Thesis (MScAgric)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Cellulose, a ~-1,4-linked polymer of glucose, is the most abundant renewable carbon source
on earth. It is well established that efficient degradation of cellulose requires the
synergistic action of three categories of enzymes: endoglucanases (EG), cellobiohydrolases
(CBH) and ~-glucosidases. ~-Glucosidases are a heterogenous group of enzymes that
display broad substrate specificity with respect to hydrolysis of cellobiose and different
aryl- and alkyl-ê-u-glucosides. They not only catalyse the final step in the saccharification
of cellulose, but also stimulate the extent of cellulose hydrolysis by relieving the cellobiose
mediated inhibition of EG and CBH. The ability to utilize cellobiose is widespread among
gram-negative, gram-positive, and Archaea bacterial genera. Cellobiose phosphoenolpyruvate-
dependent phosphotransferase systems (PTS) have been reported in various
bacteria, including: Bacillus species.
In this study, we have used a cellobiose chromophore analog, p-nitrophenyl-
~-D-glucopyranoside (pNPG), to screen a Bacillus pumilus genomic library for cellobiose
utilization genes that are functionally expressed in Escherichia coli. Cloning and
sequencing of the most active clone with subsequent sequence analysis allowed the
identification of four adjacent open reading frames. An operon of four genes (celBACH),
encoding a cellobiose phosphotransferase system (PTS): enzyme II (encoded by celB, celA
and celC) and a ó-phospho-f-glucosidase (encoded by celH) was derived from the sequence
data. The amino acid sequence of the celH gene displayed good homology with
~-glucosidases from Bacillus halodurans (74.2%), B. subtilis (72.7%) and
Listeria monocytogenes (62.2%). .As implied by sequence alignments, the celH gene
product belongs to family 1 of the glycosyl hydrolases, which employ a retaining
mechanism of enzymatic bond hydrolysis.
In vivo PTS activity assays concluded that the optimal temperature and pH at which the
recombinant E. coli strain hydrolysed pNPG were pH 7.5 and 45°C, respectively.
Unfortunately, at 45°C the CelBACH-associated activity of the recombinant strain was only
stable for 20 minutes. It was also shown that the enzyme complex is very sensitive to glucose. Since active growing cells metabolise glucose very rapidly this feature is not a
significant problem.
Constitutive expression of the B. pumilus celBACH genes in E. coli enabled the host to
efficiently metabolise cellobiose as a carbon source. However, cellobiose utilization was
only achievable in the presence ofO.01% glucose. This phenomenon could be explained by
the critical role of phosphoenolpyruvate (PEP) as the phosphate donor in PTS-mediated
transport. Glucose supplementation induced the glycolytic pathway and subsequently the
availability of PEP. Furthermore, it could be concluded that the general PTS components .
(enzyme I and HPr) of E. coli must have complemented the CelBACH system from
B. pumilus to allow functionality of the celBACH operon, in the recombinant E. coli host. / AFRIKAANSE OPSOMMING: Sellulose (' n polimeer van p-l,4-gekoppelde glukose) is die volopste bron van hernubare
koostof in die natuur. Effektiewe afbraak van sellulose word deur die sinnergistiese
werking van drie ensiernklasse bewerkstellig: endoglukanases (EG), sellobiohidrolases
(CBH) en P-glukosidases. p-Glukosidases behoort tot 'n heterogene groep ensieme met 'n
wye substraatspesifisiteit m.b.t. sellobiose en verskeie ariel- and alkiel-ê-n-glukosidiesc
verbindings. Alhoewel hierdie ensieme primêr as kataliste vir die omskakeling van
sellulose afbraak-produkte funksioneer, stimuleer hulle ook die mate waartoe sellulose
hidroliese plaasvind deur eindprodukinhibisie van EG en CBH op te hef. Sellobiose word
algemeen deur verskeie genera van die gram-negatiewe, gram-positiewe en Archae
bakterieë gemetaboliseer. Die sellobiose-spesifieke fosfoenolpirovaatfosfotransportsisteem
(PTS) is reeds is in verskeie bakterië, insluitende die Bacillus spesies,
beskryf.
In hierdie studie word die sifting van 'n Bacillus pumilus genoombiblioteek m.b.V. 'n
chromofoor analoog van sellobiose, p-nitrofeniel-p-o-glukopiranosied (pNPG), vir die
teenwoordigheid van gene wat moontlike sellobiose-benutting in Escherichia coli kan
bewerkstellig, beskryf. Die DNA-volgorde van die mees aktiewe kloon is bepaal en
daaropvolgende analiese van die DNA-volgorde het vier aangrensende oopleesrame
geïdentifiseer. 'n Operon (celBACH), bestaande uit vier gene, wat onderskeidelik vir die
ensiem II (gekodeer deur celB, celA en celC) en fosfo-B-glukosidase (gekodeer deur celH)
van die sellobiose-spesifieke PTS van B. pumilus kodeer, is vanaf die DNA-volgorde
afgelei. Die aminosuuropeenvolging van die celH-geen het goeie homologie met
P-glukosidases van Bacillus halodurans (74.2%), B. subtilis (72.7%) en
Listeria monocytogenes (62.2%) getoon. Belyning van die DNA-volgordes het aangedui
dat die celH geenproduk saam met die familie 1 glikosielhidrolases gegroepeer kan word.
Hierdie familie gebruik 'n hidrolitiese meganisme waartydens die stoigiometriese posisie
van die anomeriese koolstof behou word. PTS-aktiwiteit van die rekombinante E. coli ras, wat die celBACH gene uitdruk, is in vivo
bepaal. Die optimale temperatuur en pH waarby die rekombinante ras pNPG hidroliseer, is
onderskeidelik pH 7.5 en 45°C. Alhoewel die ensiernkompleks baie sensitief is vir glukose,
is dit nie 'n wesenlike probleem nie, omdat aktief groeiende E. coli selle glukose teen 'n
baie vinnige tempo benut.
Die celBACH operon het onder beheer van 'n konstitiewe promotor in E coli die
rekombinante gasheer in staat gestelom sellobiose as 'n koolstofbron te benut. Die
benutting van sellobiose word egter aan die teenwoordigheid van 'n lae konsentrasie
glukose (0.01 %) gekoppel. Hierdie verskynsel dui op die kritiese rol van fosfoenolpirovaat
(PEP) as die fosfaatdonor gedurende PTS-gebaseerde transport. Glukose speel waarskynlik
'n rol in die indusering van glikoliese, en sodoende die produksie van PEP as tussenproduk.
Verder kan afgelei word dat die algemene PTS komponente (ensiem I en HPr) van E. coli
die B. pumilis CelBACH-sisteem komplementeer en derhalwe funksionering van die
celBACH operon in E. coli toelaat.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52733 |
| Date | 12 1900 |
| Creators | Van Rooyen, Ronel, 1976- |
| Contributors | Van Zyl, W. H., La Grange, D. C., 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 | 82 p. : ill. |
| Rights | Stellenbosch University |
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