Production of antibody to detect and purify cyclodextrin glycosyltransferase and over-production of its soluble proteins using gene fusion and controlled environment

Cyclodextrin glycosyltransferase (CGTase, E.C. 2.4.1.19) converts starch to cyclodextrins which have closed-ring structures consisting of six ($\alpha$-CD), seven ($\beta$-CD), or eight ($\gamma$-CD) $\alpha$-1,4-linked-D-glucose residues. The enzyme is commercially important because CDs have various practical applications in the agrochemical, food, and pharmaceutical industries in separation and extraction processes. Bacillus macerans CGTase has been used in industries and well researched. An over-expression system of CGTase was established in a recombinant E. coli using the cgt gene cloned from B. macerans and the site-directed mutations of the cgt gene was performed. For specific and sensitive detection of CGTase and its mutants, polyclonal antibodies were produced in rabbits. The antibodies recognized both the native and denatured CGTase and no cross reactivity was observed with tested other amylolytic enzymes. Mutant CGTases were detectable using these antibodies by Western-blotting. Indirect, sandwich, and competitive ELISA methods were developed to quantify CGTase and its recombinant proteins. Competitive ELISA was proven to be an adequate method in use with accuracy and efficiency. It was found that the antibody-coupled immunoaffinity chromatography was effective to purify CGTase in a small scale. Since CGTase expressed in E. coli accumulated in the form of insoluble aggregates (inclusion bodies), thioredoxin gene fusion system as well as an expression technique under controlled environment were employed to over-produce the active soluble CGTase. The thioredoxin retained its function to fold protein as a soluble form after fusion. The fusion molecules, however, seem to have unstable structure which is susceptible to proteases and high temperature. By using low temperature expression and Ca$\sp{2+}$ supplement in medium, over-production of soluble and highly active CGTase was achieved. These research results provide an important groundwork to tailor a modified CGTase with better properties and to enhance economical production of CGTase