Cloning of bacillus gene possessing β-fructofuranosidase activity in escherichia coli

Abstract

A inulase producing spore-former had been isolated, identified as a Bacillus subtilis and used as gene dono. The purified genomic DNA from Bacillus subtilis was digested with PstI and eluted by electrophoresis to four fraction according to the size. Fractions 1, 2, 3, 4 corresponding average 13.5 kb, 3 kb, 1.5 kb, 0.6 kb in size. Fractions 1 and 2 were ligated with a vehicle, pUC9 which was digested with PstI adn dephosphorylated with CIP. The ligated DNA was transformed into competent E. coli JM 103 cells and spreaded on the tetrazolium plates. After incubation at 37$^\circ$C for 24 hours, one transformant that formed large and red color colony was selected adn assayed for inulase activity. That transformant showed 0.8 times less inulase activity than the original Bacillus subtilis JU 70. The insert size of the plasmid carrying the inulase gene was 2.1 kb. The restriction map of recombinant plasmid was constructed using restiriction enzymes AvaII, EcoRI, HindIII, PstI, SacI et al.