Dissertation (PhD)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The production, purification and functional characterisation of feruloyl
esterase from Aureobasidium pullulans were set as the primary objectives of
this study. A further objective was to investigate a possible co-operative effect
with other selected lignocellulolytic enzymes on substrates relevant to
industry.
In a comprehensive review, feruloyl esterases from various micro-organisms
were compared both functionally and with regard to their primary structure,
where applicable. Feruloyl esterases show intriguing differences in substrate
specificity and sequence structure. Enzymes that are closely related regarding
their amino acid sequence exhibit different substrate specificities. Sequence
similarities can be found with a range of other enzyme families, including
serine esterases, acetyl xylan esterases, lipases, tannases, glycosyl
hydrolases and xylanases. More data on the three dimensional structure of
feruloyl esterases as well as an examination of all available feruloyl esterases
with the same substrates is necessary before structure-function relationships
can be established and before the feruloyl esterases can be organized into
discrete families based on ancestral origins.
The highest production levels of feruloyl esterase by A. pullulans are achieved
when grown on birchwood xylan. Expression was not repressed when glucose
or xylose was present in the medium. However, free ferulic acid
supplemented to the medium affected fungal growth and therefore did not
increase feruloyl esterase activity. It is also suggested that the synthesis of
feruloyl esterase is independently regulated from xylanase synthesis. Feruloyl esterase from A. pullulans acts on a- and l3-naphthyl acetate, as well as
naphthol AS-D chloroacetate as substrates.
Feruloyl esterase from A. pullulans was purified to homogeneity using
ultrafiltration with high molecular weight cut-off, anion exchange, hydrophobic
interaction and ultimately gel filtration chromatography. With a molecular
weight of 210 kDa, the enzyme is the largest of the feruloyl esterases reported
to date. Kinetic data was produced using both synthetic and natural
substrates. A. pullulans feruloyl esterase shows properties similar to other
fungal feruloyl esterases, especially from Aspergillus niger cinnamic acid
esterase and Penicillium funiculosum feruloyl esterase B. The N-terminal
sequence of A. pullulans feruloyl esterase was identified, but no similarities to
known enzyme families were found. Peptide mass mapping did not reveal
structural information.
In an effort to evaluate the significance of feruloyl esterase from A. pullulans
in the degradation of lignocellulose, dissolving pulp and sugar cane bagasse
were selectively treated using feruloyl esterase and hemicellulolytic enzymes.
The enzymatic degradation reaction was monitored using microdialysis
sampling, anion exchange chromatography, online desalting and mass
spectrometry. It has been shown, that feruloyl esterase activity together with
xylanase activity releases monosaccharides from both substrates. Sugars of
higher degree of polymerisation were not released, giving evidence for the
recalcitrance of the material. The fibre architecture of the substrates was
apparently not accessible to the enzymes and therefore complete hydrolysis
was hindered. / AFRIKAANSE OPSOMMING: Die produksie, suiwering en funksionele karakterisering van feruloïel esterase
afkomstig van Aureobasidium pullulans was die primêre doelwitte van hierdie
studie. 'n Verdere doelwit was om vas te stelof daar 'n kooperatiewe effek
met ander geselekteerde lignosellulitiese ensieme op substrate wat industrierelevant
is, bestaan.
Die feruloïel esterase van verskillende mikro-organismes is vanuit die oogpunt
van funksie en primêre struktuur omvattend met mekaar vergelyk, waar
toepaslik. Interessante verskille tussen die substraat spesifisiteit en volgordestruktuur
van feruloïel esterase kan waargeneem word. Ensieme wat nou
aanmekaar verwant is wat hul aminosuurvolgorde betref, het duidelik
verskillende substraatspesifiteite. Volgordeverwantskap kan in 'n reeks van
ander ensiemfamilies, insluitende serienesterase, asetielxilaanesterase,
lipases, tannases, glikosielhidrolases en xilanases vasgestel word. Meer
inligting oor die driedimensionele struktuur van feruloïel esterase asook 'n
analise van al die beskikbare feruloïel esterase met dieselfde substrate is
nodig voordat struktuur-funksie verwantskappe vasgestel kan word en voordat
die feruloïel esterases in eie families op die grond van huloorsprong
georganiseer kan word.
Die hoogste produksie vlakke deur feruloïel esterase van A. pullulans word
bekom deur dit op berkhoutxilaan te groei. Ekspressie was nie onderdruk
wanneer glukose of xilose in die medium aanwesig was nie. Wanneer vrye
feruliensuur by die medium bygevoeg is, is die fungale groei beïnloed en het
die feruloïel esterase aktiwiteit nie vermeerder nie. Dit word ook voorgestel dat die sintese van feruloïel esterase onafhanklik deur xilanase sintese
gereguleer word. Feruloïel esterase van A. pullulans reageer op a- en f3-
naftolasetaat, asook naftol AS-D chloroasetaat as substrate. Feruloïel
esterase van A. pullulans is tot homogeniteit deur ultrafiltrering met .n hoë
molekulêre gewiggrens, anioonuitruiling, hidrofobiese interaksie en eindelik
gelfiltrasie-chromatografie gesuiwer. Met 'n molekulêre gewig van 210 kDa, is
die ensiem die grootste van die feruloïel esterases tot dusver beskryf.
Kinetiese data is met behulp van sintetiese en natuurlike substrate
geproduseer. A. pullulans feruloïel esterase het eienskappe wat vergelykbaar
is aan die van ander fungal feruloïel esterases, veral die wat afkomstig is van
Aspergillus niger sinnamiensuur esterase en Penicillium funiculosum feruloïel
esterase B. Die N-terminale volgorde van A. pullulans feruloïel esterase is
identifiseer maar geen ooreenkoms aan bekende ensiemfamilies kon
vasgestel word nie. Peptiedmassakaartering kon ook geen strukturele inligting
gee nie.
Oplosbare pulp en suikerrietbagasse is geselekteerd met behulp van feruloïel
esterase en lignosellulitiese ensieme behandel om die belang van feruloïel
esterase van A. pullulans in die afbraak van lignosellulose vas te stel. Die
hidroliese-reaksie is deur mikrodialise monsterneming, anioonuitruilingschromatografie,
oplyn ontsouting en massaspektrometrie gemonitor. Wanneer
die aktiwiteit van feruloïel esterase met die van xilanase gekombineer is, is
monosakkariede deur albei substrate afgeskei. Suikers met 'n hoër graad van
polimerisering is nie afgeskei nie, wat 'n bewys van die materiaal se
weerstandbiedendheid is. Dit het geblyk asof die vesel-argitektuur van die verbruikte substraat nie toeganklik was vir ensieme nie en dus is algehele
hidroliese verhinder.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53480 |
| Date | 12 1900 |
| Creators | Rumbold, Karl, 1973- |
| Contributors | Prior, Bernard, Robra, Karl-Heinz, 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 | 208 p. : ill. |
| Rights | Stellenbosch University |
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