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

ABSTRACT: 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.

SUBMITTER: Liu Z 

PROVIDER: S-EPMC8816995 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature