Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome

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dc.contributor.authorFortune, Brent
dc.contributor.authorMhlongo, Sizwe
dc.contributor.authorvan Zyl, Leonardo J
dc.contributor.authorHuddy, Robert
dc.contributor.authorSmart, Mariette
dc.contributor.authorTrindade, Marla
dc.date.accessioned2019-06-04T07:35:02Z
dc.date.available2019-06-04T07:35:02Z
dc.date.issued2019-04-18
dc.date.updated2019-04-21T03:17:32Z
dc.description.abstractBackground: The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. Results Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0–6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s− 1 and 219 s− 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside. Conclusion All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study.
dc.identifier.apacitationFortune, B., Mhlongo, S., van Zyl, L. J., Huddy, R., Smart, M., & Trindade, M. (2019). Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome. <i>BMC Biotechnology</i>, http://hdl.handle.net/11427/30190en_ZA
dc.identifier.chicagocitationFortune, Brent, Sizwe Mhlongo, Leonardo J van Zyl, Robert Huddy, Mariette Smart, and Marla Trindade "Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome." <i>BMC Biotechnology</i> (2019) http://hdl.handle.net/11427/30190en_ZA
dc.identifier.citationFortune, B., Mhlongo, S., van Zyl, L. J., Huddy, R., Smart, M., & Trindade, M. (2019). Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome. BMC biotechnology, 19(1), 22.
dc.identifier.ris TY - AU - Fortune, Brent AU - Mhlongo, Sizwe AU - van Zyl, Leonardo J AU - Huddy, Robert AU - Smart, Mariette AU - Trindade, Marla AB - Background: The importance of the accessory enzymes such as α-L-arabinofuranosidases (AFases) in synergistic interactions within cellulolytic mixtures has introduced a paradigm shift in the search for hydrolytic enzymes. The aim of this study was to characterize novel AFase genes encoding enzymes with differing temperature optima and thermostabilities for use in hydrolytic cocktails. Results Three fosmids, pFos-H4, E3 and D3 were selected from the cloned metagenome of high temperature compost, expressed in Escherichia coli and subsequently purified to homogeneity from cell lysate. All the AFases were clustered within the GH51 AFase family and shared a homo-hexameric structure. Both AFase-E3 and H4 showed optimal activity at 60 °C while AFase-D3 had unique properties as it showed optimal activity at 25 °C as well as the ability to maintain substantial activity at temperatures as high as 90 °C. However, AFase-E3 was the most thermostable amongst the three AFases showing full activity even at 70 °C. The maximum activity was observed at a pH profile between pH 4.0–6.0 for all three AFases with optimal activity for AFase H4, D3 and E3 at pH 5.0, 4.5 and 4.0, respectively. All the AFases showed KM range between 0.31 mM and 0.43 mM, Kcat range between 131 s− 1 and 219 s− 1 and the specific activity for AFase-H4, AFases-E3 and was 143, 228 and 175 U/mg, respectively. AFases-E3 and D3 displayed activities against pNP-β-L-arabinopyranoside and pNP-β-L-mannopyranoside respectively, and both hydrolysed pNP-β-D-glucopyranoside. Conclusion All three AFases displayed different biochemical characteristics despite all showing conserved overall structural similarity with typical domains of AFases belonging to GH51 family. The hydrolysis of cellobiose by a GH51 family AFase is demonstrated for the first time in this study. DA - 2019-04-18 DB - OpenUCT DP - University of Cape Town J1 - BMC Biotechnology KW - Thermostability KW - Arabinofuranosidase KW - Compost KW - Metagenomics LK - https://open.uct.ac.za PY - 2019 T1 - Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome TI - Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome UR - http://hdl.handle.net/11427/30190 ER - en_ZA
dc.identifier.urihttps://doi.org/10.1186/s12896-019-0510-1
dc.identifier.urihttp://hdl.handle.net/11427/30190
dc.identifier.vancouvercitationFortune B, Mhlongo S, van Zyl LJ, Huddy R, Smart M, Trindade M. Characterisation of three novel α-L-arabinofuranosidases from a compost metagenome. BMC Biotechnology. 2019; http://hdl.handle.net/11427/30190.en_ZA
dc.language.rfc3066en
dc.rights.holderThe Author(s).
dc.sourceBMC Biotechnology
dc.source.urihttps://bmcbiotechnol.biomedcentral.com/
dc.subjectThermostability
dc.subjectArabinofuranosidase
dc.subjectCompost
dc.subjectMetagenomics
dc.titleCharacterisation of three novel α-L-arabinofuranosidases from a compost metagenome
dc.typeJournal Article
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