Characterization of a thermostable mutant of Agaricus brasiliensis laccase created by phylogeny-based design

Yuhi Hamuro, Katsuya Tajima, Akiko Matsumoto-Akanuma, Sayaka Sakamoto, Ryutaro Furukawa, Akihiko Yamagishi, Naohito Ohno, Satoshi Akanuma*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)


Laccases are enzymes that oxidize various aromatic compounds, and therefore they have attracted much attention from the standpoints of medical and industrial applications. We previously isolated the cDNA that codes for a laccase isozyme (Lac2a) from the medicinal mushroom Agaricus brasiliensis (Matsumoto-Akanuma et al., Int. J. Med. Mushrooms, 16, 375–393, 2014). In this study, we first attempted heterologous expression of the wild-type laccase using a Pichia pastoris secretory expression system. However, the trial was unsuccessful most likely because the enzyme was too unstable and degraded immediately after production. Therefore, we improved the stability of the laccase by using a phylogeny-based design method. We created a mutant laccase in which sixteen original residues were replaced with those found in the phylogenetically inferred ancestral sequence. The resulting mutant protein was successfully produced using the P. pastoris secretory expression system and then purified. The designed laccase showed catalytic properties similar to those of other fungal laccases. Moreover, the laccase is highly thermally stable at acidic and neutral pH and is also stable at alkaline pH at moderate temperatures. We expect that the laccase will serve as a useful tool for enzymatic polymerization of di-phenolic compounds.

Original languageEnglish
Pages (from-to)623-629
Number of pages7
JournalJournal of Bioscience and Bioengineering
Issue number6
Publication statusPublished - 2017 Dec


  • Agaricus brasiliensis
  • Ancestral sequence
  • Fungal laccase
  • Heterologous expression
  • Multiple amino acid substitutions
  • Phylogenetic tree
  • Pichia pastoris
  • Polymerized polyphenol
  • Stable enzyme

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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