Cell surface expression of gamma-CGTase from Evansella caseinilytica on E. coli Application in the enzymatic conversion of starch to gamma-cyclodextrin

The gamma-cyclodextrin glycosyltransferase (gamma-CGTase) from Evansella caseinilytica was expressed on the cell surface of E. coli using pAIDA-I autotransporter and was further utilized in the conversion of starch to gamma-cyclodextrins (CDs). Maximum cyclization activity of 2.28 +/- 0.46 U/g biomass was achieved after 3 h of induction using 0.1 mM IPTG at 37 degrees C. Surface expression of gamma-CGTase was confirmed using flow cytometry employing a FITC-conjugated anti-HIS antibody. Biochemical characterization of surface-displayed gamma-CGTase revealed optima at pH 10.0 and 40 degrees C along with a t(1/2) of 24.75 mM at 50 degrees C. The K-m and V-max values on soluble potato starch were 10.94 mg/ml and 4.33 mu moles min(-1) g(-1) DCW respectively, and the activation energy was calculated to be 89.8 kJ/mol. The surface displayed gamma-CGTase was further utilized for CD production and specifically, gamma-CD conversion was obtained. The maximum conversion was achieved at 50 degrees C, pH 9.0 using soluble potato starch (2.5%; w/v) taking a final enzyme concentration of 0.6 U/g starch. The surface-displayed gamma-CGTase was able to convert soluble potato starch (2.5%) into gamma-CDs with a 72.7% specific yield and no other peaks corresponding to alpha- and beta-CDs were observed on HPLC. The enzyme was found to be similar to 100% operationally stable for up to 2 consecutive cycles of 24 h, with > 75% storage stability at - 20 degrees C even after 7 days.