Neocarratetraose 4‐ O ‐Monosulphate β‐Hydrolase from Pseudomonas carrageenovora

Neocarratetraose 4‐ O ‐monosulphate β‐hydrolase has been enriched 73‐fold from the cell‐free extract of Pseudomonas carrageenovora . This enzyme catalyses the hydrolysis of the β‐glycosidic linkage of neocarratetraose 4‐ O ‐monosulphate [3,6‐anhydro‐α‐ d ‐galactopyranosyl‐(1–>3)‐1‐ d ‐galactopyranosyl‐(1–>4)‐3,6‐anhydro‐α‐ d ‐galactopyranosyl‐(1–>3)‐ d ‐galactose 4‐ O ‐sulphate]. The products of hydrolysis are respectively neocarrabiose [3,6‐anhydro‐α‐ d ‐galactopyranosyl‐(1–>3)‐ d ‐galactose] and neocarrabiose 4‐ O ‐sulphate [3,6‐anhydro‐α‐ d ‐galactopyranosyl‐(1–>3)‐ d ‐galactose 4‐ O ‐sulphate]. During purification the χ‐carrageenase and 4‐sulphatase activities present in the bacterial extract were resolved from the title enzyme. From dodecylsalphate/polyacrylamide gel electrophoresis the enzyme has a molecular weight of 94000. Calcium stimulated the activity, K m 0.50 mmol dm −3 . Optimal activity was found at pH 7.0 and the K m determined for the nominal substrate was 68 μmol dm −3 . [ 14 C]Neocarratetraose 4‐ O ‐monosulphate was prepared from [ 14 C]carrageenan ( Chondrus crispus ) and utilized in a radiochemical assay for neocarratetraose 4‐ O ‐monosulphate β‐hydrolase. Synthesis of [1‐ 3 H]neocarratetraitol 4‐ O ‐[ 35 S]monosulphate yielded another substrate. This alditol was hydrolysed to yield a single radioactive species, thus supporting the position of sulphation of neocarratetraose 4‐ O ‐monosulphate deduced from 13 C NMR studies. Neocarratetraose 4‐ O ‐disulphate [3,6‐anhydro‐α‐ d ‐galactopyranosyl‐(1–>3)‐4‐ O ‐sulphato‐β‐ d ‐galactopyranosyl‐(1–>4)‐3,6‐anhydro‐α‐ d ‐galactopyranosyl‐(1–>3)‐ d ‐galactose 4‐ O ‐sulphate] was not hydrolysed by the purified glycosidase. Tentative evidence for transglycosylation was obtained with both monosulphated substrates. A pathway is described for the sequential degradation of neocarratetraose 4‐ O ‐disulphate to neocarrabiose.