Purification and Characterization of Recombinant β-CGTase From Bacillus circulans STB01

Abstract:

According to the wild used of cyclodextrin，the cyclodextrin glycosyltransferase（EC 2.4.1.19，CGTase） used to product cyclodextrins industrially has become the focus of scientific research nowadays. In this study，the purification and characterization of recombinant β-CGTase from Bacillus circulans STB01 were measured.The results showed that the recombinant β-CGTase could be purified by a combination of Phenyl HP hydrophobic chromatography and Q-HP anion exchange chromatography. The recovery of the enzyme was 45.3%. The apparent molecular weights of the β-CGTase was about 76 500 and presented monodisperse，which indicated that the purified enzyme was a monomer in solution. The optimum cyclization reaction pH of the β-CGTase was 6.5，and showed more thermostable in glycine-NaOH buffer. The optimum cyclization reaction temperature was 60 ℃. The thermostability of β-CGTase increased gradually with increased concentration of the enzyme. The function of β-CGTase did not required the metal cofactor. During the whole cyclization reaction，β-cyclodextrin was the main product. The kinetics of the β-CGTase catalyzed cyclization reaction could not be described by the Michaelis-Menten equation with corn starch，potato starch or cassava starch as the substrate. However，the kinetics of cyclization reaction could be fairly well described by the Michaelis-Menten equation while used soluble starch or maltodextrins（DE 5，15，25） as the substrate，respectively.