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纳豆中纳豆枯草杆菌的筛选和纳豆激酶的分离过程研究 总被引:8,自引:2,他引:8
采用酪蛋白平板初筛一摇瓶复筛的筛选策略,从日本传统食品纳豆中筛选获得了一株高产纳豆激酶的纳豆枯草菌菌株,与国内公开报道的不同菌株相比,有更好的产酶能力。菌株经过液体发酵后获得含酶发酵液,采用硫酸铵盐析及Sephadex G-75凝胶层析、Sepharose CM Fast Flow离子交换层析对纳豆激酶进行了分离纯化,两步层析的纯化倍数和酶活回收率分别达到4.75、743%和1.32、77%,获得了纯度很高的纳豆激酶。 相似文献
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一种双水相亲和分配配基的制备过程优化--亚氨基二乙酸-聚乙二醇 总被引:4,自引:0,他引:4
考察了不同亚氨基二乙酸(IDA)取代度的亚氨基二乙酸-聚乙二醇(IDA-PEG)的制备,并进行了条件优化(BF3浓度,NaOH浓度,反应时间,IDA结合条件等)。通过控制环氧氯丙烷与聚乙二醇的摩尔配比,得到了不同Cu( )含量的固定化金属亲和配基:Cu( )-IDA-PEG(A)(0.24molCu2 /molPEG)、Cu( )-IDA-PEG(B)(0.51molCu2 /molPEG)、Cu( )-IDA-PEG(C)(0.75molCu2 /molPEG),并初步探讨了固定化金属亲和配基的添加对PEG4000-(NH4)2SO4-H2O双水相系统相图的影响。 相似文献
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硫酸铵沉淀和层析法分离纯化纳豆激酶的研究 总被引:9,自引:1,他引:9
利用生产纳豆激酶的Bacillus subtilis进行深层发酵.采用发酵液离心除菌,20%~60%饱和度的硫酸铵沉淀,Superdex 75凝胶过滤层析和SP Sepharose Fast Flow离子交换层析对活性组分进行分离提纯.用纤维平版法测定了活力,并用十二烷基磺酸钠-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.结果表明在SDS-PAGE中观察到单一条带,分子量27.7 kD,最终纯化倍数和酶活回收率分别为8.4和49%. 相似文献
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本文采用七种活性染料为亲和配体,在双水相系统中分离人血清白蛋白、α球蛋白和β-球蛋白。实验结果表明这七种染料配体对白蛋白和β-球蛋白具有一定的亲和作用,对α-球蛋白的亲和力不显著。其中,配体活性金黄K-MG对所研究的蛋白质具有良好的亲和作用和选择性,是一种具有较高应用价值的染料配体。 本文还探讨了pH值、盐及温度等因素对三种蛋白质分配效果的影响。 相似文献
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采用改进悬浮聚合法制备了磁性聚醋酸乙烯酯微球,经水解、氨基化修饰,以戊二醛作间隔臂偶联配基对氨基苯甲脒,制得磁性PVA亲和微球,用于纳豆激酶粗酶液的分离纯化,对所制微球进行了表征,研究了纳豆激酶的吸附平衡、吸附和解吸动力学、酶的纯度和酶活回收. 结果表明,磁性微球具有较高的比饱和磁化强度(32.4 emu/g),40 min即可达到纳豆激酶的吸附平衡,15 min内完成酶的解吸,酶活回收率接近75%,纯化因子为10.3,酶的分子量为28 kDa,电泳分析为一条带. 相似文献
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膨胀床离子交换吸附分离纳豆激酶 总被引:5,自引:0,他引:5
对膨胀床分离纳豆激酶过程的各个阶段进行了考察。在填充床上的探索性实验表明,吸附时最佳的pH为6.0,电导率应低于6.2mS/cm。在膨胀床上样吸附阶段,考察了保持流速不变和保持床层膨胀率不变2种操作方式,结果表明采用控制膨胀率不变的方法更适合纳豆激酶这种料液的粘度和平衡缓冲液的粘度相差不大的情况;在冲洗阶段,通过考察颗粒冲洗的效率确定采用平衡缓冲液冲洗;洗脱阶段采用填充床模式洗脱。与传统方法相比,用膨胀床对纳豆激酶进行分离和纯化,操作步骤从6步减少为2步,操作时间缩短了8—10h,回收率提高了约50%。 相似文献
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纳豆激酶的研究进展 总被引:2,自引:0,他引:2
丁有学 《中国生物制品学杂志》2008,21(12):1129-1131
纳豆激酶是一种具有强烈溶栓功能的碱性丝氨酸蛋白酶,近年来,国内外学者对其进行了广泛的研究。本文介绍了纳豆激酶的理化性质、主要生物学活性、溶栓机理及其生物学活性体外检测方法的研究进展。 相似文献
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Preparation of Iminodiacetic Acid-Polyethylene Glycol for Immobilized Metal Ion Affinity Partitioning 总被引:2,自引:0,他引:2
1 INTRODUCTIONImmobilized metal ion affinity chromatography(IMAC) is a powerfu tool for the separation ofproteinsI1'2]. Metal amnity separation exPloits theaffinity of transition metal ions for electron-rich aminoacid residues, such as histidine and cysteine, which areaccessible on the surface of proteins. Metal recogni-tion can also be engineered into prOteins for aPplication such as protein purification[3]. Recelltly, immobi- 相似文献
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The synthesis route was investigated and optimized for the preparation of iminodiacetic acid polyethylene glycol (IDA-PEG) for immobilized metal ion affinity partitioning in aqueous two-phaze systems. IDA PEG was synthesized from PEG in two steps by the reaction of iminodiacetic acid with a monosubetituted derivative of epichlorohydrin-activated PEG. The Cu2+ content combined with IDA-PEG was determined by atomic absorp tion spectrometry as 0.5 mol.mol-1 (PEG). Furthermore, the affinity partitioning behavior of lactate dehydrogenase in polyethylene glycol/hydroxypropyl starch aqueous two-phaze systems was studied to clarify the affinity effect of the Cu(Ⅱ)-IDA-PEG. 相似文献
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双水相萃取技术分离纯化蛋白质的研究 总被引:6,自引:0,他引:6
阐述了双水相萃取原理,详细分析了影响双水相萃取分离纯化蛋白质的各种因素,探讨了双水相萃取技术在蛋白质分离纯化中的应用并对其前景进行了展望. 相似文献
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双水相萃取分离苦参生物碱 总被引:1,自引:0,他引:1
用乙醇、聚乙二醇和Triton X-100与硫酸铵等无机盐构建双水相体系,研究了各体系的形成过程与性质及对氧化苦参碱和苦参碱的萃取性能,发现乙醇/硫酸铵双水相体系稳定、分相快、萃取率高,更适合苦参生物碱的萃取;对其萃取条件的优化结果表明,乙醇和硫酸铵分别为38%(w)和18%(w),体系pH值为7.0,30℃下搅拌萃取15 min,氧化苦参碱与苦参碱的分配系数和萃取率分别为4.46和5.10,96.17%和95.74%. 在苦参的水提取液中直接用该体系萃取分离氧化苦参碱、氧化槐果碱、槐定碱、苦参碱及槐果碱5种生物碱,萃取率为91.03%~94.46%,三级错流总萃取率为97.22%~98.78%;该体系用于苦参生物碱的纯化,可显著减小杂质含量,苦参碱含量可提高1倍以上,其焓变DH0和熵变DS0均大于0,且DG0<0,表明萃取过程为吸热熵增的自发过程. 相似文献
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《分离科学与技术》2012,47(10):1483-1499
Abstract The systematic partitioning of metal ions in the presence of polyethylene glycol (PEG) chelate derivatives in PEG/salt two-phase systems is described. Results show that the partitioning is enhanced significantly, and this effect is apparently based on the characteristics of binding of the chelate and the nature of the metal ion in addition to the effect of the attached PEG molecule. Previous work with PEG-iminodiacetic acid has been extended with the chelating derivatives PEG-TED [tris(carboxymethyl)ethylene diamine], PEG-TREN-Cm [carboxy-methylated-tris(2-aminoethyl)amine], and PEG-ASP (aspartic acid). Their effectiveness to selectively partition the metal ions is demonstrated and discussed. The partition experiments were performed using two-phase systems of PEG (8000 MW), sodium sulfate, the PEG-chelating derivatives, and different concentrations of copper, nickel, and cobalt as model metal ions. Partition coefficients (ratio of top to bottom metal concentrations) greater than 1 were obtained for the metal ions with all the chelating derivatives tested. Selective enhancement of partitioning was observed in all cases. Based on the experimental results and theoretical principles, the method shows potential as an alternative mode to remove efficiently and selectively metal ions from solutions using aqueous two-phase systems and PEG-chelate derivatives. 相似文献