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 KM and kcat/KM 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.
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We would like to thank Yangtze University for laboratory and equipment.
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ZL: methodology, investigation, formal analysis, visualization, writing-original draft. GW: investigation, formal analysis, writing—review and editing. HW: methodology, formal analysis, resources, writing-review and editing.
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Accession numbers: The molecular data accession numbers for 16S rRNA gene and CGTase sequence of the strain T1 are MZ221050 and UBS25994.1 in the NCBI database, respectively.
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Supplementary Figure 1. Optimized expression conditions of CGTase-T1 by SDS-PAGE analysis. a. Cell induction at 20 ºC for 0-10 h. Lanes 1-6 were 0, 2, 4, 6, 8, and 10 h of intracellular supernatant, and Lanes 7-12 were 0, 2, 4, 6, 8, and 10 h of intracellular sediment, respectively. b. Cell induction at 25 ºC for 0-10 h. Lanes 1-6 were 0, 2, 4, 6, 8, and 10 h of intracellular supernatant, and Lanes 7-12 were 0, 2, 4, 6, 8, and 10 h of intracellular sediment, respectively. c. Cell induction at 30 ºC for 0-10 h. Lanes 1-6 were 0, 2, 4, 6, 8, and 10 h of intracellular supernatant, and Lanes 7-12 were 0, 2, 4, 6, 8, and 10 h of intracellular sediment, respectively. d. Cell induction at 35 ºC for 0-10 h. Lanes 1-6 were 0, 2, 4, 6, 8, and 10 h of intracellular supernatant, and Lanes 7-12 were 0, 2, 4, 6, 8, and 10 h of intracellular sediment, respectively. e. OD600 of induction conditions at 25 ºC for 10 h. Lanes 1-6 were OD600 reached 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 of intracellular supernatant, and Lanes 7-12 were 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 of intracellular sediment, respectively. f. Induction culture of IPTG concentration at 25 ºC for 10 h with OD600 reached 0.8. Lanes 1-7 were 0, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 mM IPTG of intracellular sediment, and Lanes 8-14 were 0, 0.1, 0.3, 0.5, 0.7, 0.9, and 1.1 mM IPTG of intracellular supernatant, respectively (DOCX 183 kb)
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Liu, Z., Wu, G. & Wu, H. Molecular cloning, and optimized production and characterization of recombinant cyclodextrin glucanotransferase from Bacillus sp. T1. 3 Biotech 12, 58 (2022). https://doi.org/10.1007/s13205-022-03111-8
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DOI: https://doi.org/10.1007/s13205-022-03111-8