Purification and characterisation of a secreted glycosidase, from the extreme xerophile Wallemia ichthyophaga : a thesis presented in partial fulfilment of the requirement for the degree in Master of Science in Biochemistry at Massey University Palmerston North, New Zealand
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Date
2014
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Massey University
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Abstract
With recent pressure to reduce the environmental impact of leather production, research has been focused on the development of an alternative depilation method, as the conventional method for depilation contributes up to 60% of the total pollution produced. Contaminated salted ovine pelts stored at LASRA were easily depilated when drum washed, and the resultant leather was of good quality. The pelts were visibly contaminated with microorganisms, and it was thought that these may be secreting enzymes that loosened the wool fibre without damaging key structural skin components. Identification of the enzyme or enzymes was thus of interest.
The microorganism/s responsible for the secretion of the depilation enzyme/s were isolated and identified through sequencing the 16S/18S ribosomal RNA genes. Depilation, using the crude secretome solutions, was then assessed using fresh ovine skin as well as SACPIC, a micro scale staining method used to assess skin structure. Unfortunately, none of the secretomes from either a single or a combination of the microorganisms isolated, had depilation activity.
The secretome of W.ichthyophaga, a xerophilic filamentous fungus, which was consistently isolated from the contaminated pelts, was chosen to be characterised using proteomic methods. 1D SDS-PAGE gel/CHIP separation of the proteins in the secretome showed it contained mainly glycosidases, with no lipases, esterases, or proteases identified. Some of the proteins identified had suggested roles in resistance to osmotic pressure, while the remaining proteins were intracellular. Overall, 21 proteins were identified.
A purification procedure involving AEX and SEC was successfully developed for the isolation of one of the glycosidases from the secretome. The resultant purified fractions formed a doublet band when analysed by SDS-PAGE. The reason for this remains unknown, but was shown not to be due to an impurity or heterodimerisation.
The purified glycosidase was identifed as belonging to the GH3 family by mass spectrometry. It was found to have a pH optimum of pH 6.0, was optimally active at 10% NaCl, and was itself glycosylated. The glycosidase was able to hydrolyse both a- and ß- linked glycosidic bonds in di- and polysaccharides. Interestingly, both the disaccharide and artifical p-nitrophenol forms of galactose were not cleaved by the enzyme.
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Keywords
Glycosidase, Purification, Industrial applications, Leather, Hair removal, Research Subject Categories::TECHNOLOGY::Bioengineering::Biochemical process engineering