Mechanistic studies on mannuronan C5-Epimerase

Whittaker, Susan Michelle (2001) Mechanistic studies on mannuronan C5-Epimerase. Doctoral thesis, University of Central Lancashire.

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The Pseudomonas aeruginosa algG gene product is a mannuronan C5-epimerase, which converts mannuronate to guluronate residues within the polysaccharide, alginate. The aim of this work was to purii' the epimerase and determine optimum reaction conditions for its enzymic activity.
The epimerase was produced as an over-expressed fusion protein, GST/algG, which was purified by affinity chromatography. Subsequent cleavage of algO from GST produced a yield of 36 % purified epimerase.
A poly mannuronate substrate for the epimerase was produced from a mutant strain of Ps. aeruginosa (FRD462). To enable the poly mannuronate to act as substrate for the epimerase it was deacetylated, deacetylation was confirmed by infrared spectroscopy.
Activity of the purified epimerase was demonstrated by NMR spectroscopy, and by a coupled assay, which coupled the epimerase activity to the activity of a guluronate specific lyase. Lyase activity was shown to be dependent on environmental conditions and could therefore not be used in the coupled assay to determine optimal conditions for epimerase activity.
An assay for epimerase activity was developed from the micro-phenol/sulphuric acid assay of Chang (Chang et aL, 1998). This assay showed activation of the epimerase at low levels of Ca2 (in the range 3 mM -18 mlvi) with higher concentrations showing inhibition. In contrast, higher concentrations of Mg 2 (of the order of 18 mlvi) were needed to induce epimerase activity but no inhibitory effects were observed. Activation of the enzyme was induced by K concentrations in the range 1.5 mM - 8 mlvi, whilst the presence of Ne appeared to have no effect on epimerase activity.
These results also suggested that the mannuronan C5-epimerase brings about conversion of the mannuronate residues within the alginate chain by a preferred or processive attack on substrate. Substrate viscosity was found to have an effect on the rate of epimerisation; increased viscosity led to lower activity.

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