D2-4放线菌抗真菌病害活性成分研究

从长白山森林土壤中分离的放线菌D2 4产生的抗生素对蕃茄灰霉病等蔬菜及经济作物的某些真菌病害有很好的防治效果。通过离子交换和吸附层析对抗生素进行了分离纯化。此抗生素为一种弱碱性水溶性抗生素 ,在酸性和中性条件下稳定 ,在碱性条件下不稳定 ,温度耐受性较强 ,在适宜pH条件下 ,1 0 0℃处理0 .5h ,其活性基本不变。     

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。     

肽抗生素及其基因工程研究

抗生素的滥用带来严重的病菌抗药性问题。肽抗生素能杀死多种微生物及特异性杀死癌细胞,通过穿膜打洞的方式使细胞死亡,不会导致抗药菌株的产生。从昆虫及高等动物中分离纯化了许多种类的肽抗生素,寻找到一些杀菌和杀癌细胞能力强的种类。由于分离天然产物产量极微,采用人工合成则成本太高,科研人员致力于用基因克隆与表达的技术研究与开发肽抗生素。     

微藻对常用抗生素敏感性的研究进展

在微藻培养过程中,需在藻液中加入抗生素以达到除菌、抑菌的目的。对近年来微藻的抗生素纯化技术进行了综述,对选择抗生素的方法、抗生素抑菌机理及常用抗生素对微藻培养的影响等方面进行了归纳和分析,并对无菌化培养的研究前景做了展望。     

蛋白质纯化技术研究进展

蛋白质组学研究的基础就是蛋白质的分离。对于天然蛋白来说,可能需要一系列的纯化步骤才能获得纯度满足研究要求的蛋白质,但是蛋白质在分离过程中常常由于溶液环境变化或外力作用造成构象变化而引起失活。本文首先介绍了常用的蛋白质分离纯化技术及其研究进展,包括膜分离技术、沉淀分离技术、电泳分离技术以及层析分离技术等常用的蛋白质纯化技术,总结了现有技术存在的问题,并对近年来发展的新型蛋白质分离技术--非对称流场流分离技术进行了介绍和展望。     

分枝杆菌核糖体的制备与初步结构研究

核糖体是抗生素的主要靶点,而获得足量高纯度的核糖体是进行结构和药物研究的基础。结核分枝杆菌壁厚且生长缓慢,制备足量高纯度的核糖体具有挑战性。本研究改进并优化了核糖体纯化制备方法,通过大量培养和安全处理致病菌,应用高效破碎厚壁革兰阳性菌的技术,结合传统的蔗糖密度离心分离和蛋白液相色谱纯化技术,经多步纯化和分离,获得了高纯度和较高产率的耻垢分枝杆菌与结核分枝杆菌的核糖体样品,为后续生化实验和结构生物学研究提供了保证。该分枝杆菌核糖体制备方法也可应用于其他革兰阳性致病菌复合物样品的直接提纯,以及复合物特异性的进一步研究,特别是利用晶体学与冷冻电镜结合的高精度复合物结构研究,有助于揭示细菌耐药性机制及用于新型抗生素的研发。     

抗生素的分离和纯化

抗生素的提炼一般是指将在发酵液中积累的抗生素经过分离、浓集、纯化,获得符合使用要求制品的全过程。是一门利用各种化工单元操作有机组合进行生物技术产品后处理的技术。由于抗生素的生源、性质各异,在发酵液中存在形式、杂质的干扰情况不一,以及制品的使用要求不同,所以抗生素的提炼过程不可能有一     

浅紫灰链霉菌CQ01819及其次级代谢产物的定向发掘

【目的】采用特征次级代谢产物生物合成的保守功能基因探针,定向分离土壤中产生特征次级代谢产物的菌种资源,借助基因转录分析为导向的培养基优化方法,获得目标次生代谢产物。【方法】首先,根据5种特征次级代谢产物保守的合成功能基因设计简并引物,定向从土壤样品中筛选、分离并纯化菌株。然后,以RT-qPCR为指导开展目标产物的发酵培养基优化;最后,对菌株进行发酵,利用多种色谱技术分离纯化目标天然产物,并结合高分辨质谱与核磁共振等技术对所获得的化合物进行结构鉴定。【结果】从土壤中筛选得到了一株AHBA合酶基因和环氧化酶基因均为阳性的链霉菌菌株(编号为CQ01819),根据转录分析优化发酵培养基,最终从该菌株分离纯化得到了含有AHBA结构单元的丝裂霉素C、聚醚类抗生素莫能霉素A和缬吲霉素。【结论】本研究通过菌株的定向分离纯化,筛选得到了产生预期抗生素的浅紫灰链霉菌菌株CQ01819;基于RT-qPCR指导的发酵培养基优化,确定了菌株的发酵条件;获得发酵粗提物后,采用多种色谱整合技术和光谱分析策略,快速分离并鉴定了目标产物。该研究为目标菌种资源的定向筛选、菌株的发酵条件的快速优化和化合物的定向分离提供了较...     

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。     

线虫共生致病杆菌PacYellow的吲哚抗生素

【目的】研究特殊生境的微生物——嗜线虫致病杆菌(Xenorhabdus bovienii Pac Yellow)的次级代谢产物,为微生物药物或农用抗生素开发提供结构多样的化合物。【方法】通过16S r RNA基因比对鉴定菌株;利用薄层层析、硅胶柱层析、凝胶层析、液相制备等技术进行分离纯化;利用质谱、红外光谱与核磁共振等光谱技术鉴定化合物结构。【结果】Pac Yellow菌株被鉴定属于致病杆菌属bovienii种。从该菌株中分离纯化了4个单体化合物,它们的结构被鉴定为吲哚类衍生物,可能源于色氨酸与缬氨酸或异亮氨酸的缩合产物。【结论】从嗜线虫致病杆菌——Pac Yellow菌株中分离鉴定了4个吲哚生物碱类抗生素,它们具有中等到高的抗菌活性。     

New principles of ion exchange preparative chromatography and their applications for isolation, purification and super purification of antibiotics]

New types of structurally segregated heteronetwork biosorbents with given parameters of heterogeneity and porosity have been developed. Physico-chemical characteristics of the biosorbents on the basis of which one can predict optimal structures of ion exchangers to be used in preparative chromatography of biologically active compounds were studied. A new principle of sequential displacement of ions of organic compounds, in particular antibiotics, adsorbed on selective biosorbents with a high adsorption capacity was developed, which enables purification and superpurification of the desired compound. The method is based on the effect of small thermodynamic shifts in physico-chemical parameters of the elution system, which results in preparative separation of substances with close properties and in purification of the desired compound from microadmixtures. The "small shift effect" is realized in the case of limiting thermodynamic and kinetic parameters of heterogeneous mass exchange and with biosorbents possessing a highly selective adsoprtion capacity.     

Purification and culture characteristics of 36 benthic marine diatoms isolated from the Solthörn tidal flat (Southern North Sea)

Marine benthic diatoms growing in biofilms on sediment surfaces generally occur associated with heterotrophic bacteria, whereas modern molecular techniques and analyses of species‐specific physiology create a demand for axenic cultures. Numerous benthic diatoms were isolated from surface sediments during a monitoring of the Solthörn tidal flat (southern North Sea, Germany) from May 2008 to May 2009. Of these, around 50% could be purified from the accompanying heterotrophic bacteria using different antibiotics combined with physical separation methods (vortexing, ultrasound). Overall, seven different antibiotics were tested at different concentrations, and a best working protocol was developed. The axenic strains were stable on average for only around 15 months, indicating a symbiotic interaction between the benthic diatoms and the associated bacteria. While most short‐term effects during the purification process were restricted to differences in growth rates among xenic and axenic diatom strains, long‐term cultivation led to distinct changes in cell volumes and growth characteristics of the axenic strains.     

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。     

PROTOCOLS FOR THE REMOVAL OF BACTERIA FROM FRESHWATER BENTHIC DIATOM CULTURES1

In this study, we describe different combinations of physical separation and antibiotic treatment to remove associated bacteria from freshwater diatoms. Diatoms were purified either from natural epilithic biofilms or from unialgal cultures. We determined that for most strains, different purification procedures have to be combined individually. In a new approach, we show that for some diatom strains, the substitution of associated aquatic bacteria by an antibiotic‐sensitive Escherichia coli strain and subsequent treatment with antibiotics may be a successful strategy to obtain axenic diatom cultures. Axenic diatom cultures are essential to study the physiology and biochemistry of individual strains as well as their responses to environmental changes without interference of accompanying bacteria.     

溶菌酶分离纯化方法的研究进展

对近年来溶菌酶分离纯化的方法,如离子交换法、色谱法、膜处理技术、反胶团萃取法、亲和层析等进行了综述,并讨论了分离纯化方法的应用前景。     

Specific berenil-DNA interactions: an approach for separation of plasmid isoforms by pseudo-affinity chromatography

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3 M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6 M and then to 0 M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.     

芽孢杆菌(Bacillus sp.)脂肽抗生素发酵工艺及分离纯化

微生物脂肽具有抗菌谱广、热稳定性高、低毒、低抗药性等优点,近年来受到国内外广泛关注。综述了芽孢杆菌脂肽抗生素的发酵和分离纯化工艺的最新研究进展。在发酵工艺中,培养基营养组成、发酵温度、搅拌转速和通气量等参数对脂肽的产量至关重要,碳源、氮源和金属离子的组成与配比都会影响芽孢杆菌的生长与产物的合成,适当控制搅拌转速和通气量可提高脂肽产量。此外,近年来一些新型发酵工艺,如泡沫回流、固定化细胞、无泡发酵、固态发酵等被用于脂肽生产,通过改进发酵方式,在降低成本的同时提高了脂肽抗生素产量。抗菌脂肽分离纯化的主要方法包括超滤、吸附、泡沫分离及色谱法等,这些方法相对于传统的酸沉和萃取,具有可连续生产、脂肽提取量高及成本低等优点。同时,多种纯化方法的组合应用大幅度提高了抗菌脂肽的提取效果,有效降低了成本,是脂肽抗生素分离提取的发展方向。     

Poly-N-acetylglucosamine and poly(glycerol phosphate) teichoic acid identification from staphylococcal biofilm extracts using excitation sculptured TOCSY NMR

We report the successful application of selective excitation sculptured TOCSY NMR (SXS-TOCSY) to identify individual solution components from a heterogeneous system using selectively acquired (1)H NMR spin system patterns. SXS-TOCSY application is illustrated by detection of the simultaneous presence of poly-beta-(1,6)-N-acetylglucosamine (PNAG) and poly(glycerol phosphate) teichoic acid (TA) carbohydrate polymer components in crude biofilm extracts from Staphylococcus epidermidis without the need for further sample purification and component separation. Biofilms are implicated in the barriers for resistance of microbes toward antibiotics and immune responses, therefore efficient rapid detection and quantification of key components are important to assist in the design of a clinical infection response.     

Purification of antibiotics by ultrafiltration through semi-permeable membranes

The process of purification of benzylpenicillin, penicillin V and erythromycin solutions with the use of semi-permeable membranes was studied. Practically complete separation of high-molecular admixtures and microbial colonies and separation of significant amounts of the colored compounds were shown to be possible. The purification process may be used effectively at the early stages of antibiotic production.