Development of screening strategies for the identification of paramylon-degrading enzymes

Alexander Gissibl, Andrew Care, Yu-hsin Sun, Graham Hobba, Helena Nevalainen, Anwar Sunna*

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Enzymatic degradation of the β-1,3-glucan paramylon could enable the production of bioactive compounds for healthcare and renewable substrates for biofuels. However, few enzymes have been found to degrade paramylon efficiently and their enzymatic mechanisms remain poorly understood. Thus, the aim of this work was to find paramylon-degrading enzymes and ways to facilitate their identification. Towards this end, a Euglena gracilis-derived cDNA expression library was generated and introduced into Escherichia coli. A flow cytometry-based screening assay was developed to identify E. gracilis enzymes that could hydrolyse the fluorogenic substrate fluorescein di-β-d-glucopyranoside in combination with time-saving auto-induction medium. In parallel, four amino acid sequences of potential E. gracilis β-1,3-glucanases were identified from proteomic data. The open reading frame encoding one of these candidate sequences (light_m.20624) was heterologously expressed in E. coli. Finally, a Congo Red dye plate assay was developed for the screening of enzyme preparations potentially able to degrade paramylon. This assay was validated with enzymes assumed to have paramylon-degrading activity and then used to identify four commercial preparations with previously unknown paramylon degradation ability.

    Original languageEnglish
    Pages (from-to)769-781
    Number of pages13
    JournalJournal of Industrial Microbiology and Biotechnology
    Volume46
    Issue number6
    Early online date26 Feb 2019
    DOIs
    Publication statusPublished - Jun 2019

    Keywords

    • Euglena gracilis
    • Paramylon degradation
    • Fluorescence-activated cell sorting
    • Proteomics
    • Congo Red plate assay

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