Enzymes from Pseudomonas atlantica in the analysis of porphyran structure
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.