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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/52875 |
| Date | 12 1900 |
| Creators | Setati, Mathabatha Evodia |
| Contributors | Van Zyl, W. H., Stalbrand, 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 | xi, 128 p. : ill. |
| Rights | Stellenbosch University |
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