Studies on the cyclodextrin glycosyltransferase (CGTase) production capacity of alkaliphilic bacteria isolated from Salda lake and on the CGTase gene

Cyclodextrin glycosyltransferase (CGTase) enzyme is used industially to obtain cyclodextrins. Also, this enzyme has a catalytic function which is utilized in the modification of some food additives. This study aims to isolate potential bacterial strains from the soda lakes in Turkey, amplification of the respective gene, cloning into Pichia pastoris for recombinant production and characterization of the enzyme. Four novel CGTase producing alkalophilic bacteria were isolated from lakes Salda and Van and coded SG2, SG3, V3, SD5. The isolates were gram(+) rods and 16S rDNA sequencing revealed these were novel Bacillus strains. The gene encoding for CGTase was amplified using the DNA of the SD5 strain and cloned into the yeast P. pastoris. PCR was conducted seperately with three different sets of primers in order to amplify and/or modify the gene sequence. The fragments obtained were sequenced and cloned into P.pastoris X33 host strain using the pPICZαA vector allowing for methanol induced expression and extracellular secretion of the protein. Thus, three recombinant strains were obtained; N2 (with natural gene sequence), S3 (also encoding the c-myc epitope and polyhistidine tail) and M5 (encoding the polyhistine tail). Extracellular CGTase activity was monitored throughout fermentation for each strain. The molecular weight of the enzymes were ~85 kDa. The CGTase GLYCOSYLTRANSFERASE obtained from the M5 strain was purified and displayed optimum activity at 50 °C and pH 6, producing γ-β-cyclodextrin. Investigation of the thermal and pH stability of showed that the enzyme retained about 100% activity after 30 minutes at 30-50 °C and pH 9.