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

Abstract

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 90h 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 degrees C, and half-lives are approximately 8h at 40 degrees C, 1.25h at 45 degrees C and 0.5h 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 Ca(2+), Ba(2+), and Zn(2+) in a concentration-dependent manner, while it is dramatically inhibited by Hg(2+). The kinetics of the alpha-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.