Extracellular expression and biochemical characterization of alpha-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 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.
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave., Wuxi 214122, PR China.
Zhaofeng
Z
Li
Bin
B
Li
Zhengbiao
Z
Gu
Guocheng
G
Du
Jing
J
Wu
Jian
J
Chen
D003001:Cloning, Molecular; D003500:Cyclization; D003505:Cyclodextrins / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D013696:Temperature
D003505:Cyclodextrins; D008670:Metals; D005964:Glucosyltransferases; C019963:cyclomaltodextrin glucanotransferase