Purification and properties of cyclodextrin glucanotransferase synthesizing 2-O-α-D-glucopyranolsyl L-ascorbic acid from Paenibacillus sp, JB-13

A Gram-positive bacterium (strain JB-13) that was isolated from soil as a producer of cyclodextrin glucanotransferase (CGTase) [EC 2.4.1.19] was identified as Paenibacillus sp. JB-13. This CGTase could catalyze the transglucosylation reaction from soluble starch to L-ascorbic acid (AA). A main product formed by this enzyme with a-glucosidase was identified as 2-O-alpha -D-glucopyranosyl L-ascorbic acid (AA-2G) by the HPLC profile and the elemental analysis. CGTase was purified to homogeneity using ammonium sulfate fractionation, ion-exchange chromatography on DEAE-Sephadex A-50, and gel chromatography on Sephacryl S-200HR. The molecular weight was determined to be 66,000 by both gel chromatography and SDS-PACE. The isoelectric point of the purified enzyme was 5.3. The optimum pH and temperature was pH 7.0 and 45 degreesC, respectively. The enzyme was stable in the range of pH 6-9 and at temperatures of 75 degreesC or less in the presence of 15 mM CaCl2, Hg2+, Mn2+, Ag+, and Cu2+ all strongly inhibited the enzyme's activity.