Abstract
Alkalohalophilic Evansella caseinilytica produced an extracellular cyclodextrin glycosyltransferase (CGTase) with cyclization activity of 43.5 ± 4.4 U/L in M1 medium containing 1% starch and 6% NaCl in nutrient broth at 37 ºC, pH 9.0, after 48 h. This is the first report of CGTase from this bacterium. 0.1% starch was found to induce CGTase, and further optimization using one variable at a time approach followed by statistical optimization led to 5.5-fold enhancement resulting in 240.5 ± 5.46 U/L. Six parameters were identified as positive signals using Plackett–Burman (PB). Of these, yeast extract, MgSO4 and tryptone were taken further for Response Surface Methodology (RSM) by disposing beef extract and fixing starch and soya peptone. The optimized M4 medium consisted of tryptone (0.1%, w/v), yeast extract (0.25%, w/v), MgSO4 (8 mM, w/v), potato starch (0.1%, w/v) and soya peptone (0.2%, w/v). CGTase was further purified with 6.44-fold purification and 19.32% yield employing starch affinity. It was found to be monomeric, corresponding to a size of 68 kDa as estimated by SDS-PAGE and was further confirmed to be 65 kDa by size exclusion chromatography. γ-Cyclodextrins were produced as the major product with a conversion of 5% soluble starch into 20.38% γ-cyclodextrins after 24 h reaction, as determined by HPLC. Peptide fingerprint after LC–MS analysis matched with IPT/TIG domain-containing protein within the genome of E. caseinilytica. Further blastp analysis revealed the closest homology with γ-CGTase from an alkalophilic E. clarkii, thereby confirming CGTase from E. caseinilytica as γ-CGTase.
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Abbreviations
- %:
-
Percentage
- ˚C:
-
Degree Celsius
- H:
-
Hours
- Min:
-
Minutes
- U:
-
Units
- µl; ml:
-
Microlitres; Millilitres
- L:
-
Litres
- Rpm:
-
Revolutions per minute
- w/v:
-
Weight by volume
- v/v:
-
Volume by volume
- nm:
-
Nanometer
- M:
-
Molar concentration
- µm:
-
Micron
- kDa:
-
Kilodaltons
- mg:
-
Milligrams
- NaCl:
-
Sodium Chloride
- NaOH:
-
Sodium Hydroxide
- Na2CO3 :
-
Sodium Carbonate
- BSA:
-
Bovine Serum Albumin
- SDS:
-
Sodium Dodecyl Sulphate
- MgSO4 :
-
Magnesium Sulphate
- CaCl2 :
-
Calcium Chloride
- K2HPO4 :
-
Dipotassium hydrogen phosphate
- PAGE:
-
Polyacrylamide Gel Electrophoresis
- RID:
-
Refractive Index Detector
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Acknowledgements
Kuldeep Saini and Amuliya Kashyap would like to acknowledge Council of Scientific and Industrial Research (CSIR) for providing Senior Research Fellowship, and Meenu Saini would like to acknowledge financial assistance (SRF) from Indian Council of Medical Research (ICMR). The authors also thank Central Instrumentation Facility (CIF) at UDSC for LC-MS analysis.
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Saini, K., Kashyap, A., Saini, M. et al. Gamma cyclodextrin glycosyltransferase from evansella caseinilytica: production, characterization and product specificity. 3 Biotech 12, 16 (2022). https://doi.org/10.1007/s13205-021-03077-z
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DOI: https://doi.org/10.1007/s13205-021-03077-z