柞蚕蛹溶菌酶的分离和纯化

本文报道热空气,大肠杆菌Ｋ１２Ｄ３１诱导柞蛹血淋巴,提高溶菌酶活力的方法。应用选择性热变性,等电点沉淀,ＤＥＡＥ－纤维素离子交换层析,ＣＣ－纤维素亲和层析等分离技术从柞蚕蛹血淋巴中提取溶菌酶,溶菌酶比活力达３６７００ｕ／ｍｇ蛋白,提高了１５０倍,活力回收达４８．６％。     

蛋白质分离纯化方法的研究进展

蛋白质作为生命物质基础之一,存在于自然界中复杂的混合体系中。蛋白质在细胞中的含量极低,将其从复杂体系中分离出来并保持其活性,难度较大,因此分离纯化蛋白质的方法受到生命科学领域广泛关注。本文首先阐述了蛋白质的预处理,其次阐述了蛋白质分离纯化的各种方法如沉淀、层析、离心等,重点阐述了蛋白质新纯化的各种原理,以及每种方法的适用范围。有助大家更全面、更彻底的了解蛋白纯化技术的发展,以期为今后开展蛋白质的制备及应用提供一定理论依据和实验指导。     

海洋微生物溶菌酶的纯化与性质研究

海洋微生物溶菌酶发酵上清液经超滤、CM-SepharoseFF阳离子交换层析和SephadexG-10 0凝胶过滤层析纯化得到电泳纯的溶菌酶,纯化倍数为34.7,活力回收为24.1%。对纯化溶菌酶性质研究表明,该酶分子量约为39kD ,对溶壁微球菌的最适作用温度为35℃,最适作用pH为8 0 ,在5 0℃以下和pH 5 0～10 0之间都有较好的稳定性,与常见金属离子和化学试剂有良好的配伍性,广谱杀菌,对多种致病菌也有较强的溶菌作用。     

疫苗分离纯化研究进展

综述了国内外关于疫苗分离纯化的研究进展,系统介绍了目前可用作各类疫苗(尤其是基因工程疫苗)分离纯化的方法。沉淀和离心等传统分离技术在各类疫苗的分离纯化中应用广泛,层析技术和其它分离技术的结合已成为疫苗分离纯化的主流,新型膜技术和亲和层析在基因工程亚单位疫苗分离纯化中的作用引人注目。     

工程菌人溶菌酶的纯化和性质

将人溶菌酶工程菌株在发酵培养、菌体经超声破碎、变性和复性后所得的粗酶液经ExpressIon S阳离子交换柱层析,得到电泳纯的酶,比活达到48KG*4]000u/mg。此酶的最适pH为6.5;等电点为8.91;对溶壁微球菌的米氏常数Km=0.0311mg/mL;60℃保温30〖KG*4]min,酶活力剩余48.3%。N末端氨基酸序列除了第一个Met,其余4个与预期相符。一些重金属离子对酶的活性影响不尽相同,在0.01     

细菌荚膜多糖的分离纯化研究进展

荚膜多糖是细菌的保护性抗原和毒力因子,也是细菌疫苗最重要的靶抗原之一,其分离纯化是制作疫苗的首要步骤。本文从去除菌体、收集总糖、去除菌体核酸和蛋白质、去除内毒素等基本工艺步骤,对现有的工艺和目前的工艺进展进行了综述,重点阐述了中空纤维、深层过滤、超滤、酶水解、柱层析等方法在荚膜多糖分离纯化中的应用进展。     

工程菌噬菌体T7溶菌酶的纯化和性质

用崔道珊等构建的噬菌体T7溶菌酶工程菌株,培养物经超声破碎和DE52,CM52柱层析纯化,我们得到电泳纯的T7溶菌酶,分子量为17000,最适反应pH为8.0.其热稳定性欠佳,保温37℃,5min即丧失酶活21%.     

多肽的分离纯化技术研究进展

随着各种活性多肽的发现,多肽分离纯化技术的研究和开发日益受到专家的关注。介绍多肽的理化性质与活性,讨论分离多肽的色谱、层析、电泳、膜、萃取等技术的特点和方法,并展望多肽的分离纯化前景,旨在为多肽的分离纯化提供参考。     

木聚糖酶分离纯化技术

木聚糖酶应用广泛,其分离纯化是进行酶学性质研究、分子研究的前提条件,是成功确定氨基酸序列和三维结构的基础。综述微生物木聚糖酶分离纯化的方法,分析了常用方法在其分离纯化中的优缺点;强调了特异性分离纯化技术是高效的分离纯化方法;并对其它方法进行了概括。     

果胶酶分离纯化及分析方法的研究进展

果胶酶能降解果胶质,在果汁制造、果酒酿造等方面有着广泛应用。果胶酶分子生物学的迅速发展极大地促进了分离纯化与分析方法的研究。由于不同菌种产生的果胶酶成分复杂程度不同,分离纯化手段和分析方法也不相同。本文对果胶酶分离纯化手段及其分析方法进行了综述。     

柞蚕微孢子虫孢子分离纯化方法

柞蚕微粒子病是柞蚕Antheraea pernyi(Guérin-Méneville)的主要胚胎传染性病害,病原为柞蚕微孢子虫Nosema pernyi(Wenet Ding),其病原分离提纯技术研究对于柞蚕微粒子病的防治具有重要意义。本文利用差速离心和Percoll密度梯度离心法研究了柞蚕微孢子虫孢子的分离纯化方法,结果表明,采用不连续密度梯度分离纯化柞蚕微孢子虫孢子的效果比单一浓度的效果好,以浓度为25%、50%、75%、100%不连续梯度,15000r/min离心30min分离纯化得到的柞蚕微孢子虫孢子纯净度高。     

Study on separation of conalbumin and lysozyme from high concentration fresh egg white at high flow rates by a novel ion-exchanger

In this report, we show that it is possible to separate valuable proteins from egg-white using a Productiv(TM) CM ion-exchanger column operated at flow rates significantly higher than those than can be achieved using traditional particulate adsorbents. In the approach taken, sample pretreatment is restricted to a simple dilution of the egg-white, which can then be applied to the column at superficial velocities (V(s)) of up to 13.8 m/h. Under a loading of 220 mg total protein per milliliter of ion-exchanger, the resolution (R(s)) between the eluted conalbumin and lysozyme fractions was found to be almost constant during nine consecutive adsorption/desorption cycles. For all nine consecutive batches, the column average adsorption capacity was greater than 30 mg/mL, with 90% recovery of adsorbed protein being achieved in each run. The overall productivity achieved was 12.6 kg/m(3) h for lysozyme and 31.2 kg/m(3) h for conalbumin. (c) 1993 John Wiley & Sons, Inc.     

淋巴细胞分离液在植物细胞原生质体分离纯化中的应用

本文报道了用淋巴细胞分离液分离纯化植物原生质体的实例,并就其方法原理和运用技巧进行了讨论。     

Characterization of superporous cellulose matrix for high‐throughput adsorptive purification of lysozyme

Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100–300 μm, mean pore size: 1–3 μm, pore volume: 57–59%, and bulk density: ～1,438 kg/m³) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for high‐throughput purification of lysozyme from chicken egg white's extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads‐CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high‐throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m³ h) and specific activity (2.2 × 10⁵ U/mg) were higher than that obtained with traditional particulate resins. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Process evaluations and economic analyses of recombinant human lysozyme and hen egg-white lysozyme purifications

Human lysozyme and hen egg-white lysozyme have antibacterial, antiviral, and antifungal properties with numerous potential commercial applications. Currently, hen egg-white lysozyme dominates low cost applications but the recent high-level expression of human lysozyme in rice could provide an economical source of lysozyme. This work compares human lysozyme and hen egg-white lysozyme adsorption to the cation exchange resin, SP-Sepharose FF, and the effect of rice extract components on lysozyme purification. With one exception, the dynamic binding capacities of human lysozyme were lower than those of hen egg-white at pH 4.5, 6, and 7.5 with ionic strengths ranging from 0 to 100 mM (5-20 mS). Ionic strength and pH had a similar effect on the adsorption capacities, but human lysozyme was more sensitive to these two factors than hen egg-white lysozyme. In the presence of rice extract, the dynamic binding capacities of human and hen egg-white lysozymes were reduced by 20-30% and by 32-39% at pH 6. Hen egg-white lysozyme was used as a benchmark to compare the effectiveness of human lysozyme purification from transgenic rice extract. Process simulation and cost analyses for human lysozyme purification from rice and hen egg-white lysozyme purification from egg-white resulted in similar unit production costs at 1 ton per year scale.     

溶菌酶的研究进展

对溶菌酶的稳定性的改善、制备、基因工程表达产物的复性处理作了介绍,并且对溶菌酶在医药、食品工业和生物工程上的应用作了概述。     

Stabilization of Micrococcus lysodeikticus cells towards lysis by lysozyme using glutaraldehyde: application as a novel biospecific ligand for the purification of lysozyme

Micrococcus lysodeiekticus was stabilized against the lytic action of lysozyme by cross-linking with 5% (v/v) glutaraldehyde for 24 h but still retained its ability to bind lysozyme. An immobilized, biospecific ligand was prepared by covalently binding the cells to glutaraldehyde activated amino-Sepharose followed by stabilization of the cells with glutaraldehyde. Lysozyme bound specifically to this column and could be eluted by glycine/NaOH buffer (50 mM, pH 10.0) containing 2 M KCl.     

Evaluation of alternatives for human lysozyme purification from transgenic rice: impact of phytic acid and buffer

Producing economically competitive recombinant human lysozyme from transgenic rice demands an inexpensive purification process for nonpharmaceutical applications. Human lysozyme is a basic protein, and thus, cation exchange chromatography was the selected method for lysozyme purification. Similar to other protein production systems, the identification of critical impurities in the rice extract was important for the development of an efficient purification process. Previous adsorption data indicated that phytic acid was probably responsible for an unacceptably low cation exchange adsorption capacity. In this study, we confirm that reducing phytic acid concentration improves lysozyme binding capacity and investigate alternative process conditions that reduce phytic acid interference. Compared with the previous best process, the adsorption capacity of human lysozyme was increased from 8.6 to 19.7 mg/mL when rice extract was treated with phytase to degrade phytic acid. Using tris buffer to adjust pH 4.5 extract to pH 6 before adsorption reduced phytic acid interference by minimizing phytic acid-lysozyme interactions, eliminated the need for phytase treatment, and increased the binding capacity to 25 mg/mL. Another method of reducing phytic acid concentration was to extract human lysozyme from rice flour at pH 10 with 50 mM NaCl in 50 mM sodium carbonate buffer. A similar binding capacity (25.5 mg/mL) was achieved from pH 10 extract that was clarified by acidic precipitation and adjusted to pH 6 for adsorption. Lysozyme purities ranged from 95 to 98% for all three processing methods. The tris-mediated purification was the most efficient of the alternatives considered.     

A simple method for efficient separation and purification of c-phycocyanin and allophycocyanin from Spirulina platensis

c-Phycocyanin and allophycocyanin were separated and purified from Spirulina platensis by precipitation with ammonium sulphate, ion exchange chromatography and gel filtration chromatography. Pure c-phycocyanin and allophycocyanin were finally obtained with an A₆₂₀/A₂₈₀value of 5.06 and an A₆₅₅/A₂₈₀ value of 5.34, respectively.     

Solvent effects on the structure,dynamics and activity of lysozyme

Kuntz and Kauzmann have argued that dehydrating a protein results in conformational changes. In contrast, Rupleyet al. have developed a hydration model which involves no significant change in conformation; the onset of enzyme activity in hen egg-white lysozyme at hydration values of about 0.2 g water/g protein they ascribe rather to a solvation effect. Using a direct difference infra-red technique we can follow specific hydration events as water is added to a dry protein. Conformational studies of lysozyme using laser Raman spectroscopy indicate changes in conformation with hydration that are complete just before measurable activity is found. Parallel nuclear magnetic resonance measurements of exchangeability of the main chain amide hydrogens, as a function of hydration from near dryness, suggest a hydration-related increase in conformational flexibility which occurs before-and is probably necessary for-the Raman-detected conformational changes. Very recent inelastic neutron scattering measurements provides direct evidence of a flexibility change induced by hydration, which is apparently necessary before the enzyme can achieve adequate flexibility for it to begin to function.