Purification and Some Properties of Agarase from Pseudomonas sp. PT-5

抄録

An agarase (agarose 4-glycanohydrolase, EC 3.2.1.81) was purified from the culture fluid of Pseudomonas sp. PT-5 by ammonium sulfate precipitation followed by Cellulofine GC-700m, Hydroxyapatite, Butyl-Toyopearl 650M, and Toyopearl-HW 508 column chromatography. The purified enzyme gave a single band on polyacrylamide gel disc electrophoresis and its molecular weight was 31, 000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was 3.6. The amino-terminal sequence was H•Ala-Asp-Trp-Asp-Gly-Leu-Ala-Val-Pro-Ala-Asp-Ala-Gly-Asp-Gly-. The enzyme was stable from pH 6 to 9 and had its maximum activity at pH 8.5. The enzyme rapidly reduced the viscosity of agarose solution and its activity was greatly inhibited by metal ions such as Zn²⁺, Cu²⁺, Co²⁺, Fe²⁺, and Al³⁺ at 1 mM concentration. The enzyme activity was elevated by 75% in the presence of 0.1 M NaCl.