Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding alpha-cyclodextrin glycosyltransferase (alpha-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of alpha-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant alpha-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 C, and half-lives are approximately 8 h at 40 degrees C, 1.25 h at 45 degrees C and 0.5 h at 50 degrees C. The recombinant alpha-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca2+, Ba2+, and Zn2+ in a concentration-dependent manner, while it is dramatically inhibited by Hg2+. The kinetics of the alpha-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation. (C) 2010 Published by Elsevier Ltd.