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

Journal of Industrial Microbiology & Biotechnology
Alexander GissiblAnwar Sunna

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...Continue Reading

References

Oct 5, 1990·Journal of Molecular Biology·S F AltschulD J Lipman
Nov 4, 1969·Biochimica Et Biophysica Acta·D R Barras, B A Stone
Nov 1, 1968·The Journal of protozoology·K Vogel, A A Barber
Jun 26, 2003·Nucleic Acids Research·Elisabeth GasteigerAmos Bairoch
Jun 1, 1964·Experimental Cell Research·D B MALKOFF, D E BUETOW
Oct 14, 2004·European Journal of Biochemistry·Ulrich NowitzkiWilliam Martin
Oct 27, 2004·Bioscience, Biotechnology, and Biochemistry·Rika NobeMasahito Suiko
Dec 15, 2004·Journal of Proteome Research·Robertson CraigRonald C Beavis
Nov 30, 2006·Cellular and Molecular Life Sciences : CMLS·H Hashimoto
Aug 10, 2007·Journal of Agricultural and Food Chemistry·Gajendra Sunnamada NaikaVishweshwaraiah Prakash
Aug 30, 2008·Annual Review of Microbiology·Piet Borst, Robert Sabatini
Jun 12, 2009·Journal of Hematology & Oncology·Godfrey Chi-Fung ChanDaniel Man-Yuen Sze
Oct 17, 2009·Applied Biochemistry and Biotechnology·Elizabeth HardimanPeter Bergquist
Jul 6, 2010·New Biotechnology·M D GibbsP L Bergquist
Jul 16, 2010·Biotechnology Advances·Solange I MussattoJosé A Teixeira
Sep 22, 2010·Journal of Applied Microbiology·J S Rodríguez-ZavalaR Moreno-Sánchez
Jan 18, 2011·Natural Product Reports·Laura BarsantiPaolo Gualtieri
Oct 19, 2012·Biotechnology Letters·Hassan Ahmadi GavlighiJ Dalgaard Mikkelsen
Nov 14, 2012·Analytical Biochemistry·Raluca OstafeRainer Fischer
Dec 15, 2012·New Biotechnology·Anwar SunnaPeter L Bergquist
Feb 16, 2013·Enzyme and Microbial Technology·Nongnuch SutivisedsakStephen W Peterson
Nov 10, 2013·Methods in Molecular Biology·F William Studier
Nov 26, 2013·Nucleic Acids Research·Vincent LombardBernard Henrissat
Jan 1, 2012·Computational and Structural Biotechnology Journal·Maria DimarogonaPaul Christakopoulos
Jul 16, 2015·Preventive Nutrition and Food Science·Sang Hoon LeeHeon Sang Jeong
Dec 3, 2016·Nucleic Acids Research·Aron Marchler-BauerStephen H Bryant
Apr 20, 2017·FEBS Letters·Yuji TanakaTakahiro Ishikawa
Apr 27, 2017·Scientific Reports·Valentina CappelloMauro Gemmi
Nov 8, 2017·Carbohydrate Polymers·Darrel Sarvesh NaiduMaya Jacob John
Jan 11, 2018·The Journal of Biological Chemistry·Sakonwan KuhaudomlarpRobert A Field

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Methods Mentioned

BETA
fluorescence-activated cell sorting
FACS
Fluorescence
electrophoreses
size-exclusion chromatography

Software Mentioned

ExPASy
Compute pI / M W tool
BLAST
Global Proteome Machine

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