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

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

Research output: Contribution to journalArticleResearchpeer-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.

LanguageEnglish
Pages769-781
Number of pages13
JournalJournal of Industrial Microbiology and Biotechnology
Volume46
Issue number6
Early online date26 Feb 2019
DOIs
Publication statusPublished - Jun 2019

Fingerprint

Screening
Enzymes
Euglena gracilis
Assays
Escherichia coli
Degradation
Glucan 1,3-beta-Glucosidase
Flow cytometry
Congo Red
Substrates
Biofuels
Amino acids
Fluorescein
Gene Library
Fluorescent Dyes
Proteomics
Dyes
Open Reading Frames
paramylon
Amino Acid Sequence

Keywords

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

Cite this

@article{10b7d4dc0b174049813d78e935390568,
title = "Development of screening strategies for the identification of paramylon-degrading enzymes",
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.",
keywords = "Euglena gracilis, Paramylon degradation, Fluorescence-activated cell sorting, Proteomics, Congo Red plate assay",
author = "Alexander Gissibl and Andrew Care and Yu-hsin Sun and Graham Hobba and Helena Nevalainen and Anwar Sunna",
year = "2019",
month = "6",
doi = "10.1007/s10295-019-02157-7",
language = "English",
volume = "46",
pages = "769--781",
journal = "Journal of Industrial Microbiology and Biotechnology",
issn = "1367-5435",
publisher = "Springer, Springer Nature",
number = "6",

}

Development of screening strategies for the identification of paramylon-degrading enzymes. / Gissibl, Alexander; Care, Andrew; Sun, Yu-hsin; Hobba, Graham; Nevalainen, Helena; Sunna, Anwar.

In: Journal of Industrial Microbiology and Biotechnology, Vol. 46, No. 6, 06.2019, p. 769-781.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Development of screening strategies for the identification of paramylon-degrading enzymes

AU - Gissibl, Alexander

AU - Care, Andrew

AU - Sun, Yu-hsin

AU - Hobba, Graham

AU - Nevalainen, Helena

AU - Sunna, Anwar

PY - 2019/6

Y1 - 2019/6

N2 - 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.

AB - 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.

KW - Euglena gracilis

KW - Paramylon degradation

KW - Fluorescence-activated cell sorting

KW - Proteomics

KW - Congo Red plate assay

UR - http://www.scopus.com/inward/record.url?scp=85062455785&partnerID=8YFLogxK

UR - http://purl.org/au-research/grants/arc/IC130100009

U2 - 10.1007/s10295-019-02157-7

DO - 10.1007/s10295-019-02157-7

M3 - Article

VL - 46

SP - 769

EP - 781

JO - Journal of Industrial Microbiology and Biotechnology

T2 - Journal of Industrial Microbiology and Biotechnology

JF - Journal of Industrial Microbiology and Biotechnology

SN - 1367-5435

IS - 6

ER -