Gene cloning and characterization of the cyclodextrin glycosyltransferase from thermotolerant Paenibacillus sp. BT01

Abstract

Cyclodextrin glycosyltransferase (CGTase) catalyzes the conversion of starch to a mixture of cyclic oligosaccharides, cyclodextrins(CDs). A cyclodextrin glycosyltransferase gene from Paenibacillus sp.BT01 isolated from waste of a starch factory in Thailand, was cloned into Escherichia coli BL21(DE3) using pET19b vector. Determination of the nucleotide sequences of both wild type (BT01) and recombinant plasmid showed the presence of 2142 bp open reading frame which encodes a polypeptide of 713 amino acid residues, with 686 amino acids for mature enzyme. The nucleotide and amino sequences showed highest homology of 99% to Bacillus circulans A11. About 99.83% of total CGTase was secreted into the LB medium after induction with 0.2 mM IPTG for 24 hours and the specific activity was 6.6 folds higher than wild type. The wild type and recombinant CGTase was purified to homogeneity by starch adsorption and DEAE-cellulose with 12.18 %yield. 24 purification fold in wild type and 70.98% yield, 8 purification fold in recombinant pBT. The molecular weight of both enzymes were estimated to be 71 kDa by SDS-PAGE. Both enzymes showed one major band at pI 4.74 and two minor bands at 4.86 and 4.62 on isoelectric focus gel electrophoresis. The enzymes from BT and pBT exhibited optimum pH and temperature for dextrinizing activity at pH 5.0 at 50 degree Celsius.Optimum conditions of cyclization activity were pH 6.0 at 50 degree Celsius.The enzymes were stable at pH 6.0-10.0 up to 50 degree Celsius.In the presence of 2% soluble starch the enzymes were stable up to 70 degree Celsius.CGTase catalyzed the conversion of starch to mixure of cyclodextrins with the ratio of alpha-:beta-:gamma-CDs at 1.0 : 2.0 :0.72, when incubated with 10% soluble starch at 60 degree Celsius,pH6.0 for 24 hours. The total beta-CD produced were 22.52 g/l in wild type and 19.33g/l in recombinant.The source of starch did not significantly affect the CGTase action and the ratio of alpha-:beta:-gamma-cds remained constant.Kinetic parameter were determined by used cyclization activity.Calculated the Km and kcal/Km were 5.36mg/ml and 40.82(min)[superscript -1](mg/ml)[superscript -1] in wild type and 4.90 mg/ml and 91.35(min)[superscript -1](mg/ml)[superscript -1] in recombinant CGTase,respectively.