Thesis (MSc)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: Esterases play a variety of roles in nearly every aspect of life ranging from cellular
metabolism, signal transduction to defence mechanisms in plants. One aspect of
esterases that recently is receiving more attention is the role esterases play in the
.degradation of plant material. With fossil fuels (coal and oil) estimated to run out in
the next 20 to 30 years, renewable sources such as plant biomass are becoming
increasingly important. Plant biomass contains hemicellulosic and cellulosic
materials that need to be degraded to their different constituents before they can be
optimally used for the production of commodities.
Although the enzymes needed to hydrolyse the xylan backbone (xylanases and
P-xylosidases) are important, enzymes that remove side chains from the polymer are
equally important. They facilitate hydrolysis by xylanases and P-xylosidases and will
improve the availability of monomeric sugars for utilisation when used in conjunction
with other xylanolytic enzymes. Many of these side-chains are esters and they need
to be removed through the action of esterases, either directly from the xylan backbone
or from shorter xylo-oligomers.
An existing genomic DNA library of Bacil/us pumilus in Escherichia coli was
screened for the presence of an acetyl esterase encoding gene. Positive clones were
identified by the formation of clearing zones on plates containing glucose
pentaacetate. Plasmid DNA was isolated from a positive E. coli clone. The DNA
insert was sequenced and found to contain two open reading frames, one of which
encoded a novel esterase (estA). Using different primers the gene was amplified by polymerase chain reaction and inserted into an inducible expression vector (pKK223-
3) for expression in E. coli. The plasmid was introduced into E coli and the esterase
activity determined, using the chromogenic substrate a-naphthyl acetate. Activity
levels decreased shortly after induction with IPTG and therefore plasmid pAP4 was
used for enzymatic assays. Cultures containing plasmid pAP4 produced extracellular
activity of 2.5 nkatal/ml. The pH and temperature optima as well as temperature
stability of the enzyme was determined. The enzyme exhibited optimal activity at pH
6 and 60°C and was stable at 60°C after 2 h. Enzyme assays on different substrates
yielded activity on methylumbelliferyl butyrate and methylumbelliferyl acetate in
addition to the glucose pentaacetate and a-naphthyl acetate. The estA gene was
cloned into a yeast expression vector between the PGK promoter and terminator
sequences for expression of the gene in Saccharomyces cerevisiae. The estA open
reading frame was also fused to the MFa 1 secretion signal for secretion of the protein
from S. cerevisiae. The expression vector was successfully transformed into S.
cerevisiae, but no extracellular activity was detected. Only low intracellular activity
of 0.260 nkatal/ml was detected in S. cerevisiae. / AFRIKAANSE OPSOMMING: Esterases speel 'n verskeidenheid van rolle in feitlik elke aspek van lewe, van sel
metabolisme, sein transduksie tot verdedigingsmeganismes in plante. Een aspek van
esterases wat al hoe meer aandag geniet, is die rol wat esterases in die afbraak van
plant en plantmateriaal speel. Met olie- en steenkoolbronne wat na beraming oor 20
tot 30 jaar tot niet sal gaan, raak die rol wat hernubare bronne speel al hoe
belangriker. Plantbiomassa bevat sellulose en hemisellulose wat tot die verskillende
komponente afgebreek moet word voordat dit optimaal vir die vervaardiging van
produkte aangewend kan word.
Alhoewel die ensieme wat vir die hidrolise van die xilaanruggraat benodig word,
(xilanases en ~-xulosidases) belangrik is, is die ensieme wat die sygroepe vanaf die
polimeer verwyder ewe belangrik aangesien hulle die hidrolise deur xilanases en
~-xulosidases bevorder. Wanneer hulle saam met die ander xilanolitiese ensieme
gebruik word, sal hulle die beskikbaarheid van monomeriese suikers vir fermentasie
verhoog. Baie van hierdie sygroepe is esters en hulle word deur die aksie van
esterases verwyder, of direk van die ruggraat, ofvanafkorter xilo-oligosakkariede.
'n Bestaande genoom DNA biblioteek van Bacillus pumilus in Escherichia coli is vir
die teenwoordigheid van 'n asetielesterase-koderende geen gesif. Positiewe klone is
deur die vorming van 'n sone op plate wat glukose pentaasetaat bevat, geïdentifiseer.
Die DNA-invoeging van die positiewe E. coli-kloon se DNA-volgorde is bepaal en
twee oopleesrame is gevind waarvan een vir 'n unieke esterase (estA) kodeer. Met
behulp van verskillende inleiers is die geen met die polimerasekettingreaksie (PKR) geamplifiseer en in 'n induseerbare promotor vir uitdrukking in E. coli gekloneer. Die
plasmied is getransformeer in E. coli en aktiwiteit is bepaal deur cc-naftielasetaatte
gebruik. Vlakke van aktiwiteit het kort na induksie met IPTG weer gedaal en daarom
was plasmied pAP4 vir ensiematiese toetse gebruik. E. coli-transformante met
plasmied pAP4 het ekstrasellulêre aktiwiteit van 2.5 nkatal/ml gelewer. Die pH en
temperatuur optima sowel as die temperatuurstabiliteit van die ensiem was bepaal.
Die ensiem toon optimale aktiwiteit by pH 6 en 'n temperatuur van 60°C.
Aktiwiteitstoetse op verskillende substrate het aktiwiteit op metielumbelliferielasetaat
en metielumbelliferielbutiraat bo-en-behalwe die glukosepentaasetaat en
c-naftielasetaar getoon. Die estA geen is in uitdrukkingskasette bevattende die PGKpromotor
en-termineerder vir uitdrukking in Saccharomyces cerevisiae gekloneer.
Dit is ook agter die MFal-sekresiesein gekoppel vir sekresie vanuit S. cerevisiae.
Geen ekstrasellulêre aktiwiteit is gevind nie. Slegs intrasellulêre aktiwiteit van 0.26
nanokatal per mililiter was bepaal.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51654 |
| Date | 03 1900 |
| Creators | Pieterse, Anton |
| Contributors | Van Zyl, W. H., Stellenbosch University. Faculty of Science. Dept. of Microbiology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 111 p. : ill. |
| Rights | Stellenbosch University |
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