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Title: Enzymes from Pseudomonas carrageenovora in the analysis of carrageenan structure
Author: McLean, M. W.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1981
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x-Carrageenan was degraded sequentially and quantitatively to neocarrabiose with three enzymes purified from Pseudomonas carrageenovora. A x-carrageenase catalysed the hydrolysis of ideal x-carrageenan sequence to neocarrabiose 4-O-sulphate and neocarratetraose 4-O-disulphate. It also slowly cleaved the tetrasaccharide to the disaccharide. A novel purification procedure for this enzyme was developed. A glycosulphatase hydrolysed the ester of neocarrabiose 4-O-sulphate, and the non-reducing end sulphate of neocarratetraose 4-O-disulphate. An [³⁵S] radioassay was devised for this enzyme and the first complete purification then achieved. Neocarratetraose 4-O-monosulphate β-hydrolase ('tetrasaccharidase'), the third enzyme was discovered and found to catalyse the β-hydrolysis of the substrate. A [¹⁴C]radioassay was devised for this activity and the enzyme was functionally purified. β-Neocarrabiose was crystallized and characterized. The distinctive C-1' and H-1' NMR chemical shifts were explained in terms of 'steric compression'. Partial relief of this constraint was observed after alditol formation. An assay for x-carrageenan structure was developed, and based on the quantitative formation of neocarrabiose by the concerted action of these three enzymes. A non-homologous hexasaccharide was produced from non-ideal regions of native x-carrageenan by digestion with x-carrageenase. It was shown to be tetrasulphated, and the non-reducing terminal disaccharide unit was sequenced by the glycosulphatase and tetrasaccharidase. A fourth enzyme activity was discovered that was involved in the degradation of the non-homologous hexasaccharide.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available