Flavobacterium heparinum 3‐O‐sulphatase for N‐substituted glucosamine 3‐O‐sulphate

A novel bacterial sulphatase has been discovered in an extract of Flavobacterium heparinum. The enzyme hydrolyses the 3‐O‐sulphate from 2‐deoxy‐2‐sulphamido‐3‐O‐sulpho‐d‐glucose and 2‐acetamido‐2‐deoxy‐3‐O‐sulpho‐d‐glucose.The activity was purified 10800‐fold by chromatography successively on CM‐Sepharose CL‐6B, hydroxy‐apatite, taurine‐Sepharose CL‐4B and CM‐Sepharose CL‐6B. Sodium dodecylsulphate/polyacrylamide gel elec‐trophoresis showed the enzyme to be homogeneous and of relative molecular mass 56000. Two novel assays were developed using 2‐[14C]acetamido‐2‐deoxy‐3‐O‐sulpho‐d‐glucose and 2‐deoxy‐2‐sulphamido‐3‐O‐sulpho‐d‐glucose as respective substrates.The purified 3‐O‐sulphatase was shown to be free of all other known heparin‐degrading enzymes. Optimal activity was at pH 7.5 for the disulphated substrate and pH 8.0 for the N‐acetylated substrate. Enzyme activity was virtually unaffected by Na+, K+ or Mg2+ ions. A 1.2‐fold enhancement of activity was effected by 0.002 mol dm−3 Ca2+. Inorganic phosphate and sulphate inhibited 3‐O‐sulphatase activity. The Km value of the N‐acetylated substrate was determined to be 42 μmol dm−3. No activity was detected with 2‐amino‐2‐deoxy‐3‐O‐sulpho‐d‐glucose.