Thesis (PhD)--University of Stellenbosch, 2000. / ENGLISH ABSTRACT: White rot fungi are of interest in the paper and pulp industry because of their
removal of lignin from wood. In this study over 600 Basidiomycete fungi were
isolated from indigenous forests as well as from commercial Eucalyptus spp.
and Pinus spp. plantations in South Africa. One hundred isolates were
identified to genus level. Biochemical tests were done to screen the fungal
cultures for characteristics that are favourable for biopulping, e.g. low
cellulase activity with concomitant high activity of ligninolytic enzymes.
Various Phanerochaete isolates with potentially high ligninolytic activity were
identified.
Although Phanerochaete chrysosporium Burds. has previously been isolated
from the indigenous forest at Knysna in South Africa, this study showed that
P. chrysosporium was a natural coloniser of wood chip piles in South Africa,
indicating potential for application in industry. A possible new species of
Phanerochaete, viz. Phanerochaete pseudomagnoliae nom. provo (strain
PP25) from decayed wood collected in Stellenbosch, South Africa, was
described and illustrated. It differs from previously described Phanerochaete
species in having smaller basidiospares, and in the formation of few
chlamydospores on malt extract agar but more on xylose containing media.
The potential of using internal transcribed spacer DNA sequences (ITS) to
infer phylogenetic relationships among species of the genus Phanerochaete
was investigated. Consensus phylogenetic trees could be presented, but the
presence of ambiguous aligning sequences within the ITS made inferring of
phylogenetic relationships within the whole genus difficult.
Fifty-five South African strains of P. chrysosporium were screened for lignin
peroxidase (liP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX)
enzymes. Manganese peroxidase activity was quantified on agar media. The
liP and GLOX activities of 13 selected strains, including control strains and
P. pseudomagnoliae (PP25), were also quantified on agar media. Differences
in MnP and GLOX activities existed among the strains. Preliminary
biochemical characterisation of strain PP25 indicated that the most important
difference was the apparent unique regulation of ligninolytic enzymes. Under
low nitrogen, liP activity of the selected strains showed no significant variation, whereas strain PP25 had significantly increased liP levels under
high nitrogen conditions. Restriction fragment length polymorph isms of the
lignin and manganese peroxidase gene DNA fragments showed variability
among strains, whereas there was probably only allelic variation for the glox
gene DNA fragments.
Previous research has indicated xylose oxidation activity within
P. chrysosporium. To investigate whether GLOX can oxidise xylose, a purified
recombinant GLOX (rGLOX) from P. chrysosporium BKM-F-1767 Burds. was
used in this study. This rGLOX oxidised D-xylose and D-glucose (D-xylose >
D-glucose) to produce H202. Xylose was oxidised to xylono-1 ,4-lactone with a
1:1 stoichiometric relationship between H202 produced and xylose used.
Xylono-1,4-lactone was converted non-enzymatically to xylonate. This
suggested that the furanose form of xylose, rather than the pyranose form, is
a substrate of GLOX. The production of H202 and the removal of inhibitory
compounds by GLOX could enhance ligninolytic activity. .
To conclude, unique strains of P. chrysosporium have been isolated from
South Africa with potential biotechnological use in paper manufacturing. The
relationship of P. pseudomagnoliae nom. provo to other Phanerochaete
species was evaluated and light was shed on the possible role of GLOX in
lignin degradation. / AFRIKAANSE OPSOMMING: Witvrot swamme is van belang vir die papier en pulp industrie omdat hulle
lignin vanaf hout kan verwyder. Meer as 600 Sasidiomiseet fungi, afkomstig
vanaf inheemse woude asook kommersiële Eucalyptus spp. en Pinus spp.
plantasies, IS geïsoleer. Een honderd isolate is tot op genusvlak
geïdentifiseer. Die isolate is biochemies vir eienskappe wat voordelig vir
"bioverpulping" kon wees, bv. die gelyktydige produksie van lae sellulosemaar
hoë ligninolitiese ensiemaktiwiteit, getoets. Verskeie isolate met
potensieel hoë vlakke van ligninolitiese aktiwiteit is verkry.
Alhoewel Phanerochaete chrysosporium Surds. vantevore in die Knysna
inheemse woud in Suid-Afrika geïsoleer is, het hierdie studie gewys dat
P. chrysosporium natuurlik op hope houtblokkies voorgekom, met moontlike
toepasing in die industrie. Isolaat PP25, geisoleer vannaf verrottende hout te
Stellenbosch, Suid Afrika, is as 'n potensieel nuwe spesie van die genus
Phanerochaete beskryf en as Phanerochaete pseudomagnoliae nom. provo
benoem. Hierdie isolaat verskil van ander Phanerochaete-spesies daarin dat
dit kleiner basidiospore vorm en nie klamydospore op moutekstrakagar
produseer nie, maar wel op media wat xilose bevat. Die potensiaal van intern
getranskribeerde spasieerder ONS opeenvolging vir die aflei van filogenetiese
verhoudings tussen spesies van die genus Phanerochaete is ondersoek.
Konsensus filogenetiese bome kon bepaal word, maar die teenwoordigheid
van varieerbare areas het die afleiding van filogenetiese verwantskappe vir
die hele genus bemoeilik.
Vyf-en-vyftig Suid-Afrikaanse isolate van P. chrysosporium is vir die
teenwoordigheid van lignienperoksidase- (liP), mangaanperoksidase- (MnP)
en glioksaaloksidase (GLOX)-aktiwiteit getoets. Vlakke van MnP-aktiwiteit is
op agarplate gekwantifiseer. Vlakke van LiP- en GLOX-ensieme op agarplate
is vir 13 geselekteerde isolate, insluitend kontroles en ras PP25,
gekwantifiseer. Aktiwiteit van MnP en GLOX het statisties betekenisvol tussen
isolate verskil. Lignienperoksidase-aktiwiteit onder lae stikstof toestande het
nie statisties betekenisvol van mekaar verskil nie. Onder hoë stikstof
toestande het isolaat PP25 wel verhoogde liP-aktiwiteit getoon. Restriksie
fragment polimorfismes van die lignien- en mangaanperoxidase-gene het variasie getoon, terwyl waarskynlik slegs alleliese variasie vir die glox geen
waargeneem IS.
Rekombinante GLOX (GLOX vanaf P. chrysosporium BKM-F-1767) het xilose
en glukose (D-xilose > D-glukose) geoksideer met meegaande produksie van
H202. Xilose is na xilono-1,4-laktoon geoksideer met 'n 1:1 stoigiometrie
tussen H202-produksie en xilose verbruik. Xilono-1,4-laktoon is nieensiematies
na xilonaat omgeskakel. Bogenoemde resultaat dui aan dat die
furanose vorm van xilose die werklike substraat vir GLOX is. Deur die
meegaande produksie van H202 en die verwydering van inhiberende produkte
word lignoliese aangehelp.
Ten slote, unieke P. chrysosporium rasse met potensiële gebruik in
papiervervaardiging is in Suid-Afrika geisoleer. Die genetiese diversiteit van 'n
nuwe spesie, P. pseudomagnoliae, is bepaal en nuwe lig is op die potensiële
rol van GLOX in lignienafbraak gewerp.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/51775 |
| Date | 03 1900 |
| Creators | De Koker, T. H. (Theodorus Hermanus), 1965- |
| Contributors | Janse, B. J. H., 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 | 164 p. : ill. |
| Rights | Stellenbosch University |
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