Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1.

Cyclodextrin glucosyltransferase (CGTase) is an enzyme which degrades starch to produce cyclodextrins (CDs). In this study, the β-CGTase producing strain T1 was identified as Bacillus sp. by its morphological characteristics and 16S rDNA sequence analysis. The cgt-T1 gene was cloned and expressed in Escherichia coli. CGTase-T1 was purified by Ni-nitrilotriacetic acid agarose column and the molecular weight was determined as approximately 75 kDa using SDS-PAGE analysis. For the expression of soluble proteins, the optimal induction conditions were 10 h at 25 °C with OD600 at 0.8. The purified CGTase-T1 exhibited maximum activity with an optimal pH and temperature of 6.0 and 65 °C. The enzyme was stable in a pH range of 7.0-10.0, retaining over 85% relative activity for 1 h. CGTase-T1 activity can be significantly enhanced by adding 1 mM Ba2+. Using a soluble starch substrate, the kinetic parameters were revealed with K M and k cat/K M values of 2.75 mg mL-1 and 1253.97 s-1 mL mg-1, respectively. Additionally, the four enzyme activities of CGTase-T1 were determined. The highest conversion rate to CDs (40.9%) was achieved from soluble starch after 8 h of enzyme reaction, where mainly β-CD was produced (79.1% of the total CDs yield), indicating that CGTase-T1 potentially has industrial application prospect. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03111-8. © King Abdulaziz City for Science and Technology 2022.