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Title: Enzymes from Pseudomonas atlantica in the analysis of porphyran structure
Author: Morrice, L. M.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1982
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Three hydrolytic enzymes from Pseudomonas atlantica were resolved by gel filtration. Novel assays for two of these activities were devised. Two distinct endo-β-agarases (I & II) were detected in the medium of this bacterium. The predominant activity (β-agarase I) corresponding to that isolated by Yaphe [Yaphe, W. (1966) in Proc. 5th Int. Seaweed Symp. pp. 333-335.] was purified 674-fold to homogeneity. This enzyme was found to specifically cleave at the reducing side of units of β-neoagarobiose (3,6-anhydro-α-L-galactopyranosyl-(1+3)-β-D-gaiactopyranose). Porphyran, a substituted agarose from Porphyra umbilicalis, was degraded by highly purified β-agarase I and the digestion products characterised by ¹³C-NMR spectroscopy. From the data several novel porphyran sequences were discovered. The oligosaccharides of porphyran digestion were divided into low and high molecular weight fractions by dialysis. The permeate (23% of starting carbohydrate) was separated by ion-exchange into neutral and anionic fractions. Gel filtration of the neutral fraction (19%) resolved two major oligosaccharides. These were shown by ¹³C-NMR to be 6³-0-methyl-neoagarotetraose and 6³,6⁵-di-0-methyl-neoagarohexaose. Gel filtration of the anionic oligosaccharides (3.3%) revealed two monosulphated tetrasaccharides, 6-0-sulphato-α-L-galactopyranosyl-(1+3)β-D-galactopyranosyl-(1+4)-3,6-anhydro-α-L-galactopyranosyl-(1+3)-β-D galactopyranose and its 6³-0-methylated derivative. The ¹³C-NMR data from the sulphated tetrasaccharides provided a novel reference which was used to characterise higher sulphated fragments in the dialysis permeate. The fraction retained on dialysis (77%) was of average degree of polymerisation 40 and was found to be homologous to the high molecular weight anionic permeate. Porphyran was found to comprise 49% sulphated disaccharide units and these were calculated to occur in stretches averaging 2.0-2.5 contiguous units.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry