Submitted in complete fulfilment for Masters Degree in Technology: Biotechnology, Durban University of Technology, 2013. / A comparative thermostability analysis of different partially purified xylanases from
Rhodothermus marinus, Bacillus halodurans, Thermomyces lanuginosus and Pulpzyme HC
was studied using differential scanning fluorometry (DSF), fluorescence spectroscopy and
circular dichroism (CD). The R. marinus xylanase was found to have an optimum
temperature and pH of 90oC and 6 respectively while the B. halodurans xylanase was
optimally active at 70oC and a broad range of alkaline pH of 8 - 10. The commercially
available xylanase from T. lanuginosus showed optimal activity at 50oC and pH 7 while the
Novozyme xylanase Pulpzyme HC showed optimal activity at 60oC and pH 7.
Fluorescence spectroscopy monitored the microenvironment and fluorescence
emission of Trp residues. In their native folded state, Trp are generally located in the core of
the protein but during unfolding they become exposed. The fluorescence changes as the
enzyme undergoes denaturation due to conformational changes and exposure of Trp residues.
Differential scanning fluorometry (DSF) monitors thermal unfolding of proteins in the
presence of a fluorescent dye such as Spyro Orange. A wide range of buffers were tested for
their ability to increase the xylanase stability. T. lanuginosus had the greatest increase in
melting temperature with 0.73M Bis Tris pH 6.5 and peaked highest at 78°C. The B.
halodurans xylanase exhibited high pH stability (pH 4-10) and exhibited very little change in
melting temperature, from 74°C-77°C over the twenty four different conditions. The R.
marinus xylanase had no increase in melting temperature showing a maximum melting
temperature of 90oC.
Circular dichroism (CD) measures unequal absorption of right- and left-handed
circularly polarized light by the molecule. The xylanase from R. marinus exhibited the lowest
ΔG of 34.71kJ at 90°C as was expected. The B. halodurans xylanase showed a much higher
ΔG of -52.71 at its optimum temperature of 70°C when compared with the xylanases from R.
marinus and T. lanuginosus. When comparing the three xylanases activities at 70°C, it can be
seen that the B. halodurans xylanase exhibited a lower relative activity then both R. marinus
and T. lanuginosus xylanases.
All three techniques offered different information on the structure and function
relationship. Fluorescence spectroscopy, the change in conformation due to fluorescence
emission as a result of increased temperature and salt concentrations. DSF, optimal
conditions for increased stability and activity at higher temperatures and CD, conformational
changes, the fraction of folded protein and change in Gibbs free energy over a range of
temperature. / National Research Foundation
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:dut/oai:localhost:10321/873 |
| Date | 30 July 2013 |
| Creators | Tabosa-Vaz, Sacha |
| Contributors | Singh, Suren, Permaul, Kugen, Kumar, Ajit |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 114 p |
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