利用聚合脂质体的沉淀可逆性固定STI及其PL—STI对胰蛋白酶 …

通过双功能试剂戊二醛、二甘醇二酸酐和１,４－丁二醇双缩水甘油醚等将大豆胰蛋白栈抑制剂ＳＴＩ固定到由二十五－１０,１２－二块－１－醇磷脂乙醇胺形成的聚合脂质体上,同时探讨了其作亲和沉淀吸附剂对对胰蛋白酶的亲和作用。在ＰＬ沉淀过程中,胰蛋白酶有２０％～６０％从ＰＬ－ＳＴＩ 解离下来。ＰＬ－ＳＴＩ可作为酶的亲和沉淀吸附剂。     

新型磁性亲和吸附剂的制备及其在尿激酶纯化中的应用研究

合成了磁性聚乙烯醇和磁性聚甲基丙烯酸甲酯微球,并以它们为基质,用环氧氯丙烷、羰基二咪唑或溴化氰活化后,分别键合氨基乙酸、6-氨基己酸、乙二胺或己二胺为间隔臂,用1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐或羰基二咪唑为偶联试剂,分别偶联对氨基苯甲脒、L-精氨酸甲酯、胍基乙酸或胍基己酸配体,合成了17种磁性亲和吸附剂,并用于尿激酶粗品的纯化.与前文制备的8种磁性亲和吸附剂作对照,研究了基质、活化试剂、间隔臂分子、偶联试剂及配体等因素对尿激酶纯化效果的影响.这些磁性亲和吸附剂在尿激酶的纯化中取得了较好的效果,大多数磁性亲和吸附剂的活性回收率在40%～70%之间,纯化倍数为15～40,吸附容量为0.08～0.2mg/mL.     

纳米SiO_2亲和吸附剂的制备及性能

采用水热法一步合成了巯基纳米二氧化硅(SiO_2-SH),随后在其表面修饰亚氨基二乙酸基团(-IDA)得到了SiO_2-SH/IDA,利用-SH和-IDA双官能团更多的吸附溶液中的Ni~(2+),从而得到SiO_2-SH/IDA-Ni~(2+)纳米亲和吸附剂.制备的亲和吸附剂可直接用于六聚组氨酸为标签的(His-tagged)融合蛋白的分离纯化.利用TEM、FT-IR、TG、SDS-PAGE等大型仪器表征了样品的形貌、结构及亲和分离能力.结果表明制备的SiO_2-SH/IDANi~(2+)纳米亲和吸附剂平均粒径为60nm,对His-tagged蛋白具有较好的特异性和较低的检测限(约为1.9×10~(-5)mol/L),且该吸附剂再生能力较强,再生3次后对目标蛋白仍具有较好的分离效果.     

N-去甲万古霉素亲和吸附剂的合成及性能

将N,N二甲基丙烯酰胺N,N′乙撑双丙烯酰胺共聚物部分水解,在共聚物中引入适量的羧基.含羧基的聚合物与氨基酸甲酯缩合,然后使酯基皂化,将氨基酸引入聚合物.将革兰氏阳性菌细胞壁粘肽的三种类似物(-Gly,-Gly-DAla,-Gly-DAla-DAla)分别引入上述聚合物,合成了3种万古霉素系列抗菌素的亲和吸附剂(Ⅰ,Ⅱ和Ⅲ).结果表明,吸附剂Ⅱ和吸附剂Ⅲ对N去甲万古霉素的吸附量分别为0.80和0.86mmol/g;最佳吸附pH值为6左右;吸附剂Ⅰ的吸附量随着吸附液中盐浓度的增加而显著降低,而吸附液中盐浓度对吸附剂Ⅱ的吸附量影响较小.说明亲和作用在吸附剂Ⅱ的吸附中贡献较大.用0.4mol/LNa₂CO₃(pH9.5)/CH₃CN(体积比为7/3)作为洗脱剂可完全脱附被吸附的N去甲万古霉素.     

胰蛋白酶定向固定化亲和配基的理性设计

在胰蛋白酶三维(3D)结构的基础上, 首先利用分子对接从ZINC 数据库中筛选获得了与胰蛋白酶具有较高亲和性的小分子配基2-硝基苯基-β-D-葡糖苷, 并分析了该配基与蛋白质之间的相互作用力主要为范德华和氢键相互作用. 并利用分子动力学模拟进一步验证了2-硝基苯基-β-D-葡糖苷与胰蛋白酶之间具有较强的亲和作用. 分子动力学(MD)模拟结果表明, 配基-目标蛋白质之间形成稳定的复合物且它们之间的距离基本没有变化. 此外, 一个水分子通过氢键在配基和目标蛋白质的结合腔之间架桥. 最后制备了偶联有该配基的亲和载体, 进行了胰蛋白酶的定向固定化, 并考察了该固定化酶的活性. 研究结果表明, 利用修饰2-硝基苯基-β-D-葡糖苷配基的亲和载体固定化胰蛋白酶的酶活达到340.8 U·g^-1, 比活达到300.3 U·mg^-1, 分别是未修饰亲和配基载体的10倍和5倍, 具有明显的优势. 上述结论证明了结合分子对接和分子动力学模拟理性设计定向固定化亲和配基的方法是可行的, 具有一定的理论和实用价值.     

膜亲和色谱用于胰蛋白酶抑制剂的分离

分别采用酰化-胺化和氯甲基化-胺化对聚砜进行化学修饰,用相转化法制成平均孔径为450nm的超滤膜,经重氮化反应,共价键合上具有活性的胰蛋白酶。其相对活力可达10200 u/g。每克化学改性聚砜膜上可固载化上15 mg胰蛋白酶。用此酶膜对胰蛋白酶抑制剂进行了亲和分离,一次可得6.5mg纯胰蛋白酶抑制剂。     

以聚乙烯基咪唑为配基的内毒素亲和吸附剂的研究

通过乙烯基咪唑(VI)在硅胶粒子表面的自由基接枝聚合制备了一种以聚乙烯基咪唑为配基的新型内毒素亲和吸附剂. 用FTIR检测样品中咪唑基的特征吸收, 用热重分析法(TGA)测定了PVI的接枝率. 实验发现, PVI在吸附剂中的含量对内毒素的吸附率影响很大. 当PVI的接枝率为2.5％左右时, 吸附剂对内毒素的去除率最大. 在离子强度小于1 mol/L和pH=7的中性条件下, PVI吸附剂对内毒素具有最佳的吸附性能. 该吸附剂具有良好的血液相容性. 内毒素在该亲和吸附剂上的吸附等温线符合Freundlich吸附方程, 其吸附动力学为二级反应.     

N-去甲万古霉素的亲和吸附

通过交联聚丙烯酸甲酯与乙醇胺反应,形成聚(N-羟乙基丙烯酰胺)树脂,在酸催化作用下与环氧氯丙烷反应,形成含有α-羟基氯乙基的树脂.含α-羟基氯乙基的树脂与D-丙氨酸、L-丙氨酸或甘氨酸反应,分别得到含有这3种氨基酸的吸附剂.这3种吸附剂吸附N-去甲万古霉素的结果表明,含D-丙氨酸的吸附剂的吸附量最大,含甘氨酸的吸附剂的吸附量次之,而含L-丙氨酸的吸附剂不吸附N-去万古霉素.说明前两种吸附剂对N-去甲万古霉素存在亲和吸附作用.含D-丙氨酸吸附剂的最佳吸附pH值为5.8,当吸附液中的盐(NaCl)浓度增加时,吸附量降低.用0.4mol/LNa₂CO₃/CH₃CN(摩尔比7∶3,pH=9.5)作为洗脱剂可完全脱附被吸附的N-去甲万古霉素.     

聚合脂质体固定对氨基苯甲脒及其对胰蛋白酶的亲和吸附作用

将对氨基苯甲脒是直接法和间接法固定到二十五－１０，１２－二炔－１－醇磷脂乙醇胺的聚合脂质体上，研究了聚合脂质体－对氨基苯甲脒对胰蛋白酶的亲和吸附作用。     

铅离子双印迹吸附剂的制备及其在原子吸收光谱法测定水中痕量铅中的应用

采用分子印迹技术,以3-疏基丙基三甲氧基硅烷为功能单体,铅离子和十六烷基三甲基溴化铵(CTMAB)为双模板形式络合体系,并加入由四乙氧基硅烷和甲醇所形成的溶胶,用氢氧化钠作催化剂制得铅离子双印迹吸附剂。经红外光谱法和氮气吸附-脱附系统对此吸附剂的结构特征及表面性能进行表征和分析。结果表明:在印迹吸附剂中除去铅(Ⅱ)离子模板后,恢复了-SH官能团;CTMAB的存在具有提高表面积和孔径的倾向。此双印迹吸附剂在静态条件下,对铅(Ⅱ)离子的吸附经10min,吸附率达95%。吸附容量达545.6mg·g-1。在镉(Ⅱ)离子共存下,相对选择性系数为192。用0.5mol·L-1硝酸溶液5mL即可从吸附剂上洗脱94.4%铅(Ⅱ)。以此吸附剂作为萃取材料分离富集了环境水样中痕量铅(Ⅱ),洗脱后用原子吸收光谱法测定其中铅(Ⅱ)量,测定值的回收率在104%~106%之间。     

Predigestion of soybean proteins with immobilized trypsin for infant formula

Soybean protein isolates of low soybean trypsin inhibitor (STI) residue were prepared by acidic precipitation of soybean flour water extracts (0.8–1.2%) at pH 5.0, followed by acidic washing at this pH and affinity adsorption of residual STI with immobilized trypsin on polystyrene anion-exchange resin GM 201. After heat treatment, soybean protein isolates were subjected to controlled hydrolysis with the immobilized trypsin. Then, the predigested soybean protein was prepared. The predigested soybean protein was free of STI activity, and its solubility at acidic pH range was greatly increased. Sedimentation test showed that it formed a much finer clot at pH 4.5 than that of untreated soybean protein. The pepsin digestibility index at pH 4.0 and chymotrypsin digestibility index at pH 8.0 were obviously improved. These results suggested that the predigested soybean protein prepared by this method may be used in infant formulas.     

Antioxidant Peptides from Protein Hydrolysate of Bovine Serum Colostrum

Chemistry of Natural Compounds - Casein was removed from defatted bovine colostrum by precipitation at pH 4.3. Serum proteins were hydrolyzed by trypsin, alkalase, and papain. The degree of...     

Phospholipase D-mediated aggregation, fusion, and precipitation of phospholipid vesicles

Large unilamellar vesicles with a diameter of 100 nm were prepared from the zwitterionic phospholipid POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) at pH 8.0. After addition to these vesicles of the enzyme phospholipase D (PLD) from Streptomyces sp. AA586 at 40 degrees C, the terminal phosphate ester bond of POPC was hydrolyzed, yielding the negatively charged POPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid) and the positively charged choline. While the reaction yield in the presence of 1 mM Ca2+ reached 100%, the yield was only approximately 68% in the absence of Ca2+. Furthermore, in the absence of Ca2+, the size of the vesicles did not change significantly with time upon PLD addition, as judged from turbidity, dynamic light scattering, and electron microscopy measurements. In the presence of 1 mM Ca2+, however, PLD addition resulted in vesicle aggregation, fusion, and precipitation, originating from the interaction of Ca2+ ions with the negatively charged phospholipids formed in the membranes. Vesicle fusion was monitored by using a novel fusion assay system involving vesicles containing entrapped trypsin and vesicles containing entrapped chymotrypsinogen A. After vesicle fusion, chymotrypsinogen A transformed into a-chymotrypsin, catalyzed by trypsin inside the fused vesicles. The alpha-chymotrypsin formed could be detected with benzoyl-L-Tyr-p-nitroanilide as a membrane permeable chymotrypsin substrate. The observed vesicle precipitation occurring after vesicle fusion in the presence of 1 mM Ca2+ was correlated with an increase of the main phase transition temperature, Tm, of POPA to values above 40 degrees C.     

Imidazole—a new ligand for metal affinity precipitation

Kunitz soybean trypsin inhibitor (STI) was specifically coprecipitated during precipitation of Cu(II)-loaded copolymers induced by increase in temperature and ionic strength. The copolymers used consisted of 1-vinylimidazole andN-vinylcaprolactam orN- isopropylacrylamide. The elution of STI was achieved by solubilization of the STI-Cu(II)-polymer complex in the presence of an excess of the competing ligand, imidazole, and a subsequent precipitation of the polymer with STI remaining free in solution in a purified form as judged by Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To the best of our knowledge this is the first reported successful metal affinity precipitation of protein in a heterobifunctional format.     

Reassemblable quasi-chip free-flow electrophoresis with simple heating dispersion for rapid micropreparation of trypsin in crude porcine pancreatin

An increasing number of small biosamples (e.g. proteins and enzymes) need micropreparation in lab. However, neither large-scale free-flow electrophoresis (LS-FFE) nor chip FFE (C-FFE) could fit the growing demands. Herein, a simple quasi-chip FFE (QC-FFE) was constructed. In contrast to C-FFE, the features of QC-FFE are as follows: (i) its separation chamber is reassemblable and rewashable avoiding discard of C-FFE due to blockage of solute precipitation in chamber; (ii) its chamber size is 45 mm × 30 mm × (80-500) μm (108-654 μL volume) having function of micropreparation; (iii) there are up to 16 outlets in QC-FFE bestowing fine fraction for micropurification. The QC-FFE was used for the micropurification of model enzyme of self-digestible trypsin in crude pancreatin. Under the given conditions, the purification factor of enzyme was 11.7, the specific activity reached 6236 U/mg, the run time for 19 μL sample purification was 45 s and the throughput of trypsin was 3.34 mg/h, and the yield of pure trypsin was 55.2%. All of the results show the feasibility of enzyme micropreparation via QC-FFE. The developed device and procedure have potential use to other micropurification of protein or peptide sample.     

Precipitation of trypsin with polymethacrylic acid

Conclusions It has been shown that the precipitation of trypsin with polymethacrylic acid results in a reversible or irreversible (after an incubation period) formation of a complex and, in the latter case, a stable, nonactive addition product can be isolated.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 8, pp. 1895–1896, August, 1970.     

壳聚糖固定化胰蛋白酶的研究

以壳聚糖为载体，戊二醛为交联剂，采用两种方法制备了固定化胰蛋白酶。考察了固定化反应中pH值，戊二醛的浓度，以及给酶量对固定化胰蛋白酶活力的影响，并研究了这两种固定化胰蛋白酶的性质。实验结果表明，以戊二醛预交联的网状壳聚糖为载体制备的固定化胰蛋白酶具有更加优良的性能，在最佳固定化反应条件下，酶的活性加收率可达56％。此固定化胰蛋白酶的最适pH为7．0－8．5，最适温度为60℃，Km值为2．52mol/L，固定化胰蛋白酶表现出较好的热稳定性，pH贮存稳定性，以及在乙醇水溶液中的稳定性。     

聚季铵盐离子液体材料制备及其对蛋白质吸附性能评价

通过一步合成法制备了两种可聚合季铵盐离子液体功能单体,并通过沉淀聚合法合成了相应的聚离子液体聚合物。对产物进行了核磁共振、扫描电镜、热重分析等表征。结果表明:所制备的两种材料粒径均匀,约为600 nm的椭球形颗粒,颗粒之间有相互粘连。通过对牛血清白蛋白(BSA)、卵清蛋白(OVA)、牛血红蛋白(BHb)、溶菌酶(Lys)、胰蛋白酶(Try)5种蛋白质的吸附性能实验,考察了聚季铵盐离子液体材料对蛋白质的吸附性能。考察结果表明:两种聚离子液体材料均对蛋白质具有一定的吸附性能。其中以4-乙烯基苄氯季铵盐离子液体为功能单体制备的聚离子液体材料对胰蛋白酶的吸附性能最好,是一种具有良好应用前景的材料。     

Analysis of staphylococcal enterotoxin A in milk by matrix-assisted laser desorption/ionization-time of flight mass spectrometry

Staphylococcal enterotoxin A (SEA) is an exotoxin excreted mainly by Staphylococcus aureus and nowadays is the most prevalent compound in staphylococcal food poisoning worldwide. SEA is highly heat-resistant, and usual cooking times and temperatures are unlikely to completely inactivate it. A procedure for extraction of this toxin based on protein precipitation with a mixture of dichloromethane and acidified water was used before SDS-PAGE separation of soluble proteins. Finally, bands of interest were excised from the gel and in-gel enzymatic digestion was done. SEA from pasteurized milk was detected with matrix-assisted laser-desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Nineteen peptides (range 800-2400 Da) were identified as products of trypsin cleavage of the SEA standard with a score of 204 and 73% coverage of the protein sequence, whereas thirteen peptides were revealed for SEA extracted from milk with a score of 148 and 58% sequence coverage obtained. This procedure has been applied successfully for identification of SEA in milk.     

Activity of immobilized trypsin in the layer structure of polyelectrolyte microcapsules (PEMC)

Polyelectrolyte microcapsules composed by using the LbL technique on stabilized RBC as templates were coated with up to ten layer pairs of trypsin/PSS or trypsin/alginate. The trypsin layer growth was confirmed by particle electrophoresis, confocal laser scanning microscopy, flow cytometry, and protein determination according to Lowry. In the coating series with trypsin/PSS, the amount of immobilized enzyme was larger than that with trypsin/alginate. The enzyme immobilization led to activity reduction of up to 90% compared to that of the same enzyme amount in the solution. No significant differences between the activities of trypsin immobilized in combination with PSS and with alginate were found.