Direct cloning and expression of putative esterase genes from environmental DNA

Takeshi Terahara, Kazutaka Yamada, Shinya Kurata, Toyokazu Yokomaku, Satoshi Tsuneda, Shigeaki Harayama

    Research output: Contribution to journalArticle

    6 Citations (Scopus)

    Abstract

    Putative esterase genes were isolated from environmental DNA by using pre-amplified inverse PCR. The sequence analysis of the isolated genes showed 32-80% amino acid sequence identities to known esterases/lipases in public databases. The isolated genes were subsequently expressed in recombinant Escherichia coli. Insoluble proteins were noted in the expression of the majority of the isolated genes. The findings suggest that it is difficult to isolate these genes by using activity-based screening with construction of metagenome library. For the enzymes characterized, we examined substrate specificity, optimal temperature, optimal pH, and thermal stability. The substrate specificity of all the enzymes was high for p-nitrophenyl acetate, but almost undetectable for p-nitrophenyl decanoate. The results indicate that the obtained enzymes are defined as esterases. The enzymes were active in a broad range of temperature. The optimum activity was observed at 25-70°C and at pH 8.0-9.0. Some enzymes have moderate thermostability and would be useful for industrial enzymes. This study illustrates that pre-amplified inverse PCR, which is one of the sequence-based approach, is potentially applicable to the isolation of diverse genes from environmental DNA.

    Original languageEnglish
    Pages (from-to)17-23
    Number of pages7
    JournalEnzyme and Microbial Technology
    Volume47
    Issue number1-2
    DOIs
    Publication statusPublished - 2010 Jul

    Fingerprint

    Cloning
    Esterases
    Organism Cloning
    DNA
    Enzymes
    Genes
    Substrate Specificity
    Metagenome
    Decanoates
    Polymerase Chain Reaction
    Temperature
    Lipases
    Substrates
    Lipase
    Escherichia coli
    Sequence Analysis
    Amino acids
    Amino Acid Sequence
    Screening
    Thermodynamic stability

    Keywords

    • Esterase
    • Inverse PCR
    • Metagenome
    • Sequence-based approach

    ASJC Scopus subject areas

    • Biochemistry
    • Biotechnology
    • Applied Microbiology and Biotechnology

    Cite this

    Direct cloning and expression of putative esterase genes from environmental DNA. / Terahara, Takeshi; Yamada, Kazutaka; Kurata, Shinya; Yokomaku, Toyokazu; Tsuneda, Satoshi; Harayama, Shigeaki.

    In: Enzyme and Microbial Technology, Vol. 47, No. 1-2, 07.2010, p. 17-23.

    Research output: Contribution to journalArticle

    Terahara, Takeshi ; Yamada, Kazutaka ; Kurata, Shinya ; Yokomaku, Toyokazu ; Tsuneda, Satoshi ; Harayama, Shigeaki. / Direct cloning and expression of putative esterase genes from environmental DNA. In: Enzyme and Microbial Technology. 2010 ; Vol. 47, No. 1-2. pp. 17-23.
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