Cloning and expression of isomannanase gene and characterization of the enzyme

A gene (isoman K-6) encoding an isomannanase of Bacillus subtilis K-6 was cloned using the plasmid vector pET-30 (a) and expressed in Escherichia coli BL21 (DE3) . Genbank accession number of isoman K-6 was HQ902141, full length of isoman K6 was 1083bp, encoding 360 amino acid residues, activity of recombinant strain was 415.9U/mL. SDS-PAGE demonstrated the molecular mass of recombinant enzyme was 45000u. The isomannanase was purified by IDA HisBind resin column, enzymatic characteristics analysis showed that the enzyme could be activated by Al3+ and Ba2+, the optimum temperature was 55℃, the optimum pH for the enzyme was 6.0 and was stable from pH4.5 to 7, The apparent Km and Vmax values for locust bean gum were 1.3U/mg and 7.3mg/mL, respectively.