The agarose-degrading system of Pseudornorzas atlantica has been re-examined. In addition to the previously reported extracellular endo-/-agarase [Y aphe, W. (1966) in Proceedings 5th International Seaweed Symposium, pp. 333 -3351 a second, membrane-bound endo-enzyme activity, [hgarase I1 has been discovered. These two enzymes act in concert to degrade agarose to neoagarobiose [3,6-anhydro-a-i.-galactopyranosyl-( 1 -+ 3)-~-galactose] and also to degrade partially 6-0-methylated agarose to neoagarobiose and 61-0-methyl-neoagarobiose. Novel assays were devised for fi-agarase I1 and the associated disaccharidase, neoagarobiose hydrolase. These allowed the critical purification of P-agarase I and 11. P-Agarase I was purified 670-fold from the bacterial medium by a new method using ammonium sulphate precipitation and gel filtration on Sephadex G-100. The enzyme was resolved from the small amount of extracellular /I-agarase IT. Dodecylsulphate/polyacrylamide gel electrophoresis indicated a homogeneous protein and a molecular weight of 32 000. Activity was observed against agar over the pH range 3.0 ~ 9.0 and optimally at pH 7.0. The enzyme could be used indefinitely at 30 C but only for up to 2 h at 40 ,C.fi-Agarase 11 was partially purified (5-fold) from the soluble fraction of disrupted cells by chromatography on Sephadex G-I 00, hydroxyapatite and DEAE-Sepharose CL-6 B. This preparation was free of /I-agarase I and disaccharidase. P-Agarase TI was stimulated by NaCl, optimally in the range 0.10-0.20 mol dm-3 (2.4-fold the activity at 0.010 mol d m -3 NaCI). Alkali earth metal (0.002 mol d m -3 CaC1, or 0.005 mol dm-3 MgCI,) gave 1.2-fold the normal activity. Optimum activity was over pH 6.5-7.5.Although some of the structural components of agar-type polysaccharides were elucidated by chemical methods [I] the final structure was deduced from enzymic studies [2, 31.Agar-degrading bacteria were first described by Gran in 1902 [4]. In 1956 Araki and Arai [2] reported the agardegrading bacterium, Pseudonzonas kyoterzsis. Using a crude, extracellular enzyme they detected four oligosaccharides, the lowest member being neoagarobiose. From the same digest they isolated 42-neoagarobiosyl-neoagarobiose, neoagarotetraose [3]. Both oligosaccharides had been derived from unsubstituted agarose sequence.In 1957 Yaphe described the same series of oligosaccharides from the digestion of agar by an extracellular [hgarase from Pseudomonas atlantica [S]. In a later publication he noted the existence of two intracellular enzyme activities [6]. One of these, '[j-neoagarotetraose hydtolase', hydrolysed neoagarotctraose to neoagarobiose whilst the other split this disaccharidc to its component monosaccharides. Later the same author and coworkers succeeded in resolving these activities [7, 81. Similar enzyme systems have been reported by Vattuone et al. [9] and by van der Meulen and Harder [lo].As a prerequisite for an investigation of the highly substituted agar, porphyran, the enzyme system of /'. utlanticn was re-examined. A second and...

A x-carrageenase was isolated from the cell-free medium of cultured Pseudomonas carrageenovora. From dodecylsulphate/polyacrylamide gel electrophoresis, a single protein (identified as the x-carrageenase) was detected in the medium. Activity against nominal carrageenan types and inspection of the products indicate the enzyme to be a x-carrageenase.Purification is described here by ammonium sulphate precipitation and subsequent CM-Sepharose C L d B ion-exchange chromatography. Molecular weight was estimated as 35 000 by dodecylsulphate/polyacrylamide gel electrophoresis. Products of degradation were analysed by gel filtration, spectrophotometric assays and 13C nuclear magnetic resonance. These results are consistent with the product of limit digest being neocarrabiose 4-O-sulphate.

Hydrated and partially hydrated films and aqueous solutions of heparin, heparans and N-desulphated preparations of these polymers were studied by near- and fundamental-region-i.r. spectroscopy in the presence of a range of countercations. The results suggest that ion binding is not explicable solely in terms of simple electrostatic theory, and that specific cation effects, and the hydration pattern of the polymer-cation complex need to be taken into account.

The sol → gel → sol transformation of aqueous K‐carrageenan occurs with complex changes in optical rotation which may be explained qualitatively in terms of the double helix model for junction zones. Evidence is given that the double helices in the gel are normally aggregated but can be kept separate within a narrow temperature range, and that gelation is a kinetic rather than an equilibrium process. An attempt has been made to predict the preferred conformations of irregular carrageenans by joining segments of simpler polysaccharides; the effect of certain structural variations on gel properties may then be explained. Model building in the computer does not exclude the possibility of double helices for the following polysaccharides which are structurally related to K‐ and i‐carrageenan: hyaluronic acid, chondroitin, chondroitin 4‐sulfate, chondroitin 6‐sulfate, dermatan sulfate, keratan sulfate, agarose, porphyran, μ‐carrageenan and furcellaran.

8-Agarase I and I1 were characterised by their action on agar-type polysaccharides and oligosaccharides. P-Agarase I, an endo-enzyme, was specific for regions containing a minimum of one unsubstituted neoagarobiose unit [3,6-anhydro-a-~-galactopyranosyl-( I -+ 3)-~-galactose], hydrolysing at the reducing side of this moiety. Yaphe demonstrated that agar was degraded by this enzyme to neoagaro-oligosaccharides limited by the disaccharide but with a predominance of the tetramer Faphe, W. (1957) Can. J. Microbiol. 3,987 -9931. fi-Agarase I slowly degraded neoagarohexaose but not the homologous tetrasaccharide. [l-3H]Neoagarohexaitol was cleaved to neoagarotetraose and [l-3H]neoagarobiitol. The highly substituted agar, porphyran was degraded to methylated, sulphated and unsubstituted neoagaro-oligosaccharides which were invariably terminated at the reducing end by unsubstituted neoagaro biose.The novel enzyme, /3-agarase 11, was shown to be an endo-enzyme. Preliminary evidence indicated this enzyme was specific for sequences containing neoagarobiose and/or 61-O-methyl-neoagarobiose. It degraded agar to neoagaro-oligosaccharides of which the disaccharide was limiting and predominant. P-Agarase I1 rapidly degraded isolated neoagarotetraose and neoagarohexaose to the disaccharide. With [l-3H]neoagarohexaitol, exo-action was observed, the alditol being cleaved to neoagarobiose and [l-3H]neoagarotetraitol. Neoagarotetraitol was hydrolysed at 4% of the rate observed for the hexaitol. Porphyran was degraded to oligosaccharides, the neutral fraction comprising 24 % of the starting carbohydrate. This fraction was almost exclusively disaccharides (22.4 %) containing neoagarobiose (7.4 %) and 61-O-methyl-neoagarobiose (3 5 %). P-Agarase I1 is probably the 'P-neoagarotetraose The agarose-degrading enzyme system of Pseudomonas atlantica was initially described by Yaphe and co-workers [l-41. The polymer was degraded to neoagaro-oligosaccharides by an extracellular P-agarase [I, 2,5]. The same school reported an oligosaccharidase, 'P-neoagarotetraose hydrolase', which further digested the oligomers to neoagarobiose by exo-scisson [3]. A third enzyme, neoagarobiose hydrolase, split the disaccharide to its monosaccharide constituents [4].Recently a study of the highly-substituted agaroid, porphyran, necessitated the critical isolation of agarose-degrading enzymes and this Pseudomonad system was examined. Two extracellular activities (I and 11) were detected and resolved. fi-Agarase I was used in the study of porphyran primary structure [6]. The respective purifications of agarases I and I1 from the bacterial culture medium and the disrupted cells have been described [7].The present report describes the specificity of P-agarase I1 and contrasts it with that of P-agarase I. Preliminary findings on the action of P-agarase I1 on porphyran are presented. MATERIALS AND METHODSBio-Gel P-2 and AG 1 x 8 (chloride) were from Bio-Rad Laboratories (Richmond, USA). Agar (Special Agar Noble)Enzymes. P-Agarase (EC. 3.2.1 .Sl); neoagarobiose hydrol...