Extractive Bioconversion of Gamma-Cyclodextrin and Recycling of Cyclodextrin Glycosyltransferase in Liquid Biphasic System Using Thermo-Separating Polymer.

An extractive bioconversion conducted on soluble starch with cyclodextrin glycosyltransferase (CGTase) enzyme in ethylene oxide-propylene oxide (EOPO)/potassium phosphates liquid biphasic system (LBS) to extract gamma-cyclodextrin (γ-CD) was examined. A range of EOPO (with potassium phosphates) molecular weights was screen to investigate the effect of the latter on the partioning efficency of CGTase and γ-CD. The results show that the optimal top phase γ-CD yield (74.4%) was reached in 35.0% (w/w) EOPO 970 and 10.0% (w/w) potassium phosphate with 2.0% (w/w) sodium chloride. A theoretical explanation for the effect of NaCl on γ-CD was also presented. After a 2 h bioconversion process, a total of 0.87 mg/mL concentration of γ-CD was produced in the EOPO/ phosphates LBS top phase. After the extraction of top phase from LBS, four continuous repetitive batches were successfully conducted with relative CGTase activity of 1.00, 0.86, 0.45, and 0.40 respectively.

Production of γ-cyclodextrin by Bacillus cereus cyclodextrin glycosyltransferase using extractive bioconversion in polymer-salt aqueous two-phase system.

Aqueous two-phase system (ATPS) extractive bioconversion provides a technique which integrates bioconversion and purification into a single step process. Extractive bioconversion of gamma-cyclodextrin (γ-CD) from soluble starch with cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) enzyme derived from Bacillus cereus was evaluated using polyethylene glycol (PEG)/potassium phosphate based on ATPS. The optimum condition was attained in the ATPS constituted of 30.0% (w/w) PEG 3000 g/mol and 7.0% (w/w) potassium phosphate. A γ-CD concentration of 1.60 mg/mL with a 19% concentration ratio was recovered after 1 h bioconversion process. The γ-CD was mainly partitioned to the top phase (YT=81.88%), with CGTase partitioning in the salt-rich bottom phase (KCGTase=0.51). Repetitive batch processes of extractive bioconversion were successfully recycled three times, indicating that this is an environmental friendly and a cost saving technique for γ-CD production and purification.

Direct recovery of cyclodextringlycosyltransferase from Bacillus cereus using aqueous two-phase flotation.

Purification of cyclodextrin glycosyl transferase (CGTase) from Bacillus cereus using polyethylene glycol (PEG)-potassium phosphates aqueous two-phase flotation (ATPF) system was studied in this paper. The effects of varying PEG molecular weight, tie-line length (TLL) value, volume ratio (VR), pH value, crude concentration and gas nitrogen flotation time were investigated. The optimal condition for purification of CGTase was attained at 18.0% (w/w) PEG 8000, 7.0% (w/w) potassium phosphates, VR of 3.0, 20% (w/w) crude load at pH 7, and 80 min nitrogen flotation time at a flow rate of 5 L/min. With this optimal condition, purification factor (PFT) of 21.8 and a yield (YT) of 97.1% were attained. CGTase was successfully purified in a single downstream processing step using the ATPF.