Mutation of a Bacillus Cyclodextran Glucanotransferase to Increase Its Reaction Velocity.

概要

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Amino acid residues of Cyclodextran glucanotransferase from Bacillus circulans T-3040 were replaced using site-directed mutagenesis to improve the productivity of cyclodextrans. The computer program of random-centeroid optimization for genetics was applied for selecting the positions of mutations to change hydrophobicity or bulkiness of the enzyme protein. Two mutants, namely A452N and V744L, produced larger amounts of CIs per hour than the wild-type enzyme. A452N and V744L mutant enzymes increased the reaction velocities 3- and 2-fold, and Km values 9- and 3-fold, respectively. Activation by Ca2+ and inactivation by Cu2+ were reduced in both mutants. Optimum temperature and pH, and stability against changes in heat and pH of the two mutants were not affected. Wild-type and mutant enzymes were all inactivated with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide in the absence of Ca2+, however, inactivation was reduced by an additional 10 mM of Ca2+.