The ENZYMIC HYDROLYSIS OF CARRAGEENAN BY PSEUDOMONAS CARRAGEENOVORA: PURIFICATION OF a Κ-Carrageenase

Weigl, Josef; Yaphe, W.

doi:10.1139/m66-127

Cited by 82 publications

(40 citation statements)

References 8 publications

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“…2). Because the original K-carrageenan was isolated with care and there is little likelihood of contamination by ,u-carrageenan itself, our enzyme evidence adds support to the conclusion of Weigl and Yaphe (16), based on chemical conversion, that the ERF represents oligomeric segments of metabolically unfinished ec-carrageenan. The higher sulfohydrolase activity obtained with ERF as compared with,ucarrageenan is a reflection ofthe lower viscosity, smaller molecular size, and higher molar concentration of the ERF.…”

supporting

confidence: 72%

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Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

Wong¹,

Craigie²

1978

Plant Physiol.

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An enzyme catalyzing the conversion of i-to X-carrageenan Cell walls of red seaweeds frequently contain conspicuous amounts of galactans among which the carrageenans are a commercially important group. These polysaccharides are composed of regularly alternating a-1,3; 1-1,4-linked D-galactose residues which differ in degree and position of sulfate esterification, and in their content of 3,6-anhydrogalactose (12). As early as 1963 Rees (1 1) postulated that a "A-like" fraction from Chondrus crispus may be the biological precursor of K-carrageenan in a manner analogous to that seen in the biosynthesis of porphyran. In the latter case L-galactose-6-sulfate residues in the polymer were converted to 3,6-anhydrogalactose by an enzyme present in Porphyra umbilicalis (10). The predicted enzyme activity was detected in extracts of Gigartina stellata (6), but similar preparations from C. crispus appeared to be inactive (Rees, personal communication).The present paper is the first characterization of an enzyme from C. crispus which eliminates sulfate from p-carrageenan to form the 3,6-anhydrogalactose of K-carrageenan. The enzyme was not sufficiently characterized to be listed in the 1972 Enzyme Nomenclature. Because the mechanism ofthe reaction is unknown the name "sulfohydrolase" used in the present report is based on the release of sulfate from the polymer. Enzyme Extraction from Haploid Plants. Apical 2-to 3-cm pieces of C. crispus (100 g) were frozen in liquid N2 and pulverized in a ball mill for 2 min. The resulting flour was allowed to thaw and 150 ml of cold buffer containing 0.05 M Tris-HCl, 0.5 M KCI, and 0.01 M 2-mercaptoethanol (pH 9.5) were added. The suspension was stirred for 2 hr and left overnight in crushed ice. The crude homogenate was recovered after centrifugation at 15,000g for 20 min. Cold saturated (NH4)2SO4 was added and the fraction precipitating between 2.65 and 4.24 M was collected by centrifugation. The precipitate, containing some polysaccharides as well as the active protein, was dissolved in 25 ml of 0.05 M Tris-HCl with 2-mercaptoethanol (pH 7.1) and dialyzed overnight in the same buffer. This enzyme preparation was routinely used. MATERIALSPurification of DEAE-CeUlulose. The protein (150 mg) from the (NH4)2SO4 precipitation was applied to a column (25 x 3 cm) of DEAE-cellulose previously equilibrated with 0.05 M borate containing 0.01 M 2-mercaptoethanol (pH 7.5). The column was washed with 250 ml of the same buffer followed with I M KCl.Three-ml fractions were collected every 1.6 min. Fractions 141 to 159 containing the active enzyme were pooled and treated with saturated (NH4)S04 until 4.24 M was reached. The precipitate collected by centrifugation was redissolved in 0.05 M Tris-HCl containing 0.01 M 2-mercaptoethanol (pH 7.1) and dialyzed overnight in the same buffer.Preparation

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confidence: 72%

“…A culture of Pseudomonas carrageenovora was kindly provided by W. Yaphe, McGill University, Montreal. K-Carrageenase was prepared to the first DEAEcellulose step as described by Weigl and Yaphe (16).…”

mentioning

confidence: 99%

Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

Wong¹,

Craigie²

1978

Plant Physiol.

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“…Therefore to extend this study, we report here the cloning and mechanistic characterization of the first λ-carrageenase. Up to now, the marine bacterium P. carrageenovora is the only micro-organism known to degrade λ-carrageenan [18]. This activity was initially proposed to be due to an extracellular complex involving three hydrolases [19].…”

Section: Introductionmentioning

confidence: 99%

“…This marine bacterium was grown in the presence of 10 g of λ-carrageenan purified from the tetrasporophytic plant of G. skottsbergii at 20 • C for 48 h in 5 litres of the Y-2 modified medium [18]. The culture medium was centrifuged (10 000 g for 30 min) and the cell-free supernatant was concentrated five times by tangential ultrafiltration (Pellicon system, 10 kDa; Millipore).…”

Section: Introductionmentioning

confidence: 99%

Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases

Guibet¹,

Colin²,

Barbeyron³

et al. 2007

Biochem. J.

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Carrageenans are sulfated galactans found in the cell walls of red seaweeds. They are classified according to the number and the position of sulfate ester groups. lambda-Carrageenan is the most sulfated carrageenan and carries at least three sulfates per disaccharide unit. The sole known depolymerizing enzyme of lambda-carrageenan, the lambda-carrageenase from Pseudoalteromonas carrageenovora, has been purified, cloned and sequenced. Sequence analyses have revealed that the lambda-carrageenase, referred to as CglA, is the first member of a new family of GHs (glycoside hydrolases), which is unrelated to families GH16, that contains kappa-carrageenases, and GH82, that contains iota-carrageenases. This large enzyme (105 kDa) features a low-complexity region, suggesting the presence of a linker connecting at least two independent modules. The N-terminal region is predicted to fold as a beta-propeller. The main degradation products have been purified and characterized as neo-lambda-carratetraose [DP (degree of polymerization) 4] and neo-lambda-carrahexaose (DP6), indicating that CglA hydrolyses the beta-(1-->4) linkage of lambda-carrageenan. LC-MALLS (liquid chromatography-multi-angle laser light scattering) and (1)H-NMR monitoring of the enzymatic degradation of lambda-carrageenan indicate that CglA proceeds according to an endolytic mode of action and a mechanism of inversion of the anomeric configuration. Using 2-aminoacridone-labelled neo-lambda-carrabiose oligosaccharides, in the present study we demonstrate that the active site of CglA comprises at least 8 subsites (-4 to +4) and that a DP6 oligosaccharide binds in the subsites -4 to +2 and can be hydrolysed into DP4 and DP2.

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“…that degrade carrageenan have been isolated from a marine environment (9). The carrageenase produced by a marine Cytophaga has been examined (10), as no one has reported such an enzyme except for that produced by Pseudomonas carrageenovora (3,5,7,(13)(14)(15).…”

mentioning

confidence: 99%

Potentiality of Artificial Sea Water Salts for the Production of Carrageenase by a Marine Cytophaga sp.

Sarwar

Oda

Sakata

et al. 1985

Microbiology and Immunology

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Production of an extracellular enzyme complex (carrageenase) was studied by examining cell-free fluids from cultures of a marine Cytophaga, 1 k-C783, growing on different media.Among artificial sea water salts, only NaCl and MgCl2 were utilized by the organism to produce carrageenase.The minimal concentrations of suitable com2 binations of NaCl and MgCl2 were found to be 0.05 M NaCl plus 0.25 M MgCl2, and 0.15 M NaCl plus 0.15 M MgCl2.KCl and CaCl2 did not have any role in carrageenase production in ZoBell 2216 E broth medium.Carrageenase was synthesized continuously within the resting cells and was released from the cells as well as in the growing cells, when nutrient had been supplied.

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The ENZYMIC HYDROLYSIS OF CARRAGEENAN BY PSEUDOMONAS CARRAGEENOVORA: PURIFICATION OF a Κ-Carrageenase

Cited by 82 publications

References 8 publications

Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

Sulfohydrolase Activity and Carrageenan Biosynthesis in Chondrus crispus (Rhodophyceae)

Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to κ- and ι-carrageenases

Potentiality of Artificial Sea Water Salts for the Production of Carrageenase by a Marine Cytophaga sp.

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