| Cyclodextrin, one kind of modified starches synthetized via glycosyl transferase catalytic reaction, has a wide range of industrial applications due to non-reducing-end groups, a larger cavity, hydrophobic and hydrophilic characteristics. However, only a few cyclodextrins with low water solubility (i.e.,α-, β-, y-cyclodextrin, DP9, DP10, DP14, DP26) have been industrialized. Other large cyclodextrins are in limited supply and are costly to obtain on a large scale because of their similar structures, high water solubility and difficulty to obtain monomer by crystallization separation. Since E.coli DH5a-MalQ strains reportedly produce amylomaltase enzyme which can catalyze the amylose maize starch, they may serve as potential sources for high-yield cyclodextrins production. In this research, we investigated the effects of ethanol, DMSO, CaCl2 on the conversion rate of starch and cyclic ratio of large cyclodextrins, and obtained an evidence for the monomer with polymerization degree of 16-20 separated by polyacrylamide gel column chromatography and silica gel column chromatography. The main results are as follows:1. The recombinant vector was extracted from E.coli DH5α-MalQ strains preserved at-80℃ and thus analyzed by agarose gel electrophoresis after double digestion. Then two single strip with the molecular weight of about 2.1kbp and 3.6kbp, respectively, were obtained. It indicated the existence of recombinant vector TpET-DsbA-MalQ that can express amylomaltase. The recombinant vector TpET-DsbA-MalQ could been transferred to E.coli BL21 competent cell to prepare recombinant strains with high expression of amylomaltase and long-term storage stability. All enzyme assays were performed using TLC and the enzymatic activity of transglycosylase was 1.7 U.2. Large cyclodextrin was produced from amylase corn starch by the catalyze reaction of amylomaltase at the condition of 20 U/g starch,50℃ and 5 h. The effects of ethanol, DMSO, CaCl2 on the conversion rate and cyclic ratio were investigated in the preliminary experiments. The results showed that the effect of CaCl2 on conversion rate increased with the increase of CaCl2 concentration while the effect of ethanol, DMSO on conversion rate was negligible. But all of these three chemicals has a significant effect on cyclic ratio. The results have shed light on the mechanisms that regulate the synthesis, mostly because of the change of cyclodextrins conformation caused by the formation of inclusion complexes, and the stimulation of the conformation of the enzymes and activity of enzyme in response to three chemicals. Subsequently, the effects on the conversion rate and cyclic ratio of using ethanol, DMSO, CaCl2 were optimized by response surface method with a 17-run Box-Behnken design. The optimal conditions for conversion rate and cyclic ratio were 19%(v/v) of ethanol,9.7%(v/v) of DMSO, and 44.5 mmol/L of CaCl2. The conversion rate and cyclic ratio reached 29.4% and 94.07%, respectively, which are exceeded by the amount (30% and 20%, respectively) before optimization.3. The optimal conditions obtained by RSM led to not only higher conversion rate and cyclic ratio in large cyclodextrins production, but also a higher production of large cyclodextrins of single degree of polymerization 16-20 with 3.66,9.2,6.62 and 6.33 times, respectively. A new polyacrylamide gel column with an overall effective column height of 160*1.2 cm was prepared by using 4cm long thin tube to connect two Bio-GeL P-4 gel columns with specifications of 100 cm* 1.2 cm i.d and 70 cm* 1.2 cm i.d, respectively. The large cyclodextrins crude products were separated by the new column. Finally, the large cyclodextrins (DPI6-20) monomer was separated by silica gel column (20*1.2 cm i.d) with an effective column height of 18 cm. |
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