Cloning, Overexpression and Immunological Detection/Purification of Recombinant B. Macerans; Cyclodextrin Glycosyltransferase in E.coli

摘要

Cyclodextrins (CDs) are high value molecular chelating agents produced by enzymatic synthesis from starch, which have applications in foods, pharmaceuticals, enantiomeric separations and cosmetics. This paper highlights our work on B.macerans cyclodextrin glycosyltransferse (CGTase, E.C. 2.4.1.19) involving cloning and expression fo the B. macerans cgt gener in E.coli, characterization of its kientic activity, development of immunological detection and purification techiques and synthesis of high expression fusion proteins with E. coli thioredoxin. The cgt gene from B. macerans was amplified by polymerase chain reaction, cloned and expressed in E.coli, as a mixture of soluble protein and as inclusion body proteins in the pET-21(+)- cgt/BL21(DE3)pLysS system. Using differnet fermentation media conditions and promoers provided overexpression ranging from a 39-fold increase (54.9 mg/L) to 115-fold increase (160.4 mg/L) after renaturation of the inclusion proteins. Recombinant genetic fusion fo the cgt gene with the E coli thioredoxin gene at the N-terminus significantly increased the expression of soluble CGTase, up to 1.2 g/L, when expressed at low temperature with the addition of Ca~2+. Kinetic studies of the enzyme demonstrated classical substrate inhibition, with an inhibition constant of 5.89+-0.27 mg/mL and optimal substrate concentration at 0.55 mg/mL. Product inhibition studies, using alpha-, beta-, and gamma-CDs, were fitted to a uncompetitive kinetics model, with inhibition constants of 0.077 +_0.005 mg/ml for alpha-CD, and +-0.01 mg/ml for both beta- and gamma-CDs. The mean maximum rate of formation of CD and the mean Michaelis-menten constant were determined to be 0.0046+-0.0001 mumol/min-mu, and 0.054+-0.005 mg/mL respectively. These models support the hypothesis that the catalytic structure of the CGTase is composed of at least two binding sites. Polyclonal antibodies raised agaisnt the enzyme were used to develop a series of rapid enzyme-linked immunosorbent assays (ELISA) with sensitivities down to 0.2 mug/mL for both native and denatured proteins, as well as recombinant mutations. No cross-reactivity was detected to alpha-, beta-amylases or pullulanase. Immunoaffinity chromatographic columns were developed for purification of recombinant CGT as from cell lysates, along with procedures which allow stable re-use of these columns.