纳豆激酶的分离纯化及酶学性质研究

目的　分离纯化纳豆激酶并进行酶学性质研究。方法　从纳豆中分离纯化具有纤溶活性的纳豆激酶 ,采用氯化钠溶液浸提、硫酸铵盐析及 sephadex G- 1 5 0凝胶过滤分离纯化纳豆激酶 ,纯化倍数 5 0倍 ,纤维蛋白平板法测其比活。结果　纳豆激酶比活为 6 0 0 U·mg-1,回收率 40 %。酶学性质研究表明 ,最适反应温度为 5 0℃ ,5 5℃以下稳定 ,最适反应 p H为 8.0 ,在 p H 6～ 1 0溶液中基本稳定 ,分子量约 2 8k D。酶作用机理初探表明 ,纳豆激酶不仅具有纤溶性 ,还有抗凝血性。结论　纳豆激酶有可能成为新型溶栓药物     

纳豆激酶的活性测定

建立快速、简单测定纳豆激酶活性的方法。本实验以TAME为底物测定纳豆激酶的效价,根据扫描发现蓝色复合物在574nm处有最大吸收,故在574nm处测定吸收度,依照公式,TAME(u.mL-1)=(甲醇微克分子数×样品稀释倍数)/(样品量mL×时间min),计算样品的TAME单位。结果 TAME法灵敏度高,操作简便,适合作为常规的分析检测手段。结论实验证明,TAME法能准确测定纳豆激酶的活性,且设计简单,操作方便,重现性好,是一个切实可行的测定纳豆激酶的方法。     

纳豆激酶分离纯化与鉴定

为了提高纳豆激酶的溶栓活性，从纳豆粗提物中分离纯化了纳豆激酶。采用Butyl-Toyopead柱层析对纳豆粗提物进行了分离纯化，以试管法确定和收集了活性部位，用纤维蛋白平板法测定了活力，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(SDS-PAGE)对分离纯化的效果进行了检验。考察了纳豆激酶粗提物在不同温度和湿度条件下的稳定性。结果表明在SDS-PAGE中观察到三条明显的蛋白谱带，其中相对分子质量为19275和27101的区带具有与文献报道类似的纳豆激酶的相对分子质量和纤溶活性，而相对分子质量为14400的蛋白区带与文献报道的纳豆激酶的相对分子质量大有差异。纯化倍数为50倍，活性回收率为75．57％。初步稳定性实验结果表明：其外观在40℃和60℃保存条件下发生变化，纤溶活力在60℃保存条件下发生显著变化。而4℃条件下活力和外观却很少有变化；纳豆粗提物在不同湿度条件下均具有较强吸湿性。纳豆粗提物对高温和高湿稳定性较差。     

纳豆激酶的研究进展

纳豆激酶是一种在日本发现的高效血栓溶解酶，综述了纳豆激酶的发现、活性测定、分离纯化、溶栓作用研究及开发现状.     

纳豆激酶的提取分离纯化及研究新进展

 纳豆激酶是一种由纳豆芽胞杆菌产生的碱性丝氨酸蛋白酶,具有高效抗血栓、口服易吸收、无副作用等优点.本文对纳豆激酶的分子结构、生化特性、提取、分离与纯化等方面进行了综述,重点讨论了纳豆激酶的固体和液体发酵及层析法分离纯化,并介绍了纳豆激酶的最新研究进展.     

纳豆和纳豆激酶的药理学研究进展

纳豆激酶(Nattokinase)是1987年日本须见洋行博士从200多种食品中筛选出的纳豆中提取纯化到一种纤溶酶。纳豆是日本的传统发酵食品,以大豆为原料,经浸泡,蒸煮和接种枯草杆菌(Bacillus subtilis natto),发酵而成为纳豆。纳豆作为日本的传统食品已有千年历史,含有丰富的营养成分,具有多种生物学功能。自从发现纳豆中的纳豆激酶之后,不但日本人非常重视食用纳豆,同时全世界各地的人民也开始     

纳豆菌糖苷酶的分离纯化及酶学性质研究

目的：通过对纳豆糖苷酶的酶学性质研究,获得其适宜条件,为其各方面研究应用提供理论基础。方法：根据不同蛋白质分子理化和生物学性质差异,应用SDS-PAGE凝胶电泳技术和DEAE-cellulose DE-52柱分离方法建立纳豆糖苷酶的分离纯化方法,确定相对分子量;以酶活力为指标研究其酶学性质。结果：纳豆糖苷酶的相对分子质量约为65.6kDa。最适温度为40℃,最适pH为5.0,在20℃~40℃、pH5~pH7、低金属离子浓度条件下稳定性较好;,金属离子浓度达到5mmol/L时Na 、K 、Ca2 对酶有激活作用,Mn2 、Cu2 对酶有抑制作用。结论：纳豆糖苷酶的热稳定性较好;对较强酸碱性环境敏感,耐受性较差;较高浓度金属离子对该酶有明显激活或抑制作用。     

纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备

目的实现纳豆激酶(NK)在巴斯德毕赤酵母中的分泌表达,以纯化产物为抗原制备兔抗NK血清。为建立双抗夹心酶联免疫吸附反应法测定生物体内NK含量,进一步研究NK在体内代谢与功能奠定基础。方法将NK基因克隆到毕赤酵母表达载体pHBM905A中,鉴定后的重组质粒用SalⅠ酶切线性化后电转化到毕赤酵母宿主菌GS115中,甲醇诱导表达,经盐析和Millipore超滤膜过滤分离纯化表达产物。纤维蛋白法检测纯化产物的活性,并以纯化产物为抗原免疫新西兰大白兔制备兔抗NK血清。结果SDS-PAGE结果显示NK成功表达,其相对分子质量(Mr)约为27 000,纤维蛋白平板实验显示表达产物具有较好的纤溶活性。酶联免疫法测得多克隆抗体效价约为1∶8 000,Western blot结果显示在Mr27 000附近有1条特异带出现。结论NK在巴斯德毕赤酵母中实现了分泌表达,表达产物具有较好的纤溶活性和免疫原性。     

纳豆激酶体外活性测定及影响因素考察

目的建立纳豆激酶(NK)体外活性测定方法,考察影响因素。方法采用对甲基苯磺酰-L-精氨酸甲酯(TAME)作为底物,用变色酸反应显色,在574 nm波长处比色测定酶活性。结果NK在0.05~0.25 mg/mL浓度范围内具有良好的线性关系;NK在pH 8.0,4℃,活性保持最久。结论TAME法测定NK活性操作简便,可较准确测定NK体外活性。温度、pH、某些溶剂对NK的活性有影响。     

蚯蚓纤溶酶的分离纯化研究

目的从赤子爱胜蚓(Eisenia Foelide)中分离纯化出一种相对分子质量(Mr)较小的纤维蛋白溶解组分———蚯蚓纤溶酶(EFE),为其注射剂的研制开发打下基础。方法采用盐析、透析、凝胶过滤色谱、离子交换色谱等方法分离纯化EFE,用SDS-PAGE对纯化的活性组分进行Mr测定,用酶谱分析方法初步探讨蚯蚓中存在的其它纤溶酶活性组分。结果分离纯化得到了EFE单一组分,经SDS-PAGE EFE呈单一条带,Mr约为25 000。经酶谱分析发现纤溶酶活性组分Mr分布在25 000~50 000之间。结论反复使用色谱技术可分离纯化EFE,并得到单一组分。     

纳豆激酶活性测定方法的研究

从专属性、灵敏度及实际操作的可行性等几个方面,对TAME法和纤维蛋白平板法进行了比较研究.结果：TAME法灵敏度高,操作简便,适合作为常规的分析检测手段,而平板法虽专属性高,却操作繁琐,适宜于定性分析.结论：利用TAME法与平板法各自的优点,将两者结合起来作为纳豆激酶的活性检测方法,可达到高效灵敏、专属性强的目的.     

亲和共沉淀从红芸豆中提取红细胞凝集素及其性质研究

目的研究一种从红芸豆中提取红细胞凝集素(PHA-E)的新方法。方法红芸豆经过粗提,然后和甲状腺球蛋白的粗提取液进行共沉淀,利用超滤制得PHA-E产品,采用电泳和质谱进行鉴定,并研究其凝血活力。结果所获样品的纯度高,其亚基相对分子质量为32 000,等电点为6.5。经质谱鉴定,纯化产品为PHA-E。使兔红细胞50%凝集的PHA-E的最低浓度为2.45μg/mL,单糖不影响PHA-E凝血活力,EDTA抑制其凝血活力,Zn2+促进其凝血。结论通过亲和共沉淀的方法能快速简便的提取活力高的PHA-E。     

溶栓素的分离纯化及特性测定

目的研究一种高效分离纯化溶栓素的方法并测定其pI和相对分子质量(Mr)。方法采用阴、阳离子交换色谱等技术分离纯化溶栓素并通过对缓冲体系的pH值、离子强度的调节及对初始供试品pH值的调节来优化纯化过程,以等电聚焦聚丙烯酰胺凝胶电泳(IEF PAGE)和SDS PHGE方法分别测定纯品的pI和Mr。结果溶栓素达到简捷、高效分离纯化的目的,电泳鉴定为一条蛋白质带,并测定溶栓素的pI为4 .5 0、Mr 为35 10 0。结论溶栓素的纯化工艺达到高纯度纯化程度,为进一步研究溶栓素奠定了基础     

纳豆激酶的发酵工艺及其制剂学研究

目的研究纳豆激酶的发酵及其胶囊的制备工艺。方法考察纳豆激酶发酵提取液的热稳定性,并对纳豆激酶的制剂工艺进行研究。结果通过简单的制备工艺所制得的纳豆激酶胶囊,装量差异、含量均匀度、崩解时限均符合《中国药典》的要求。结论将纳豆激酶转化成可供临床应用的制剂具有重要意义。     

黑曲霉产纤维素酶系中内切酶的纯化和性质

目的从黑曲霉产纤维素酶系中分离纯化一种内切酶,并对其性质进行研究。方法硫酸铵盐析、Sephadex G100脱盐、DEAE FF弱阴离子交换柱层析,再经SDS-PAGE电泳标定其纯度及分子量。结果从黑曲霉发酵粉中分离纯化出了一种电泳纯的内切β-葡聚糖苷酶,SDS-PAGE分析该内切酶的分子量为26.4kD。结论实验采用的分离纯化方案稳定可行,酶学性质表明,该酶的最适反应温度是55℃,最适pH为4.8。     

A physiological model for tert-amyl methyl ether and tert-amyl alcohol: hypothesis testing of model structures.

The oxygenate tert-amyl methyl ether (TAME) is a gasoline fuel additive used to reduce carbon monoxide in automobile emissions. To evaluate the relative health risk of TAME as a gasoline additive, information is needed on its pharmacokinetics and toxicity. The objective of this study was to use a physiologically-based pharmacokinetic (PBPK) model to describe the disposition of TAME and its major metabolite, tert-amyl alcohol (TAA), in male Fischer-344 rats. The model compartments for TAME and TAA were flow-limited. The TAME physiological model had 6 compartments: lung, liver, rapidly perfused tissues, slowly perfused tissues, fat, and kidney. The TAA model had 3 compartments: lung, liver, and total-body water. The 2 models were linked through metabolism of TAME to TAA in the liver. Model simulations were compared with data on blood concentrations of TAME and TAA taken from male Fischer-344 rats during and after a 6-hour inhalation exposure to 2500, 500, or 100 ppm TAME. The PBPK model predicted TAME pharmacokinetics when 2 saturable pathways for TAME oxidation were included. The TAA model, which included pathways for oxidation and glucuronide conjugation of TAA, underpredicted the experimental data collected at later times postexposure. To account for biological processes occurring during this time, three hypotheses were developed: nonspecific binding of TAA, diffusion-limited transport of TAA, and enterohepatic circulation of TAA glucuronide. These hypotheses were tested using three different model structures. Visual inspection and statistical evaluation involving maximum likelihood techniques indicated that the model incorporating nonspecific binding of TAA provided the best fit to the data. A correct model structure, based upon experimental data, statistical analyses, and biological interpretation, will allow a more accurate extrapolation to humans and, consequently, a greater understanding of human risk from exposure to TAME.     

降纤酶的聚乙二醇修饰及产物的纯化研究

研究了降纤酶的聚乙二醇修饰及纯化方法。采用正交法考察影响修饰的4个因素，确定了最佳条件。利用分子筛柱色谱得到了较高酶活力保留的修饰产物，SDS-PAGE检测为单一条带。     

石杉碱甲的分离纯化工艺

目的:研究一种石杉碱甲的工业化分离纯化工艺,以达到批量生产之目的。方法:通过对石杉碱甲理化性质的分析,以中性氧化铝为填料对石杉碱甲粗品进行柱色谱分离纯化,采用薄层色谱-柱色谱考察了各因素的影响,以石杉碱甲回收率和含量为考察指标,寻找最佳工艺参数。结果:柱色谱最佳分离条件为:洗脱剂体系为氯仿-甲醇(v/v)=100∶5,100∶10,流速为3mL.min-1,柱床体积比为25∶1,进料量为10 mL,柱温为室温,在此条件下石杉碱甲回收率达97.6%,含量达99.2%。结论:该工艺条件能够在简单的条件下实现石杉碱甲批量分离纯化,具有工业应用价值。     

Isolation and characterization of a thrombin-like enzyme from the venom of the snake Bothrops insularis (jararaca ilhoa)

A thrombin-like enzyme (I-SII-R) from the venom of the Brazilian snake Bothrops insularis (jararaca ilhoa) was purified to homogeneity by gel filtration on Sephadex G-150 followed by precipitation of contaminating proteins with half-saturated NaCl and two further gel filtrations under the same conditions. I-SII-R showed specific activities of 820 clotting units/mg protein and 530 TAME units/mg protein (a 23-fold purification) and a single precipitation band against antibothropic horse antiserum by immunoelectrophoresis, as well as a single band by PAGE and by SDS-PAGE with beta-mercaptoethanol. The estimated molecular weight was 45,000, of which the neutral sugars account for 9900, or 22%. Its amino acid composition, which accounts for a minimum mol. wt of 33,900 is as follows: Asx35, Thr18, Ser17, Glx15, Pro29, Gly22, Ala27, Val21, Cys18, Met7, Ile19, Leu25, Tyr11, Phe11, His7, Lys8, Arg17, Trp5. When compared to thrombin, I-SII-R is relatively stable at 45 degrees C and 60 degrees C, pH 7.4, but is inhibited by heparin at a higher rate than thrombin. Both enzymes hydrolyze the alpha and beta chains of fibrinogen and lose more than 95% of their clotting and esterase activities when treated with phenylmethylsulphonyl fluoride. In contrast to thrombin, I-SII-R does not activate factor XIII.     

Purification and characterization of a thrombin-like enzyme, elegaxobin, from the venom of Trimeresurus elegans (Sakishima-habu).

A thrombin-like enzyme, named elegaxobin, was purified from the venom of Trimeresurus elegans (Sakishima-habu) by gel filtration on Sephadex G-100, and ion-exchange chromatographies on Q-Sepharose Fast Flow and S-Sepharose Fast Flow. By this procedure, about 8.5 mg of purified enzyme was obtained from 1.1 g of the venom. The purified enzyme showed a single protein band in SDS-polyacrylamide electrophoresis under reducing condition and its molecular weight is 30,000. The specific activity of this enzyme toward tosyl-L-arginine methyl ester (TAME) was 490 TAME units/mg of protein. Elegaxobin clotted only rabbit fibrinogen whereas human and bovine fibrinogens were unaffected. In the fibrinogen-fibrin convertion, the enzyme released only fibrinopeptide A from rabbit fibrinogen, whereas fibrinopeptide B was not released. The N-terminal sequences (Val-Ile-Gly-Gly) of this enzyme was identical to typical sequence of serine proteinases.