【作者】 崔波；

【导师】 金征宇；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2011， 博士

【摘要】 麦芽糖基-β-环糊精（Mal-β-CD或者G₂-β-CD）是分支环糊精的一种,是在保留环糊精空腔结构基本不变的情况下,采用化学或生物酶法向环糊精母体的6位羟基接枝麦芽糖基而得到的改性产物。麦芽糖基-β-环糊精既具有普通母体β-环糊精所具有的包合性能,同时又具有β-环糊精所不具有的极好的溶解度,因此在食品、医药、农药、精细化工、分析检测、环保等行业具有巨大的应用前景。本论文从商品普鲁兰酶液中分离得到普鲁兰酶,对所用普鲁兰酶的性质进行了研究,详细研究了普鲁兰酶反向催化合成麦芽糖基-β-环糊精的机制,并通过计算化学阐述了反应的可行性,对普鲁兰酶逆向催化反应合成麦芽糖基-β-环糊精的工艺进行了优化,并对合成产物结构进行了表征,同时对麦芽糖基-β-环糊精的应用进行了初步研究。确定了麦芽糖基-β-环糊精的高效液相法和纸层析法检测条件。高效液相法定量检测的检测条件为:Hypersil NH₂柱ф4.6×250mm,K-2301示差折光检测器,流动相乙睛-水（60:40）,流速1 mL/min,柱温30℃,进样量10μL。纸层析法用于样品中麦芽糖基-β-环糊精的定量定性检测的检测条件为:下行法层析,展开剂为正丙醇-正丁醇-水（5:3:4）,展开时间12小时;以0.1 mol/L的HCL溶液作为洗脱剂,样品的含量在150-250μg之间。定性检测时干燥后直接显色即可。纸层析法准确性和重复性都较好,可以和HPLC互相配合,作为麦芽糖基-β-环糊精在生产和应用中的一种经济可靠的常规检测方法。对普鲁兰酶进行了分离纯化,并进行了化学修饰。商品普鲁兰酶液,通过硫酸铵分级盐析,AKTA系统凝胶柱HiPrep 26/10 Desalting脱盐、Mono Q 10/100 GL柱上的离子交换层析、Superdex^Tm 200 10/300柱和HiPrep Sephacryl S-200HR凝胶色谱,分离得到了普鲁兰酶。SDS-PAGE电泳分析呈现单一条带,这表明该酶已经达到了电泳纯,该酶的分子量约为82 kDa。用DIC（丁二酮）、PMSF（苯甲磺酰氟）、DTT（二硫苏糖醇）、NBS（N-溴代琥珀酰亚胺）、DEPC（焦碳酸二乙酯）、EDC（碳二亚胺）、Ch-T（氯氨-T）和TNBS（三硝基苯磺酸）等八种专一性氨基酸残基修饰剂修饰纯化得到的普鲁兰酶后,结果表明精氨酸、氨基酸的羟基和巯基与酶活力无关,而色氨酸残基、组氨酸中的咪唑基、氨基酸的羧基、甲硫氨酸残基和赖氨酸ε-NH₂与普鲁兰酶活性有关,可能是酶活力的必需基团。通过单因素实验和响应面法优化了麦芽糖基-β-环糊精制备条件。通过优化,麦芽糖基-β-环糊精生产的最适反应条件:温度,60℃,pH值4.5,麦芽糖和β-环状糊精摩尔比为12:1,底物浓度80%,加酶量200 U/gβ-环糊精,反应时间60 h。在此条件下,反应体系的麦芽糖基-β-环糊精浓度达到71 mg/mL,β-环糊精合成麦芽糖基-β-环糊精的转化率达到65%。阐述了反向合成麦芽糖基-β-环糊精的可行性。应用计算化学手段,用AM1方法优化了生成麦芽糖基-β-环糊精过程中各分子的构型,得到了各分子的能量及反应的热效应。从能量结果看,生成麦芽糖基-β-环糊精的反应是吸热反应,吸收热量为294.7 kJ/mol。升高温度,平衡向正反应方向移动,且反应速度加快,有利于麦芽糖基-β-环糊精的生成,与实验结果一致。对于接枝多个麦芽糖基（两个或三个以上）生成如（G₂）₂-β-CD、（G₂）₃-β-CD等麦芽糖基-β-环糊精的反应,升高温度时,反应不利于合成接枝共聚物,且这种逆反应速度加快。通过串联质谱、富立叶变换红外光谱仪、核磁共振波谱仪等仪器对合成的产物进行结构鉴定,表明产物结构为一个麦芽糖基通过α（1-6）键连接到了β-环糊精母体上;将麦芽糖基-β-环糊精用糖化酶水解,得到的产物通过串联质谱、富立叶变换红外光谱仪、核磁共振波谱仪等手段进行结构解析,发现糖化酶水解后的产物是葡萄糖基-β-环糊精（Glu-β-CD）,进一步说明合成产物为麦芽糖基-β-环糊精。利用DSC法对麦芽糖基-β-环糊精的热力学行为进行研究,按Kissinger方程和Ozawa方程对活化能等参数进行了计算。结果表明,麦芽糖基-β-环糊精的开始分解温度是328.7℃,Kissinger方程以及Ozawa方程计算出的麦芽糖基-β-环糊精的热分解活化能分别为41.44 kJ /mol和44 kJ /mol。根据Ozawa方法获得指前因子A为1.85×10³ min^-1,通过DSC曲线图,可以获得焓变（△H）为23.70 kJ /mol,以及熵变（△S）为0.0394 kJ /mol·K,降解速度常数k为0.228 min^-1,同时麦芽糖基-β-环糊精的半衰期为3.04 min,玻璃化转变温度Tg是173℃。研究了Mal-β-CD对黑胡椒精油的包合作用。首先运用GC-MS对黑胡椒精油的基本成分组成进行分析,β-石竹烯占黑胡椒精油挥发性组分的37.11%;其次研究了Mal-β-CD存在下,β-石竹烯的相溶解度曲线,建立了Mal-β-CD—β-石竹烯的定量分析方法;然后以包埋率为指标,运用正交设计优化了包合物的制备工艺;通过红外光谱及热重分析,确认了Mal-β-CD—β-石竹烯包合物的形成,热重分析表明β-石竹烯经Mal-β-CD包埋后,耐热性显著提高;黑胡椒精油的Mal-β-CD包合物在稳定性试验中呈现出较高的稳定性,产物完全溶于水,可显著提高黑胡椒精油的溶解度,包合物产物的临界湿度为67%。更多还原

【Abstract】 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 （ф4.6×250mm）, 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.△H and△S 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%.更多还原