A single mutation in cyclodextrin glycosyltransferase from Paenibacillus barengoltzii changes cyclodextrin and maltooligosaccharides production.

Cyclodextrin glycosyltransferases (CGTases) are bacterial enzymes that catalyze starch conversion into cyclodextrins, which have several biotechnological applications including solubilization of hydrophobic compounds, masking of unpleasant odors and flavors in pharmaceutical preparations, and removal of cholesterol from food. Additionally, CGTases produce maltooligosaccharides, which are linear molecules with potential benefits for human health. Current research efforts are concentrated in the development of engineered enzymes with improved yield and/or particular product specificity. In this work, we analyzed the role of four residues of the CGTase from Paenibacillus barengoltzii as determinants of product specificity. Single mutations were introduced in the CGTase-encoding gene to obtain mutants A137V, A144V, L280A and M329I and the activity of recombinant proteins was evaluated. The residue at position 137 proved to be relevant for CGTase activity. Molecular dynamics studies demonstrated additionally that mutation A137V produces a perturbation in the catalytic site of the CGTase, which correlates with a 10-fold reduction in its catalytic efficiency. Moreover, this mutant showed increased production of maltooligosaccharides with a high degree of polymerization, mostly maltopentaose to maltoheptaose. Our results highlight the role of residue 137 as a determinant of product specificity in this CGTase and may be applied to the rational design of saccharide-producing enzymes.

Isolation of two strains (S-R type) of Bacillus circulans and purification of a cyclomaltodextrin-glucanotransferase.

From wild type inoculum of Bacillus circulans DF 9 which produce cyclomaltodextrin-glucanotransferase (EC 2.4.1.19, CGTase), two different kinds of colonies were isolated, which correspond to the classical S-R variation. From the culture medium of both colonies grown together a CGTase was purified about 50 fold with a yield of 54% in two steps. From pure R-cell culture the enzyme was purified by about 38 folds with a yield of 79% in only one step, showing a complete homogeneity as judged by a native PAGE analysis.

Some properties of a cyclomaltodextrin-glucanotransferase from Bacillus circulans DF 9 R type.

The cyclomaltodextrin-glucanotransferase (EC2.4.1.19, CGTase) which was purified to homogeneity from Bacillus circulans strain DF 9, R type, showed a pI of 5.3 determined by disc-isoelectric focusing, a Mw of 78 kDa estimated by SDS-PAGE with a range of pH of optimal enzymatic activity rather large (4.5-7.5). The thermal stability of the enzyme at 55 degrees C was increased 4-5 times when calcium ion (10 to 100 mM) or alpha-cyclodextrins (10 mM) were added to the preincubation mixtures. The alpha: beta: gamma ratio determined by HPLC was about 1:0.9:0.4 and the maximal conversion to cyclodextrins with 5% soluble starch was about 36%.