Armstrong, Zachary

Briers, Yves

João de Carvalho Maurício da Fonseca, Maria

Withers, Stephen G

de Carvalho Maurício da Fonseca, Maria João

Ghent University Academic Bibliography

High-throughput substrate specificity analysis of metagenomic-derived arabinoxylan-active enzymesMaria João Maurício da Fonseca1, Zachary Armstrong2, Stephen G. Withers2, Yves Briers11 Department of Biotechnology, Ghent University, Ghent, Belgium2 Department of Chemistry, University of British Columbia, Vancouver, CanadaContact: MariaJoao.Fonseca@ugent.be Functional enzyme screening of the increasing amount of metagenomic data is oftencumbersome, especially for the discovery of enzymes with complex substratespecificities such as arabinoxylan-active enzymes. We present here our high-throughput approach based on DNA sequencer-aided fluorophore-assistedcarbohydrate electrophoresis (DSA-FACE) for the parallel analysis of carbohydratespecificities of putative arabinoxylan-active enzymes present in metagenomics data(Fig. 1).INTRODUCTIONAEXPERIMENTAL DATABCONCLUSIONSC ENVIRONMENTAL SAMPLE COLLECTIONGDNA EXTRACTIONMETAGENOMIC DNA LIBRARIESHT PROTEIN EXPRESSION AND PURIFICATIONSCREENING WITH CHROMOGENIC/FLUOROGENICSUBSTRATESHPAEC-PADMetagenomic sampling1Functional activitypre-screening2XXXXX XXXX XXX XXsubstrateenzyme 1enzyme 2enzyme 3substrateenzyme 1enzyme 2enzyme 3SPECIFICITY SCANSsubstrateenzyme 1enzyme 2enzyme 3HT ENZYMATIC REACTIONSsubstrate blanksenzyme blanks5 XOS 7 AXOS3 enzymes, 2 enzyme concentrationsDSA-FACE ELECTROPHEROGRAMSDegradation maps4Specificity scans3Fig 1. Blueprint to discover substrate specificities of new metagenomic putative arabinoxylan-active enzymes with DSA-FACE. Environmental DNA is extracted from natural sources and used toconstruct high molecular weight DNA libraries (fosmids) or small insert DNA libraries. After DNA librariesscreening and sequencing, interesting protein coding sequences are cloned and expressed in parallel.Enzymatic reactions with representative arabino(xylan)-oligosaccharides ((A)XOS) are done and analysed byHPAEC-PAD upon prediction of enzymatic activity with chromogenic/fluorogenic substrates. To be able toreveal complete specificity profiles, parallel enzymatic reactions are done with (A)XOS with differentsubstitution profiles. For example, in a 96-well plate 5 XOS and 7 AXOS are tested with 3 different enzymes at2 different enzyme concentrations. Substrate and enzyme blanks must be added for posterior interpretationof DSA-FACE electropherograms. Based on electrophoretic mobilities and peak areas, DSA-FACEelectropherograms are converted into specificity scans showing reaction products formed and allowingcomparison of activities of different enzymes or enzyme concentrations on a substrate. From specificityscans, degradation maps per enzyme are inferred allowing to better understand substrate specificitypathways.1234SPECIFICITY SCANS FOR GH43_28 FAMILY MEMBER:DEGRADATION MAPS FOR GH43_28 FAMILY MEMBER:Proposed enzymatic reaction❖ By DSA-FACE we could rapidly perform specificity scans for GH43_28 andrepresentative (A)XOS at different enzyme concentrations.❖ Based on specificity scans, it seems the preferred substrate for GH43_28 is A2XX. Atlow concentration, GH43_28 behaves as a α-L-arabinofuranosidase being able toremove O-2 arabinofuranosyl substitutions from mono-substituted A2XX and O-2and O-3 arabinofuranosyl substitutions from A2+3XX when there is no xylose at thenon-reducing end.❖ At higher concentrations GH43_28 shows endo-xylanase activity.❖ GH43_28 seems to be inhibited by non-reducing xylose monomers.xylose arabinose++++s++++++ ++++s++++++ sXA2+3XXXA3XXA2+3XXA2XXA3XXXXXXXXXXXXXXXXXXXXX++++s++++++s - substrate+ - 0,02 mg/mL++ - 0,1 mg/mL+++ - 0,4 mg/mL++++ - 2,2 mg/mL++++s++++++ ++++s++++++ ++++s++++++ ++++s++++++ ++++s++++++ ++++s++++++ ++++++++++A metagenomic study on the North American beaver (Castor canadensis) fecesrevealed the presence of a putative arabinoxylan-active enzyme fromuncharacterized subfamily 28 of the GH43 CAZy family. Specificity scans were donefor 4 enzyme concentrations and for 5 XOS and 5 AXOS.

High-throughput substrate specificity analysis of metagenomic-derived arabinoxylan-active enzymes

https://biblio.ugent.be/publication/8617235/file/8617236.pdf

High-throughput substrate specificity analysis of metagenomic-derived arabinoxylan-active enzymes

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