Purification and Characterization of Cycloisomaltooligosaccharide Glucanotransferase and Cloning of cit from Bacillus circulans U-155

Abstract

The enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) was isolated from Bacillus circulans U-155, which had a yield of 25.8%, and purified to homogeneity. The M_r was approximately 98,000, similar to that for all known CITases. Specific activity of the purified enzyme was 2.11 U/mg protein, with maximal activity at approximately pH 6.0. The enzyme was stable at pH 4.5 up to pH 9.0 and at temperatures up to 50°C. The main product of the initial enzyme reaction was cycloisomalto-heptaose. The cit gene has a 2,895-bp open reading frame and encodes CITase in B. circulans U-155. We cloned this gene into a recombinant plasmid pCI811 and expressed it in Escherichia coli. A comparison between DNA sequence data from the transformant and the N-terminal amino acid sequence of the purified enzyme from B. circulans U-155 suggested that CITase was translated as a secretory precursor with a 30-amino-acid signal peptide. The mature enzyme contained 934 residues with a predicted molecular mass of 103.93 kDa. The enzyme activity in the transformants was approximately 3.0 mU/mL, similar to that of the purified enzyme secreted by B. circulans U-155. The enzyme also showed 72% and 67% identity with CITase from Paenibacillus sp. 598K and B. circulans T-3040, respectively. These results suggest that the enzyme isolated from B. circulans U-155 shares a greater similarity with CITase expressed by Paenibacillus sp. 598K than B. circulans T-3040.