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Cited by 28Enzyme Catalysis and ImmobilizationEnzyme-mediated dye degradationEnzyme Production and Characterization

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A Semi-Rational Approach to Engineering Laccase Enzymes

Miele, Annalisa, Giardina, Paola, Notomista, Eugenio, Piscitelli, Alessandra, Sannia, Giovanni, Faraco, Vincenza·2010·Molecular Biotechnology
DOI 10.1007/s12033-010-9289-y
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Abstract

In order to develop improved laccase-based bio-catalysts, semi-rational mutagenesis of the laccase POXA1b from Pleurotus ostreatus was performed through a combination of directed evolution with elements of rational enzyme modification. The R4 laccase was prepared by joining mutations of previously selected POXA1b random variants. An enhancement of stability features was thus obtained, making the novel enzyme R4 more appropriate as scaffold for directed evolution. A library of 1000 randomly mutated variants of R4 was prepared and screened for the ability of oxidising 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). One of the variants selected (V148L) for improved activity was also proved to show higher stability than R4 at pH 5, and to retain its high stability at pH 7 and 10. In comparison with the POXA1b wild-type laccase, the semi-rational approach allowed us to develop a more efficient bio-catalyst, rising specific activity on ABTS up to around 5-fold. The new variant was also proved to be both more versatile and more durable than the wild-type enzyme, exhibiting higher activity in wide temperature and pH ranges and higher stability at acidic (t (1/2) at pH 5 = 35 days), neutral (t (1/2) at pH 7 = 38 days) and alkaline (t (1/2) at pH 10 = 62 days) pH values.

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  • Laccases have been expressed in hosts including Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Kluyveromyces lactis, Aspergillus niger, Aspergillus sojae, and Trichoderma reesei.

    “Laccases have expressed in hosts as Saccharomyces cerevisiae [228], Yarrowia lipolytica [229], Pichia pastoris [44,204,230,231,232], Kluyveromyces lactis [233] and even filamentous fungi such as Aspergillus niger [234], Aspergillus sojae [235], and Trichoderma reesei [236].”

    From A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases (2021) · cites this paper as [228]

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Research works printed but not linked: 24

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Metadata

Authors
Miele, A., Giardina, P., Notomista, E., Piscitelli, A., Sannia, G., & Faraco, V.
Affiliations
University of Naples Federico II (IT)
Corresponding author
Faraco, Vincenza
ORCID
Venue
Molecular Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2010-05-14
Volume
46
Issue
2
Pages
149-156
ISSN
  • 1073-6085 (ISSN-L)
  • 1559-0305
DOI
10.1007/s12033-010-9289-y
OpenAlex
W1996390274
Cited by
28
References
0
Citations by year
2026: 1 · 2025: 1 · 2024: 2 · 2023: 1 · 2021: 3 · 2019: 1 · 2018: 1 · 2017: 1 · 2016: 1 · 2015: 3 · 2014: 4 · 2013: 4 · 2012: 2
FWCI
1.17
Type
journal article
Language
en
Open access
closed
Licence
www.springer.com/tdm (tdm)
Locations
  • Molecular Biotechnology (publishedVersion)
  • PubMed (publishedVersion)
Funders
Ministero dell'Università e della Ricerca
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Enzyme Catalysis and Immobilization
  • Life Sciences › Immunology and Microbiology › Applied Microbiology and Biotechnology › Enzyme-mediated dye degradation
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
Keywords
laccase, Pleurotus ostreatus, directed evolution, enzyme stability, ABTS oxidation, biocatalysts, pH stability, thermal stability, enzyme engineering
MeSH terms
Enzyme Stability, Escherichia coli, Models, Molecular, Saccharomyces cerevisiae, Substrate Specificity, Sulfonic Acids, Directed Molecular Evolution, Pleurotus, Laccase, Mutant Proteins, Benzothiazoles

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