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Heterologous production of family 5 fungal endo-1,4-B-mannanases in Saccharomyces cerevisiaeSetati, Mathabatha Evodia 12 1900 (has links)
Dissertation (PhD)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Mannan polysaccharides occur in the hemicellulose fraction of plant cell walls, either as
structural polymers or as reserve carbohydrates. They are found predominantly in the
seeds of leguminous plants in the form of galactomannan, and in softwoods as
galactoglucomannan. Endo-I,4-I3-mannanases hydrolyze mannan polysaccharides to
oligosaccharides of various lengths. These enzymes are secreted as single catalytic
modules or as part of multi-modular proteins by fungi, bacteria, plants and animals. For
example, the l3-mannanase of Aspergillus aculeatus, designated Aa-Man5A, is secreted
as a single catalytic module, whereas that of Trichoderma reesei, designated Tr-Man5A,
contains a l3-mannanase catalytic module linked to a cellulose-binding module by a Pro-
Ser-Thr-rich linker.
Heterologous gene expression in yeast provides the opportunity to produce individual
hydrolytic enzymes in a host expression system devoid of related activities.
Saccharomyces cerevisiae has a well-developed expression system and has frequently
been used as a model organism for heterologous gene expression. A number of
autoselection systems have been devised so that recombinant S. cerevisiae strains can be
cultivated in any medium of choice without exerting selective pressure. An autoselection
system based on defective chromosomal ura3 andfurl genes involved in the pyrimidine
biosynthesis pathway of S. cerevisiae, and complementation of the ura3 gene with a
multicopy plasmid-borne URA3 gene, were used in this study.
The man1 of A. aculeatus gene encoding Aa-Man5A was cloned and expressed in
autoselective S. cerevisiae under the regulation of the alcohol dehydrogenase (ADH2PT)
and the phosphoglycerate kinase (PGK1PT) promoter and terminator sequences.
Expression of man1 under both promoters resulted in high production levels of Aa-
Man5A. The production levels were significantly higher than the levels of endo-l,4-13-
mannanases produced by heterologous expression in Escherichia coli, and were
comparable to the production levels of enzymes produced in Pichia pastoris, which
presumably has a higher secretion capacity than S. cerevisiae. The recombinant yeast strain expressing man1 under the regulation of the PGK1p promoter displayed stunted
biomass formation during the logarithmic phase, which was relieved when the native f3-
mannanase secretion signal was replaced with the yeast MFuis secretion signal. The
recombinant Aa-Man5A displayed biochemical properties similar to those of the native
Aa-Man5A. The recombinant enzyme hydrolyzed unsubstituted mannan to
predominantly mannose, mannobiose, and mannotriose.
The expression of the man1 and man1 ácbd gene constructs of T reesei in S. cerevisiae
fur 1::LEU2 strains under the regulation of the PGK1 PT promoter and terminator resulted
in the production and secretion of Tr-Man5A and Tr-Man5A~CBD (lacking the cellulose
binding module), respectively. However, the production levels of both proteins were
approximately I5-fold lower than the production levels of Aa-Man5A. These levels did
not improve after replacement of the native secretion signal with the MFuis secretion
signal. Interestingly, reducing the cultivation temperature from 30°C to 20°C led to a
five-fold increase in the secreted levels of Tr-Man5A, but a three-fold decrease in the
production of Aa-Man5A.
A preliminary investigation was performed to evaluate the possibility of using the
recombinant Aa-Man5A in the processing of instant coffee. Arabica coffee extracts
treated with Aa-Man5A displayed low viscosity in comparison to the untreated extract
and showed better retention of volatile/aromatic compounds than the autoclaved extract.
The results indicated that Aa-Man5A is capable of hydrolyzing coffee galactomannan
and can be used for processing instant coffee. / AFRIKAANSE OPSOMMING: Mannaanpolisakkariede kom in die hemisellulose fraksie van plantselwande as
strukturele polimere of reserwe koolstofbron voor. Mannaan word hoofsaaklik in die sade
van peulplante, III die vorm van galaktomannaan, en III sagtehout as
galaktoglucomannaan aangetref. Endo-I,4-j3-mannanase kan mannaanpolisakkariede na
oligosakkariede van verskillende lengtes afbreek. Hierdie ensieme word deur fungi,
bakterieë, plante en diere as enkele katalitiese modules of as deel van multi-modulêre
proteïene uitgeskei. Die j3-mannanase (Aa-Man5A) van Aspergillus aculeatus is
byvoorbeeld 'n enkele katalitiese module, maar die j3-mannanase (Tr-Man5A) van
Trichoderma reesei bestaan uit 'n j3-mannanase katalitiese module gekoppel aan 'n
sellulose-bindingsmodule deur middel van 'n Pro-Ser- Thr-ryke koppelstuk.
Heteroloë geenuitdrukking in gIS bied die geleentheid om individuele hydrolitiese
ensieme in 'n gasheer uitdrukkingsisteem sonder verwante aktiwiteite te produseer.
Saccharomyces cerevisiae het 'n goed ontwikkelde uitdrukkingsisteem en word as model
organisme vir heteroloë geenuitdrukking gebruik. 'n Aantal outoseleksiesisteme is
ontwikkel, waardeur rekombinante S. cerevisiae-tese in enige medium sonder selektiewe
druk gekweek kan word. 'n Outoseleksiesisteem, gebaseer op defektiewe chromosomale
ura3 en furl gene wat vir ensieme in die pirimidien biosinteseweg kodeer, en
komplementasie van die ura3-geen met die wilde-tipe URA3-geen wat op In multikopie
plasmied teenwoordig is, is vir hierdie studie gebruik.
Die manl-geen, wat vir die Aa-Man5A j3-mannanase van A. aculeatus kodeer, is
gekloneer en in outoselektiewe S. cerevisiae onder die regulering van die
alkoholdehidrogenase 2 (ADH2PT) en fosfogliseraatkinase 1 (POKl PT) promotor- en
termineerderopeenvolgings uitgedruk. Uitdrukking van die manl-geen onder albei
promotors het hoë produksievlakke van Aa-Man5A gelewer. Die produksievlakke was
aansienlik hoër as die endo-I,4-j3-mannanase-vlakke wat deur heteroloë geenuitdrukking
in Escherichia coli geproduseer was, en kon vergelyk word met die produksievlakke van ensieme in Pichia pastoris. P. pastoris is veronderstel om In hoër sekresiekapasiteit as
S. cerevisiae te hê. Die rekombinante gisras wat die manl-geen onder beheer van die
PGKl p promotor uitgedruk het, se biomassavorming was belemmer gedurende die laat
logaritmiese fase. Die belemmering is opgehef nadat die natuurlike sekresiesein van
13-mannanasemet die MFais sekresiesein vervang is. Die rekombinante Aa-ManSA het
soortgelyke biochemiese eienskappe as die natuurlike Aa-ManSA getoon. Die
rekombinante ensiem het onvertakte mannaan tot hoofsaaklik mannose, mannobiose en
mannotriose gehidroliseer.
Die uitdrukking van die manl- en manl Licbd-geenkonstrukte van T. reesei in
S. cerevisiae furl::LEU2-rasse onder regulering van die PGKlPT promotor en
termineerder het tot die produksie en sekresie van onderskeidelik die Tr-ManSA en
Tr-ManSAilCBD (sonder die sellulose-bindingsdomein) ensieme gelei. Die
produksievlakke van beide proteïene was egter ongeveer IS-voudig laer as die vlakke van
Aa-ManSA. Hierdie vlakke het egter nie verbeter nadat die natuurlike sekresiesein met
die MFais sekresiesein vervang is nie. Interessant is die feit dat 'n afname in
opkwekingstemperatuur vanaf 30°C tot 20°C tot 'n vyf-voudige toename m
sekresievlakke van die Tr-ManSA gelei het, maar tot 'n drie-voudige afname in die
produksie van Aa-ManSA.
'n Voorlopige ondersoek na die moontlike gebruik van rekombinante Aa-ManSA in
kitskoffieprosessering is ondersoek.. Arabica koffie-ekstrak wat met Aa-ManSA
behandel is, het 'n laer viskositeit in vergelyking met onbehandelde ekstrak getoon, asook
beter behoud van vlugtige/aromatiese verbindings in vergelyking met geoutoklaveerde
ekstrak. Hierdie resultate toon dat Aa-ManSA in staat is om koffee galaktomannaan te
hidroliseer en dat dit vir die prosessering van kitskoffie gebruik kan word.
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Organization of the shrimp (Metapenaeus ensis) vitellogenin gene: evidence for multiple genes and ovaryexpressionTsang, Wing-sze., 曾詠思. January 2002 (has links)
published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy
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Isolation, characterization, and expression analysis of genes encodingstarch synthesizing enzymes from grain amaranthLu, Bei., 呂蓓. January 2006 (has links)
published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy
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The effects of castration and testosterone replacement on the gene expression of adrenomedullin and its receptor component proteins inthe rat epididymis, seminal vesicle and coagulating glandWong, Pik-fan., 黃碧芬. January 2009 (has links)
published_or_final_version / Physiology / Master / Master of Medical Sciences
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Zone-specific gene expression of mandibular condylar cartilage : biological implications of regional differencesBasudan, Aishah Mohammed A January 2015 (has links)
Mandibular condylar cartilage (MCC) consists of fibrous (F), proliferative (P), mature (M) and hypertrophic (H) zones, and exhibits distinctive biological features in physiology and function. Accordingly, the genetic regulation of MCC is expected to be different from other articular cartilages. Combined lasercapture microdissection (LCM) and microarray analysis (MAA) approach allows large-scale screening of zone-specific gene expressions. A few investigators have attempted to apply this approach on different cartilages, but not on MCC yet. Therefore, this study aimed to: 1) optimize an LCM protocol for isolating homogenous cell populations from MCC zones; 2) perform a zone-specific comprehensive gene expression analysis for MCC using LCM & MAA; and 3) find a set of genes, following the validation of MAA data using in-vivo and invitro quantitative reverse transcription-polymerase chain reaction (qRT-PCR), which could potentially distinguish MCC zones from each other and from articular chondrocytes.
MCC and femoral condylar cartilage (FCC) specimens were harvested from normal 5-week-old SD rats, and formalin-fixed sections and cryosections were compared histologically. LCM samples for five groups (FCC and four MCC zones) were prepared, and then RNAs were extracted and evaluated for integrity. For MAA experiment, LCM samples were amplified before microarray hybridization. MAA data were analyzed using GeneSpring software. cDNA from unamplified LCM-RNA samples were also prepared for the five groups for in-vivo qRT-PCR validation of 48 genes selected from MAA data, 10 of which were additionally validated by cultivating ATDC5 cells and extracting RNA at different time points for in-vitro qRT-PCR validation.
Factors enhancing tissue visualization, LCM efficiency, LCM specificity, and RNA yield and integrity were optimized in the suggested LCM protocol. At a 2-fold change, 8353 (26.86%) transcripts were differentially expressed among the MCC zones and FCC. Subsequent data mining allowed the creation of seven subsets of 127 genes. Forty-eight genes were selected for validation based on their signal intensities, clustering classification, and gene ontology. In-vivo and in-vitro qRT-PCR showed high consistency with the MAA data. Results revealed robust gene expression differences among MCC zones, and between articular chondrocytes and MCC cells. The F & P zones could be characterized by upregulation of Crabp1, Dpt, Fndc1, Aspn, Tnmd, Bcl11b, Angptl1, Col14a1, and downregulation of Mug1, Foxa2, Lect1, and Matn3. Opposite modulation of the same genes may characterize M & H zones. In addition, unizonal distinct profiles were also identified; upregulated Igfbp6, Igha, Hils1, and Ptgds genes might be considered as potential markers for F, P, M, and H zones, respectively.
In conclusion, this study sets up an LCM protocol that enables isolating homogenous zone-specific cell populations from the MCC, and obtaining highquality RNAs for subsequent gene expression analysis. Comprehensive gene profiling has been successfully achieved with high fidelity; using minute RNA amounts via the LCM & MAA combined approach. The MCC cells clearly exhibit distinguishable phenotypes from the articular chondrocytes, and a set of genes has been determined as potential unizonal/bizonal markers to identify MCC zones. Generating accurate regional data enhances our understanding of MCC biology and provides invaluable insights for tissue-engineering and cellbased therapeutic strategies. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy
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Gene expression during development on Schistosoma mansoniJohnson, K. S. January 1986 (has links)
No description available.
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Growth factor production in pregnant equidsLennard, Simon N. January 1994 (has links)
No description available.
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Circles of censorship : La Censure and its metaphors in history, psychoanalysis and literary cultureHarrison, Nicholas January 1993 (has links)
No description available.
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Studies on the structure and expression of the isocitrate lyase gene of Chlorella fuscaUrwin, Nigel A. R. January 1990 (has links)
No description available.
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Studies on the expression of bFGF and IGF-I and -II in 3T3 cellsLi, Dajiang January 1997 (has links)
No description available.
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