pastoris</i>decolorizationbiotransformation |
| spellingShingle | Katarzyna M. Wiśniewska Aleksandra Twarda-Clapa Aneta M. Białkowska Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation International Journal of Molecular Sciences psychrophilic laccase <i>Kabatiella bupleuri</i> recombinant enzyme <i>Pichia pastoris</i> decolorization biotransformation |
| title | Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation |
| title_full | Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation |
| title_fullStr | Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation |
| title_full_unstemmed | Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation |
| title_short | Novel Cold-Adapted Recombinant Laccase KbLcc1 from <i>Kabatiella bupleuri</i> G3 IBMiP as a Green Catalyst in Biotransformation |
| title_sort | novel cold adapted recombinant laccase kblcc1 from i kabatiella bupleuri i g3 ibmip as a green catalyst in biotransformation |
| topic | psychrophilic laccase <i>Kabatiella bupleuri</i> recombinant enzyme <i>Pichia pastoris</i> decolorization biotransformation |
| url | https://www.mdpi.com/1422-0067/22/17/9593 |
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