Purification and characterization of stomach protease from the turbot (Scophthalmus maximus L.)

Proteolytic activity in the different parts of the digestive tract of the turbot (Scophthalmus maximus L.) were studied in this work. One pure protease was isolated from turbot stomach and its behavior was studied. Results showed the optimum pH for proteases in the different parts of the digestive tract of the turbot were pH 2.0 for the stomach, pH 8.0 for the pylorus cecum, pH 8.0 for the foregut, pH 8.5 for the midgut, and pH 8.0 for the hindgut. The activity of proteases in the different parts of the digestive tract were in the sequence pylorus cecum protease > stomach protease > foregut protease > midgut protease > hindgut protease. The stomach protease was purified by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose F.F. and Sephadex G-100. The purified enzyme gave a single band in SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Its molecular weight was found to be approximately 42,000 Da. The enzyme is stable at pH 1.0–9.0 and at temperatures below 40°C. Its activity was maximum at pH 2.0 and 40°C. When reaction time was prolonged the optimum temperature of the enzyme tended to decline. The enzyme was activated by Mn²⁺ and Cu²⁺ and inactivated by Fe³⁺. It was fully inhibited by pepstatin and partially inhibited by PMSF, TPCK, PCMB, and NBS. These results imply the enzyme is a pepsin.     

Purification and characterization of a cysteine-like protease from the body wall of the sea cucumber <Emphasis Type="Italic">Stichopus japonicus</Emphasis>

The sea cucumber (Stichopus japonicus) is able to undergo autolysis in response to a variety of environmental and mechanical cues. Within the framework of a long-term study of this phenomenon we have purified a protease from the body wall of the sea cucumber by means of ion-exchange chromatography with DE-52 cellulose and gel filtration chromatography with Sephadex G-100. The final enzyme preparation was nearly homogeneous on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 35.5 kDa. The purified enzyme exhibited a maximum activity for the hydrolysis of casein at pH 7.0 and 50°C and a remarkable stability at pH 4.0–7.0 and 40–60°C. Based on the inhibition and activation profiles obtained with numerous specific protease inhibitors and an activator, the protease purified from the body wall of the sea cucumber was defined to be a cysteine-like protease.     

南极磷虾体内胰蛋白酶的纯化及性质研究

以南极磷虾(Euphausia superb)为研究对象,通过硫酸铵分级沉淀、Phenyl-Sepharose疏水层析、DEAE-Sepharose FF离子交换层析等方法,从南极磷虾体内分离纯化出胰蛋白酶。其纯化倍数为5.44倍,比活力为38.3 U/mg,得率为26%。SDS-PAGE电泳结果显示,该酶的分子质量为28 ku。蛋白酶最适温度为37℃、最适pH为7.5,Mg2+、Ca2+、Mn2+对南极磷虾蛋白酶具有激活性,Zn2+、Cu2+、Fe3+具有酶活抑制性,其中Cu2+的抑制性最强。酶的动力学实验结果表明,以BApNA为底物测得Km为0.073 mmol/L,Vmax为1.44×10-2mmol/L·s,kcat为0.6S-1,kcat/Km为8.22×103,PMSF作为蛋白酶抑制剂,对南极磷虾蛋白酶作用机制为不可逆抑制。     

大豆异黄酮的提取纯化及抗氧化性研究

[目的]对大豆异黄酮提取纯化的最佳工艺条件及其抗氧化活性进行研究.[方法]通过单因素试验和L9（34）正交试验,确定提取大豆异黄酮的最佳工艺条件,应用D101大孔树脂技术对提取液进行进一步分离纯化,得出最佳纯化条件,并对纯化得到的染料木苷和大豆苷进行抗氧化活性研究.[结果]试验得出,提取大豆异黄酮的最佳工艺条件为乙醇浓度70％,料液比1∶15 g/ml,提取时间为3h,提取温度为60℃,最高得率达9.18％;纯化最佳条件为：上柱静态吸附时间5h,洗脱时间30 min,80％乙醇作为洗脱剂,洗脱流速为lml/min,并分离纯化得到染料木苷和大豆苷;抗氧化活性研究表明,染料木苷、大豆苷和大豆总黄酮对超氧阴离子自由基和羟自由基均具有清除作用.[结论]研究对大豆保健食品开发和天然药物研制具有重要意义.     

应用混合树脂技术从蛹虫草中提取纯化虫草素

[目的]应用混合树脂技术从蛹虫草中提取纯化虫草素.[方法]利用微波辅助提取法从蛹虫草子实体或其大米培养残基中提取虫草素,利用混合大孔树脂对虫草提取物进行分离纯化.通过比较大孔吸附树脂和阴阳离子交换树脂对虫草素的静态吸附和解吸,以吸附量和解吸附率为评价指标,从11种商品化树脂中筛选出较佳树脂,进一步进行树脂混合比例的静态吸附试验,筛选出较佳的干树脂质量比,按此比例在树脂柱中设计了几种不同的填装混合方式进行动态吸附和解吸.[结果]试验筛选出LSA-21、XAD-16、LX-28 3种大孔吸附树脂为较佳树脂,进行静态吸附试验时三者较佳的干树脂质量比为1∶1∶1.将XAD-16、LX-28、LSA-21 3种树脂按由上至下分层填装的方式进行动态吸附和解吸附效果较好,应用该混合树脂床进行一次动态吸附和解吸附后,解吸液浸膏中虫草素的含量由原生药中的0.442％提高至23.600％.[结论]该方法可应用于蛹虫草子实体或其培养残基中虫草素的提取纯化.     

柽柳转录因子基因ThbZIP1的原核表达及重组蛋白纯化

柽柳是重要的抗逆植物,提取柽柳总RNA,克隆了柽柳的16.5 k D b ZIP转录因子基因Thb ZIP1,构建原核表达载体p GEX-Thb ZIP1,导入宿主菌获得重组菌株BL21-Thb ZIP1,用异丙基-β-D-硫代半乳糖(IPTG)进行重组蛋白r Thb ZIP1诱导表达。试验结果表明:IPTG浓度为1.0 mmol/L,诱导温度为37℃条件下,诱导4 h,大肠杆菌提取液提取50 min时,所获得重组蛋白r Thb ZIP1表达量最大,占总蛋白的76.42%;经过10%SDS-PAGE电泳检测,发现重组蛋白r Thb ZIP1主要以包涵体的形式存在;并利用电纯化法成功获得纯化的目的蛋白。     

基于生物炭的生态浮床设计

根据生物炭的强吸附特性,对传统生态浮床进行改进,构建了由浮床框体、床体、内外双环圆柱结构种植单元、沉水植物生长袋4个部分组成的新型组合生态浮床,结合水下空间和水面景观重点描述了浮床的构建过程,为生态浮床的发展提供了新的思路。     

淮南麻黄鸡的提纯选择

淮南麻黄鸡原始群10只公鸡与75只母鸡交配所得子一代杂色羽率33.25%,白耳率7.27%,杂色喙率和杂色脚率分别只有3.64%和4.15%。表明原始群羽色基因纯合程度很低,耳色基因纯合程度较低,而喙、脚色基因纯合程度较高。经提纯选择后得予二、三代上述四个外貌性状分离比率分别降低到24.05%、0、2.75%、3.09%和17.25%、0、1.92%、2.24%。     

水稻雄性不育系自交结实问题的探讨

水稻雄性不育系自交结实会严重降低F_1的纯度。违成不育系自交结实的原因虽有几种可能:特球的环境条件、回复突变、胞质杂交,质核自然适应等,但这些都不足以导致不育系中出现高比例的自交结实。生物学混杂及其连锁反应才是导致上述现象的最普遍、最严重的诱发因素。本文提出不育系繁育要遵循“起点要高、防杂要严、数量要大、世代要短、程序要简”的方针。     

PBP1基因在大肠杆菌中的表达及其表达产物的纯化

将获得的拟南芥黑芥子酶结合蛋白PBP1基因表达载体pTYB2-PBP1导入大肠杆菌ER2566细胞,经IPTG诱导,SDS-PAGE电泳显示,PBP1-CBD融合蛋白获得了较好的表达。进一步对PBP1蛋白进行分离纯化,SDS-PAGE电泳结果显示分离纯化效果较好,所获得的纯化的PBP1蛋白可进一步用于研究PBP1与MBP6的酵母双杂交技术。