Characterization of tranglycosylation products of recombinant cyclodextrin glycosytransfeerase from Paenibacillus sp.BT01

Abstract

The aim of this study was to use cloned cylcodextrin glycosyltransferase from Paenibacillus sp.BT01 (pBT) in producing transglycosylation products. The enzyme was able to synthesize various products using beta cyclodextrin (-CD) as glucosyl donor and various saccharides (glucose, mannose, fructose, xylose, maltose, etc.) as glucosyl acceptors. Salicin was found to be one of the efficient acceptor with interesting glucosylated derivatives which move faster than glucose standard (G1) when analyzed by TLC. Three kinds of products, with retention time of 6.1 minute, 9.5 minute and 15.7 minute were clearly separated on HPLC and named product 1, 2 and 3. The optimal condition for salicin glycoside production was 3.0 % (w/v) salicin, 1.8% (w/v) -CD and 100 unit/ml recombinant CGTase , pH 6 at 50ºC for 3 hours. After optimization, product 1(Rt 6.1), product 2 (Rt 9.5) and product 3(Rt 15.7) were obtained with yield of 47.23 %, 20.66 % and 9.96 %, respectively. The molecular masses of transfer products 1, 2 and 3 were 417.2 dalton, 633.2 dalton and 795.2 dalton, respectively when analysed by ESI-TOF mass spectrophotometer. The results showed that recombinant CGTase transfer the glucose unit from -CD to hydroxyl group of salicin. The transglycosylation products were identified as glucosyl salicin (α-D-glucopyranosyl-(1→4)-salicin), maltosyl salicin (α-D-glucopyranosyl-1→4-α-D-glucopyranosyl-1→4-salicin) and triosyl salicin (α-D-glucopyranosyl-1→4-α-D-glucopyranosyl-1→4-α-D-glucopyranosyl1→4-salicin) by NMR analysis. The water solubility of the glucosyl salicin maltosyl salicin and triosyl salicin were 72.23, 134.46 and 207.76 mg/ml respectively compared to 23.31 mg/ml for salicin. Salicin glycoside products showed higher anti-coagulation activity than salicin. These salicin glycosides may have useful application in patients with defects in blood coagulation.