Purification and properties of inulin fructotransferase (DFA III-producing) from Bacillus sp. snu-7

Inulin fructotransferase (DPA XII-producing) [EG 2.4.1.93] secreted from Bacillus sp, snu-7 was purified 60.3-fold with a yield of 11.6% from a culture supernatant by ammonium sulfate precipitation, preparative isoelectrofocusing, anion exchange chromatography and preparative polyacrylamide gel electrophoresis. The purified enzyme gave a single band on polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 62 kDa on SDS-polyacrylamide gel electrophoresis. The N-ter minal amino acid sequence was found to be Ala-Asp-Gly-Gln-Asp-Gly-Ala-Pro-Leu-Asn-Gln-Val-Asn-Thr-Tyr-Asp. The optimal pH and temperature far the enzyme reaction were 6.0 and 40 degrees C, respectively. The enzyme was stable with a pH range of 4.0 to 7.0 and at up to 60 degrees C. As the production of di-D-fructose 1,2':2,3' dianhydride increased in the course of enzyme reaction, the K-m of the purified enzyme was estimated to be 5.4 mM. One mhz each of Cu2+, Fe2+ and Hg2+ inhibited the enzyme activity strongly. Exhaustive enzymatic digestion of inulin produced 1-kestose, 1-nystose, and 1-F-fructofuranosylnystose as well as di-D-fructose 1,2':2,3' dianhydride.