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

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Abstract
A novel bacterial sulfatase was discovered in an extract of F. heparinum. The enzyme hydrolyses the 3-O-sulfate from 2-deoxy-2-sulfamido-3-O-sulfo-D-glucose and 2-acetamido-2-deoxy-3-O-sulfo-D-glucose. The activity was purified 10,800-fold by chromatography successively on CM-Sepharose CL-6B, hydroxyapatite, taurine-Sepharose CL-4B and CM-Sepharose CL-6B. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed the enzyme to be homogeneous and of relative molecular mass 56,000. Two novel assays were developed using 2-[14C]acetamido-2-deoxy-3-O-sulfo-D-glucose and 2-deoxy-2-sulfamido-3-O-sulfo-D-glucose as respective substrates. The purified 3-O-sulfatase was shown to be free of all other known heparin-degrading enzymes. Optimal activity was at pH 7.5 for the disulfated 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+. Pi and sulfate inhibited 3-O-sulfatase activity. The Km value of the N-acetylated substrate was determined to be 42 .mu.mol .cntdot. dm-3. No activity was detected with 2-amino-2-deoxy-3-O-sulfo-D-glucose.

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