Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060

Yasuhisa Fukuda, Asuka Abe, Takashi Tamura, Takahide Kishimoto, Atsushi Sogabe, Satoshi Akanuma, Shin Ichi Yokobori, Akihiko Yamagishi, Katsumi Imada, Kenji Inagaki

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    Abstract

    Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.

    Original languageEnglish
    Pages (from-to)497-502
    Number of pages6
    JournalJournal of Bioscience and Bioengineering
    Volume121
    Issue number5
    DOIs
    Publication statusPublished - 2016 May 1

    Keywords

    • Ancestral mutation
    • Cellulomonas
    • Diagnostic enzyme
    • Epistasis effect
    • Glycerol kinase
    • Thermostabilization

    ASJC Scopus subject areas

    • Biotechnology
    • Applied Microbiology and Biotechnology
    • Bioengineering

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