【作者】 李雨虹；

【导师】 谢达平；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 辣椒素酯(Capsiate)类物质，始称类辣椒素类物质(Capsaicinoid-like Substances CLSs)，是二十世纪八十年代末从日本的一种甜椒品种中分离得到的一种结构类似辣椒素的酯类物质，即为相对应的辣椒素分子中的-NH-基团被-O-所替代，其生物活性与辣椒素类似。随着非水酶学的发展，有机相中生物催化合成天然产物的研究正在兴起，其中脂肪酶的非水相催化研究最为广泛。本文对利用生物合成方法将辣椒素转化为无辛辣味的辣椒素酯进行了研究，建立了辣椒素酯的实验室制备方法，并对合成壬酸香草醇酯的酶促反应建立了动力学模型，结果如下：第一，本文考察了与天然辣椒素酯结构最接近的壬酸香草醇酯在有机相中用脂肪酶催化合成的方法。结果表明脂肪酶Novozym 435的活性最好，最适条件为：在1mL脱水丙酮中，香草醇与壬酸甲酯的浓度分别为50mmol／L、75mmol／L，酶量为20mg，30℃下反应10h，产率可达到65.9％。所得产物经硅胶柱层析纯化后以UV、IR、1HNMR、13CNMR及MS表征了结构。第二，在上述条件下，以天然辣椒素为原料经甲醇醇解后所得混合辣椒素脂肪酸甲酯，再与香草醇在脂肪酶催化下实现了天然辣椒素酯类物质的酶法合成。产物经硅胶柱层析纯化，经HPLC检测不含辣椒素类物质，其结构以GC-MS进行了表征。第三，进行了菌种培养及发酵产酶条件、酶的固定化及催化转化方法的研究。以假丝酵母菌发酵所得的粗脂肪酶固定化，初步研究了有机相中酶促合成壬酸香草醇酯的方法，结果表明：以微晶纤维素为载体时，产物收率为0.23％；以活性炭为载体时，产物收率为0.33％。第四，研究了转酯反应的动力学，其机制属于双底物的乒乓机制。动力学参数为：Vm=44.84mmol／(min·g)；KmA=1.022mmol／L；KmB=0.056mmol／L。更多还原

【Abstract】 Capsiate has been extracted from a nonpungent cultivar of red pepper, CH-19 sweet,and shown to be acapsaicin analogue called capsinoid that has an ester bond instead of the amide bond between the vanillylmoiety and fatty acid chain. Despite non-pungency, capsiate shares certain biological activities with capsaicin.As the development of Nonaqueous Enzymology, the lipase-catalyzed synthesis of bioactive substances inorganic solvent gained increasing interest.It is possible that capsiate could be a preferable medical or nutritional application over capsaicin.On thisbasis,the aim of this work was to study the direct enzymatic biotransformations of capsaicin truns intocapsiate in laboratory scale.And the kinetic model of lipase-catalyzed transesterification of vanillyl nonanoatewas constructed. The results as follows:Firstly,vanillyl nonanoate,the analogue of natual capsinoids,was synthesized in organic media catalyzedby lipase.The results indicated that lipase Novozym 435 showed the highest activity for the reaction.Theoptimization of reaction as follows: 50 mM vanillyl alcohol and 75 mM methyl nonanoate in 1 mL of acetoneusing 20 mg of Novozyme 435 at 30℃for 10h,the conversion yields was 65.9%. After purifying theproducts by silica gel column chromatography, confirmed the structures by UV,IR,NMR and MSmeasurements.Secondly, under the aboved conditions,natural capsaicin was converted to natural capsinoids by thecombinations of chemical-enzymatic pathway.After purifying the products by silica gel columnchromatography, detected the residual capsaicin by HPLC measurement and confirmed the structures byGC-MS measurements.Thirdly, the synthesis of vanillyl nonanoate by immobilized Candida lipase was primary studied,includingthe method of strain culture,the condition of produting lipase,the immobilizati-on method of the lipase andthe synthesis condions. It indicated the conversion yields were 0.23% and 0.33% using crystallitic celluloseand active carbon as the carrier, respctively.Finally, Kinetics of lipase-catalyzed trasesterification of vanilly alcohol and methyl nonanoate have beeninvestigated.The reaction rate could be described in terms of the Ping-Pong Bi-Bi mechanism.The kineticparameters were computed as: V_m= 44.84mmol/ （min·g）; K_mA=1.022mmol/L; K_mB=0.056mmol/L.更多还原