Study On Enzymatic Synthesis Characteristics And Application Of Maltosyl(α-1â†'6)β-cyclodextrin

Maltosyl-β-cyclodextrin (Mal-β-CD or G₂-β-CD) is a kind of branched cyclodextrin, it is the modified cyclodextrin made by grafting maltose through chemical and bioenzymic method with maintance of native cyclodextrin conformation. Mal-β-CD has the properties of inclusion, which is the samilar as nativeβ-CD, but also has properties of high solubility which nativeβ-CD don't have. Thus, Mal-β-CD could be used in field of food, medicine, agricultural chemical, fine chemical engineering, analysis and detection and environmental protection. This study separated pullulanase from commerical pullulanase liquids, and the properties of pullulanase were studied, the catalysis mechanism of reverse synthesis Mal-β-CD were investigated in detail, and the feasibility of the reaction was elaborated by computational chemistry, the synthetic technology was optimized and the structure of product was characterized, we further studied the application of Mal-β-CD.We investigated the detection conditions of HPLC and paper chromatography (PC) for Mal-β-CD. Results showed that the detection conditions on HPLC were: Hypersil NH₂ column (Ñ„4.6×250mm), K-2301 refractive index detector (RID), mobile phase of acetonitrile- water (60:40), flow speed of 1 mL/min, temperature of 30℃, injection of 10μL. The detection conditions for PC were: chromatography of descending, developing solvent of 1-propyl:n-butyl:water (5:3:4), developing time of 12 h, 0.1 mol/Lof HCL as eluant, sample content of 150-250μg. The accuracy and repeatability of PC were satisfied, and could be used as an enconomic and realible detection method, which work in coordination with HPLC in Mal-β-CD production and application.We separated and purified pullulanase and made some chemical modification on pullulanase. Commercial pullulanase liquids were fractional precipitated by ammonium sulfate, then were desalted by gel column (HiPrep 26/10 desalting column), and then purified by ion exchange column chromatography (Mono Q 10/100 GL),gel chromatography (Superdex^Tm 200 10/300 and HiPrep Sephacryl S-200HR) using AKTA pufrification system. The final purified pullulanase was detected by SDS-PAGE, single band means electrophoretically pure of pullulanase, the relative molecular weight was about 82 kDa。The purified pullulanase was modified by DIC,PMSF,DTT,NBS,DEPC,EDC,Ch-T and TNB, results showed that Arg, hydroxy and sulfydryl of amino acid were independent with pullulanase activity, while Trp, Met, imidazolyl in His, carboxyl in amino acid, andε-NH₂ in Lys were related to pullulanase activity, may be the essential group for maintaining pullulanase activity.The preparation condition for Mal-β-CD were optimized by single factor experiment and response surface design, the optimum condition were set up as temperature of 60℃,pH of 4.5,molar ratio of maltosetoβ-CD were 12:1,substrate concentration of 80%,enzyme concentration of 200 U/gβ-CD,reaction time of 60 h. Under these conditions the Mal-β-CD concentration was 71 mg/mL,the conversion percent forβ-CD synthesis Mal-β-CD was 65%。 The feasibility of reverse systhesis Mal-β-CD was investigated. Using computational chemistry, the configuration of each molecule for synthesis of Mal-β-CD were optimized by AM1, and we got the energy and thermal effect of each molecule. Results showed that the reaction for synthesis of G₂-β-CD were endothermic reaction, the absorption energy was 294.7kJ/mol。The reaction balance moved to positive reaction with temperature increasing, and also the reaction speed fastened, which was suitable for G₂-β-CD preparation,this simulation results was accordance with experiments. For grafting reaction with more than two maltose such as(G₂)₂-β-CD,(G₂)₃-β-CD, the negative reaction speed were increased with temperature increasing.The structure of synthetic product was identified using MS, FTIR and NMR, results showed that the structure of the product was that maltose connected withβ-CD byα(1-6)glucosidic bond. The product was hydrolyzed by glucoamylase and the hydrolysate was identified as Glu-β-CD by MS, FTIR and NMR, which further identified the product. The thermodynamics behavior of Mal-β-CD was studied by DSC and the parameters, such as activation energy was calculated using Kissinger equation and Ozawa equation.The thermodynamics behavior of Mal-β-CD was studied by DSC, and the activation energy parameters were calculated by Kissinger equation and Ozawa equation. The results showed that decomposition temperature for Mal-β-CD was 328.7℃, the thermal decomposition activation energy calculated by Kissinger equation and Ozawa equation were 41.44 kJ /mol and 44 kJ /mol, separately. Exponential factor A was calculated as 1.85×10³ min^-1 by Ozawa method.â–³H andâ–³S was calculated from DSC diagram as 23.70 kJ /mol and 0.0394 kJ /mol·K,respectively . The degradation velocity constant (k) was calculated as 0.228 min ^-1, and half time period was calculated as 3.04 min, glass transition temperature Tg was 173℃.The inclusion properties of Mal-β-CD were studied by forming complexes essential oil from piper nigrum. Firstly, the volatile components of black pepper oil were identified by GC/MS method.β-caryophyllene was one of the main constituents of the identified terpinene compounds, up to 37.11 % of the total volatile substances detected. The complexation ofβ-caryophyllene with Mal-β-CD was investigated by phase solubility method and then the quantitative analysis method ofβ-caryophyllene from Mal-β-CD complex was established. The parameters of preparing complexes between Mal-β-CD andβ-caryophyllene were optimized by orthogonal design.The thermal behavior of the solid CDs complexes was analysized by thermogravimetric (TG). Complexes of Mal-β-CD with essential oil from piper nigrum showed high stability in the stability experiment, and the complex was totally soluble, thus the solubility of essential oil from piper nigrum could be significantly increased. The critical humidity of complex was about 67%.