In the past few decades much research has been conducted on yeast alcohol
dehydrogenases with emphasis on their role in S. cerevisiae. There are seven
known Adh proteins that were identified in S. cerevisiae. Six of them are
intensively studied on gene and translational level. After sequencing of S.
cerevisiae chromosome II (Feldman et al. 1994), ADH5 was identified which is
76% and 77% identical to ADH1 and ADH2 respectively. Later studies on
global localization on genes showed that ADH5 is localized in the cytoplasm
(Hue et al. 2003). No further research has been performed on ADH5.
In this study Adh5p was over-expressed, purified and characterized towards its
primary substrate, ethanol, and preferred co-factor NAD+. Furthermore, indepth
kinetic studies were performed using various alcohols, increasing in
chain length and branching. Adh5p is capable of oxidizing alcohols from two
carbons to ten carbons. However, the catalytic efficiency decreases with
increasing chain length. Results showed that Adh5p functions in vitro the same
as Adh1p and Adh2p, sharing a primary substrate (ethanol). Adh1p and Adh2p
are capable of converting more substrate per unit enzyme per second Adh5p.
The second part of this study was to determine if Adh5p could substitute the
catalytic function of Adh1p in vivo. For this purpose, ADH5 expression needed
to be similar to Adh1p levels in the cell. Thus an expression vector was used
containing ADH5 gene flanked with the promoter and terminator regions of
ADH1. S. cerevisiae TÎ123 was constructed with ADH4 and ADH5 still intact.
S. cerevisiae TÎ123 was transformed with a pRS413 and pRS423 construct
containing the ADH1 promoter and terminator fused to the ADH5 ORF. Growth
was monitored in chemically defined media containing 7 g l-1 ethanol or 8 g l-1
glucose. Growth parameters were also compared to the S. cerevisiae W303-1A
and the adh quadruple deletion strain (Q1) containing only ADH1. The S.
cerevisiae TÎ123::ADH5_S and S. cerevisiae TÎ123::ADH5_M constructs were
capable of limited growth on both glucose and ethanol as carbon sources. When comparing the biomass yield of S. cerevisiae TÎ123::ADH5_S and S.
cerevisiae TÎ123::ADH5_M to the biomass yield for both S. cerevisiae W303-
1A and S. cerevisiae Q1 the constructs delivered a much lower biomass yield.
To verify the expression of Adh5p, a Micro BCA⢠protein assay kit supplied by
Pierce (Smith et al. 1985) was used. Protein concentrations were determined at
various time intervals. Cell homogenization was standardized, S. cerevisiae Q1
cells were diluted to fit the OD600 obtained for both the S. cerevisiae
TÎ123::ADH5_S and S. cerevisiae TÎ123::ADH5_M strains.
To conclude, Adh5p is capable of oxidizing various alcohols, but ethanol is its
primary substrate. Furthermore Adh5p is not capable of replacing Adh1p in
cellular metabolic function. The low turnover number illustrated by Adh5p and
the lack thereof to reduce acetaldehyde is the most prominent cause of cellular
death. Unlike Adh1p, Adh5p is not capable of reducing acetaldehyde to
ethanol and thus not capable of NAD+ - NADH regeneration.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-10172011-131813 |
| Date | 17 October 2011 |
| Creators | Henn, Michael Ernst |
| Contributors | Prof JC du Preez, Dr O de Smidt, Prof J Albertyn |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-10172011-131813/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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