【作者】 杨希；

【导师】 余龙江；

【作者基本信息】 华中科技大学 ， 生物工程， 2019， 硕士

【摘要】 黄姜皂素是合成甾体激素类药物的重要前体物质,工业上生产黄姜皂素主要采用传统酸解法,存在污染大,资源浪费等问题,严重影响黄姜皂素产业的健康发展。微生物转化法因其反应温和、绿色环保,是目前最具潜力的清洁生产途径。课题组前期筛选获得的菌株塔宾曲霉HG57,在工业应用的过程中存在转化率不稳定的问题。针对此问题,本文对HG57转化黄姜皂苷进行研究,取得结果如下:(1)利用DNS法检测酶催化黄姜皂苷水解后生成的还原糖,并对检测条件进行优化,得到的最适条件为:底物浓度10 g/L,反应温度50℃,pH 5.5,反应时间4h,据此建立了黄姜皂苷水解酶的总酶活性检测方法。(2)通过硫酸铵分级沉淀、阴离子交换层析、SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对HG57产黄姜皂苷水解酶进行初步分离纯化,获得A、B、C三个蛋白组分;分子量大小约为70 kDa的蛋白为关键的皂苷水解酶。(3)分别利用分离纯化的三个蛋白组分催化黄姜皂苷,并通过HPLC法检测酶催化黄姜皂苷水解生成的皂素含量,可知A蛋白组分的皂素得率明显高于B、C蛋白;对A蛋白组分转化黄姜皂苷的条件进行优化,得到的最适条件为:反应温度50℃、酶浓度34 mg/mL、反应时间72 h;利用高分辨液质联用仪(HR-LC-MS)检测A蛋白组分转化前后皂苷的组成变化,发现有10种皂苷,其中1-5号为水溶性皂苷,经A蛋白组分催化后水解为薯蓣皂苷元及5种水不溶的皂苷。以上研究结果为后续构建高效转化黄姜皂苷生产皂素的工程菌提供重要的理论依据,为微生物转化黄姜皂苷为皂素的工业化应用奠定了基础。更多还原

【Abstract】 Diosgenin is an important precursor for the synthesis of steroid hormone drugs.At present,the traditional acid hydrolysis processof diosgeninhas problems such as high pollution and high waste,which seriously affects the healthy development of the diosgeninindustry.Microbial transformation is the most promising method because of its high efficiency and environmentalprotecting capacity.In ourprevious study,the high efficient transformed strain of Aspergillus tubigensis HG57 was obtained,while had the problem of unstable conversion rate in industrial application.To solve this problem,this paper studied the key saponin hydrolase in the process of transforming saponin with HG57.The results are as follows:(1)The reducing sugar produced from the degradation of saponin by enzymes was detected by DNS(3,5-dinitrosalicylic acid)method,and the detection condition wasoptimized,which is substrate concentration 10 g/L,reaction temperature 50 °C,pH 5.5,and reaction time 4 h.Thus a method for detecting the total enzyme activity of saponin hydrolase was established.(2)The saponin hydrolase produced by HG57 was isolated and purified by ammonium sulfate fractionation,anion exchange chromatography,and SDS-polyacrylamide gel electrophoresis(SDS-PAGE)to obtain three groups of protein constituents,namely,A,B,and C.Amolecular weight of 70 kDa is a key saponin hydrolase.(3)The diosgenin of saponin hydrolyzate catalyzed by saponin hydrolase was determined by HPLC.The diosgenin yield of protein A constituent was significantly higher than that of B and C constituents.The condition for diosgeninusing A constituent was optimized,whichis reaction temperature 50 ℃,enzyme concentration 34 mg/mL,and reaction time 72 h;the high-resolution liquid chromatography-mass spectrometer(HR-LC-MS)was applied to detect the saponin species before and after the conversion of the A constituent,and found thatfive water-soluble saponins were degraded todiosgenin andfive water-insoluble saponins.The above results provide an important theoretical basis for the subsequent construction of engineering bacteria for the efficient conversion of saponins,which lays a foundation for its industrial application.更多还原