Identification and biochemical characterisation of an aryl β-glucosidase isolated from a cellulose-acetate rich environment via a functional metagenomic approach

Pieters, Janto (2018-11)

Thesis (MScAgric)--Stellenbosch University, 2018.

Thesis

ENGLISH ABSTRACT: Functional metagenomics has established itself as an indispensable tool in the search for novel genes by accessing the genetic information of previously unculturable microbes. These novel genes are commonly identified by assessing their function through qualitative observations of their general biochemical activities, prior to nucleic acid sequencing. In this study, a fosmid library was created from a cellulose-acetate rich environment (cigarette waste bin) and functionally screened for cellulose degrading and deacetylation enzymes. Here we report on the identification of a novel aryl-β-glucosidase identified from this library. Following plate based functional screening, one putative β-glucosidase was identified (BG4). Next generation sequencing of the 40 Kb fosmid insert identified an open reading frame (ORF) which, contained two distinct glycosyl hydrolase family one domains. The BG4 coding ORF was isolated, cloned into the pSF-OXB20-NH2-10HIS-EKT bacterial expression vector and heterologously expressed in E. coli. While BG4 showed selective hydrolytic activity to β-1,4-glucosidic bonds it displays a natural substrate specificity only to aryl-β-glucosides including arbutin, esculin hydrate, gensitin and salicin. It was most active on esculin hydrate (Km= 5.15 mM, Vmax= 0.28 µmol glucose.min-1 ) and showed a temperature and pH optimum of 40 °C and pH 6. The BG4 activity was stimulated by the presence of manganese chloride and ethyl acetate and inhibited by detergents (SDS, glycerol and Triton X-100) and glucose concentrations exceeding 1.5 mM. The specificity and high activity of BG4 towards aryl-β-glucosides could make it applicable in medical industries to release the biological potent aglycone moiety for glycosylated precursors.

AFRIKAANSE OPSOMMING: Funksionele metagenomika is ‘n onontbreeklike tegniek in die soektog na onbekende gene omdat dit die gentiese inligting van onkweekbare mikrobes beskikbaar stel. Die gene word geïdentifiseer deur kwalitatiewe obserwasie van funksie voordat DNS volgorde beplaing gedoen word. In die studie is ‘n funksionele metagenomiese biblioteek is gemaak vanaf DNS geïsoleer uit ‘n selluloseasetaat ryk omgewing (sigarette stompie asdrom). Dit is daarna geëvalueer vir die teenwoordigheid van sellulose degraderende en deasetilerende gene. Hierdeur is ‘n voorlopige β-glukosidase (BG4) geïdentifiseer. Volgorde bepaling van BG4 fosmid het ‘n moontlike β-glukosidase oopleesraam geïdentifiseer. Die oopleesraam is daarna gekloneer in pSF-OXB20-NH2-10HIS-EKT bakterïele proteïn uitdrukkings vektor wat daarna in E. coli getransformeer is om BG4 te produseer. BG4 het selektiewe hidrolitiese aktiwiteit getoon op β-1,4-glukosiede verbindings en was selektief aktief op ariel-β-glukosied substrate wat arbutin, esculin hidraat, gensitin en salicin insluit. BG4 was mees aktief op esculin hidraat (Km= 5.15 mM, Vmax= 0.28 µmol glucose.min-1 ) en het optimale aktiwiteit getoon by pH 6 en 40 °C. BG4 akiwiteit was gesimuleer deur mangaan chloried en etielasetaat en sterk geïnhibeer deur SDS en glukose bo 1.5 mM. Die hoë aktiwiteit en spesifisiteit van BG4 teenoor ariel-β-glukosiede verleen dit aan moonltike toepassing vir die vrystelling van biologiese aktiewe molekules vanaf hulle onaktiewe, geglukosiseerde voorlopers.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/105187
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