| Large ring cyclodextrin (LR-CD) was produced by 4-α-glucanotransferase (4αGtase) through reacting with starch, while the yield and polymerization degree (DP) of LR-CD were different for various 4αGtase source. In this paper, the structure and basic properties of 4αGtase were studied using bioinformatics method, the novel 4αGtase activity determination method was developed, the conditions for controlling cylization reaction were studied, and the yields of LR-CD were optimized, LR-CD was identified and separated by capillary electrophoresis, and the application of LR-CD in the inclusion properties was investigated.The genetic engineering bacteria containing gene of 4αGtase was constructed. Nucleotide sequence alignment showed that the degree of nucleotide similarity for 4αGtase to maltotransferase from Thermus acquaticus was as high as 98%. The amino acid sequence was aligned using Clustal, the result showed that the product specificity amino acid was existed in substrates bonding area, instead of conserved domain. The residue in active region were Asp293, Glu340, Asp395. Disulfide bond has no contribution to maintain with 3D structure of 4αGtase. The relative molecular weight (Mw) of purified 4αGtase determined by SDS-PAGE was 57000, which was in agreement with the Mw calculated by NCBI (56793 Da). From product analysis by HPLC, 4αGtase showed great glycosyl transfer activity, and the minimum interaction saccharide was maltose.A novel method was established for 4αGtase activity determination. We firstly adopted triple wavelength colorimetry (TWC) and orthogonal function spectrophotometry (OFS) to determine the content of LR-CD and amylose and the results were compared with commonly used single wavelength colorimetry (SWC). The results showed that standard derivations of these three methods were all less than 2% and could meet the demand of experiment. When OFS was used in the determination of amylose content in a mixture, the recovery ratio was 87%-106%, which was much superior to TWC (0-106%) and SWC (109%-145%). When TWC was used in detecting LR-CD content in a mixture, the recovery ratio was 94%-104%, which was much superior to OFS (87%-106%) and SWC (132%-146%). Therefore, it was concluded that TWC was suitable for cylization activity determination and OFS was suitable for total activity determination. Besides, the results demonstrated that the cylization activity of 26 U/μl and total activity of 342 U/μl for crude enzyme, the cylization activity of 73 U/μl and total activity of 243 U/μl for purified enzyme.The factors for influencing 4αGtase cylization reaction were investigated. The rate constant was 21.85 mmol/mL and maximum rate was 0.9906 mg/mL/min for cylization, while for total reaction, the rate constant was 2.92 mmol/μl and maximum rate was 546.78 mg/mL/min; the conditions for maximum cylization rate were pH of 9.0 and temperature of 70℃,the conditions for maximum total rate were pH of 7.5 and temperature of 80℃; the addition of maltose, DMSO, ethanol and metal ions additions changed secondary structure and environmental polarity of 4αGtase, which caused variation of 4αGtase activity. For optimizing LR-CD yield, eight factors including starch content, enzyme concentration, buffer concentration, ethanol content, temperature, time, pH, DMSO content were designed through Plackett-Burman design; enzyme concentration, temperature and time were found tobe the significant factors for LR-CD yield, and the three factors were optimized by Box-Behnken design, when enzyme addition of 91.34 U/g, temperature of 71.7℃and time of 6.8 h, the final LR-CD yield was as high as 55%, and increased 2.3 folds.LR-CD with DP of 20-40 was firstly separated by iodine affine capillary electrophoresis. The separation buffer were: 0.6 mM of I2, 3.6 mM of KI, 80 mM of phosphate buffer (PBS), pH of 5.10; the optimum separation conditions were voltage of 10 kV and separation temperature of 20℃; this novel separation procedure saves much time than that of HPAEC, and could be operated more easily than HPLC, it could also be used for structure analysis and inclusion analysis for LR-CD. The LR-CD produced by 4αGtase was identified by HPLC, TOF-MS and composite enzymatic method, the results showed that molecular weight (Mw) of produced LR-CD was 3 733 Da, the cyclic ratio bonded byα-1,4 was 47%, and the polymerization degree (DP) was 20-40.Using nystatin as guest, we investigated the inclusion properties of cyclodextrins with various size. The results suggested that the phase solubility curve forβ-CD,γ-CD and LR-CD were AL type, while that ofα-CD were BS type; the embedding constant were 0.375, 0.539, 1.577 l/mmol forβ-CD,γ-CD and LR-CD, respectively; the solubility folds enhanced by saturated cyclodextrins were predicted as 6.21, 103.39, 2958.87 forβ-CD,γ-CD and LR-CD, respectively; the final complexes were identified by infrared spectrum (IR), the results were deduced that the ester linkage and diene were included in the cavity of cyclodextrins, while conjugate arachidonic, carboxyl and amino group were left outside of cyclodextrins, molecular simulation was conducted and the simulation result was accordance with that deduction; it is easier for LR-CD to form complex with large guest, the interaction mainly was space force; the store stability of nystatin was improved by forming complexes.
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