Catalytic function and affinity purification of site-directed mutant β-cyclodextrin glucanotransferase from alkalophilic Bacillus firmus var. alkalophilus

Subsites −3 and −7 in the active site of β-cyclodextrin glucanotransferase (β-CGTase) from alkalophilic Bacillus firmus var. alkalophilus were modified through site-directed mutagenesis to obtain novel mutant CGTases. Four mutant CGTases, H59Q, Y96M, 90-PPI-92, and Δ(154–160) were constructed and produced using a recombinant E. coli with a secretive expression system extracellularly. The secreted mutant β-CGTases were purified by one-step affinity adsorption chromatography using a β-cyclodextrin (CD) polymer as an adsorbent to nearly homogeneous purity. The catalytic activities were modified significantly compared to the wild-type. In particular, the Y96M and Δ(154–160) mutants increased cyclization activity around 1.5 times without any significant reduction of coupling and hydrolyzing activities. Meanwhile, the Y96M and Δ(154–160) mutants exhibited a much higher conversion yield into CDs from 28.6 to 39% without any recognizable change in the CD ratio. The conversion yield into linear maltooligosaccharides was also significantly reduced. The catalytic functions of subsites −3 and −7 in the active site of β-CGTase would appear to be related to the overall productivity of CDs rather than the product specificity.