褐藻胶寡糖制备的研究进展

褐藻胶寡糖(alginate oligosaccharides,AOS)是褐藻胶降解而形成的一种功能性寡糖,具有广泛的生物活性,如促进植物生长、提高植物抗逆性、抗氧化、抗菌、抗肿瘤等。褐藻胶寡糖的制备方法主要分为:化学法、物理法和酶解法。不同的方法制备出的褐藻胶寡糖结构亦有所不同。介绍了化学法、物理法和酶解法等各种褐藻胶寡糖制备方法的研究现状、存在的问题及发展趋势。     

褐藻寡糖的制备方法及生物活性研究进展

褐藻胶是一类多糖聚合物,由于其独特的理化性质和有益健康的作用,已被广泛应用于制药和食品工业.然而,由于褐藻胶的水溶性低、黏度大,进而限制了褐藻胶的开发和应用.褐藻寡糖(alginate oligosaccharide,AOS)是褐藻胶的降解产物,由于其分子量低、水溶性高、安全无毒等特点,近年来受到广泛关注.AOS独特的...     

产双功能褐藻胶裂解酶菌株的筛选与初步研究

目的：双功能褐藻胶裂解酶既能降解聚β-D-甘露糖醛酸,又能降解聚α-L-古罗糖醛酸,可以用一种酶来制备不同结构的褐藻胶寡糖。本文的目的是筛选能产生双功能褐藻胶裂解酶的菌株,对其产酶曲线和降解产物作初步研究。方法：利用唯一碳源培养基筛选产生褐藻胶裂解酶的菌株,通过16SrDNA序列比对进行菌种鉴定,通过在凝胶上检测褐藻胶裂解酶活性来判断发酵上清液中褐藻胶裂解酶的数量及分子量,利用薄层层析确定降解褐藻胶的终产物组成。结果：从褐藻上筛选到一株海洋细菌QY107,鉴定为弧菌属细菌。发酵120h时褐藻胶裂解酶产量为12．32U／mL,其发酵液上清中只含有一种褐藻胶裂解酶,分子量在28kDa左右,并且对聚β—D-甘露糖醛酸和聚α-L-古罗糖醛酸都能降解,降解褐藻胶的终产物主要为三糖。结论：本文筛选到一株弧菌QY107,其发酵液上清中只有一种双功能褐藻胶裂解酶,可用于大量制备褐藻胶三糖。推测该酶具有特殊的催化腔结构,对其结构与功能相互关系的研究可能会发现新的底物结合与催化机制。酶解制备褐藻胶寡糖因其环保高效而越来越受到人们的重视,因此该菌株能促进海洋寡糖类生物制品的开发,在医药、食品、农业、生物燃料等领域具有广阔的应用前景。     

植物细胞膜NADPH氧化酶的研究进展

植物细胞质膜NADPH氧化酶是植物中一种与哺乳动物嗜中性粒细胞gp91phox同源的氧化还原酶。当植物受到生物或非生物胁迫时,该酶通过短时间内大量产生信号分子活性氧(activeoxygenspecies,AOS)调节基因表达和细胞代谢,使植物及时对逆境胁迫作出反应,以适应环境的变化。NADPH氧化酶在调节植物的生长和发育方面也起着非常重要的作用。本文对其结构特征、活性调节和功能等方面的最新进展进行了综述。     

微生物来源褐藻胶(alginate)生物合成及应用前景

褐藻胶及其制品在医药、食品及化工等领域具有重要价值。除褐藻以外,微生物是褐藻胶的另一重要来源。虽然微生物来源的褐藻胶未能够实现规模化生产,但是由于微生物合成褐藻胶具有发酵条件可控、产物单一、结构稳定并且易于纯化等优势,引起广泛的关注。并且利用生物工程技术已经实现了对微生物来源褐藻胶结构的调控和改造,促进了褐藻胶的高值化利用。此文综述了微生物褐藻胶生物合成的概况,并对利用基因工程改造褐藻胶的发展趋势和应用前景进行了论述。     

产褐藻胶裂解酶交替单胞菌的发酵优化及alg2951的外源表达

褐藻胶裂解酶是制备生物活性寡糖的重要功能酶,在食品、农业、工业等行业中具有重要应用价值。本研究以交替单胞菌属新种HB161718为出发菌株,在单因素实验基础上,通过Box-Behnken设计及响应面法优化获得该菌的最佳产酶培养基:海藻酸钠7.23 g/L,蛋白胨7 g/L,NaCl 23.11 g/L,K_2HPO_40.1 g/L,MgSO_40.1 g/L,优化条件下酶活力为(54.28±3.47) U/mL,达到优化前的1.59倍。为进一步提高酶活性,通过分子生物学方法实现了褐藻胶裂解酶alg2951在大肠杆菌中的外源表达,纯化后的酶活性为636 U/mL,达到原始菌株酶活的18.6倍。本研究为褐藻胶裂解酶的工业生产提供了新的来源。     

脂肪酶催化制备生物柴油的研究进展

生物柴油作为一种可再生的清洁能源,以其良好的环境效应受到越来越多的关注。酶法生产生物柴油具有化学催化法不可比拟的优越性,是工业化生产的发展方向。本文综述了利用固定化脂肪酶、游离酶、全细胞生物催化剂制备生物柴油的研究与应用进展,并探讨了我国生物柴油产业化发展的困境和对策。     

一株产褐藻胶裂解酶的需钠弧菌筛选及酶解产物分析

[背景]褐藻胶裂解酶种类丰富、降解机制多样,是高效环保降解褐藻胶、制备褐藻寡糖的工具酶,成为褐藻植物高值化开发利用的研究热点.[目的]从海泥中筛选获得褐藻胶裂解酶高效产酶菌株,确定菌株发酵产酶最优条件,鉴定和分析酶降解产物,进而解析该酶的降解特性.[方法]以褐藻胶为唯一碳源,从海带养殖场附近海泥中筛选菌株,通过形态学观...     

褐藻胶裂解酶的分子改造研究进展

褐藻胶是由β-D-甘露糖醛酸(M)以及α-L-古罗糖醛酸(G)2种单体组成的酸性多糖。褐藻胶裂解酶作为多糖裂解酶的一种,可以温和高效地将褐藻胶降解为褐藻寡糖,并用于食品、医药和农业领域。然而天然来源的褐藻胶裂解酶通常存在活性不高、催化效率低以及热稳定性差等缺点,在一定程度上限制了其工业化应用潜力。近年来分子改造策略已经开始大量应用于褐藻胶裂解酶,使得褐藻胶裂解酶的应用性能得到极大提升。本文对已报道的褐藻胶裂解酶结构与催化机制进行总结,对改善热稳定性、提高催化效率、改变底物分布等性质的褐藻胶裂解酶分子改造策略如理性设计、定向进化、结构域截短与重组等进行系统分析与综述,并展望了未来褐藻胶裂解酶分子改造的发展方向。     

表达内皮抑素的rCHO细胞的微囊化培养

微囊化重组基因细胞移植治疗肿瘤是一种新兴的肿瘤基因治疗方法,如果将此技术应用到临床研究,就需要制备大量的细胞活性良好、重组蛋白表达量高的生物微胶囊。种子细胞是生物微胶囊治疗作用的执行者, 是构建微囊微反应器的基本元素。如何获得大量高活性的种子细胞已经成为规模化制备生物微胶囊所面临的最关键的限制因素。本实验考察了搅拌式生物反应器内扩增的重组CHO细胞进行包囊及微囊化细胞在生物反应器内规模化培养的可行性。实验结果显示:重组CHO细胞在生物反应器内可以快速生长,并且对数期细胞包囊,微囊化细胞活性良好。制备的微囊化细胞可以在生物反应器内培养,与培养板培养比较细胞生长较快、内皮抑素表达量较高。应用生物反应器培养技术能够在体外快速、大量扩增重组CHO细胞,满足微囊化细胞制备对种子细胞量与质的要求,微囊化细胞可以在生物反应器内培养。     

Determination of nitrie in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection

All photometric or HPLC methods described to date have been unable to detect nitrite, a reliable marker of NO synthase activity, in human blood because of its rapid metabolism within the erythrocytes. We now elaborate on method to prevent nitrite degradation during sample preparation which in combination with high-performance anion-exchange chromatography and electrochemical detection allows a sensitive measurement of nitrite. A linear current response in the concentration range of 10–1000 nmol/l nitrite was observed yielding a correlation coefficient of 0.99. In addition, the combination of the electrochemical with a UV detector allowed us to simultaneously quantify nitrate one analytical run, which is the end product of NO/nitrite metabolism. Basal levels for nitrate and nitrite in human blood were determined with 25±4 μmol/l and 578±116 nmol/l (n=8), respectively and thus were in the same concentration range as expected from NO measurement in saline perfused isolated organs or cultured endothelial cells. Therefore, the presented method may be used to assess activity of endothelial constitutive NO synthase in humans under physiological and pathophysiological conditions.     

Preparation of multivesicular liposomes

A novel type of liposome, named here multivesicular liposomes, was prepared by evaporation of organic solvents from chloroform-ether spherules suspended in water. Within each spherule were numerous water droplets that contained solutes to be trapped in liposomes upon solvent evaporation. Liposome preparations of different average diameters were made, varying from $\text{29 ± 10 μ}\text{m}$ to $\text{5.6 ± 1.7 μ}\text{m}$. The liposomes were morphologically characterized by light microscopy and transmission electron microscopy. Materials successfully trapped within the liposomes ranged in molecular size from glucose to nucleic acids. Extremely high percentages of encapsulation (up to 89%) were achieved.     

Separation of motile human spermatozoa by means of a glass bead column

Fertility potential of semen depends upon the presence of viable and motile spermatozoa. A variety of techniques has been reported whereby motile spermatozoa are separated from semen for use in homologous insemination. Using a column of glass beads, we tested various sizes for spermatozoa-separating efficiency. This procedure is quick and simple and results in a signficantly better recovery of motile and viable spermatozoa from poor-quality semen. Therefore it has the potential for use in intrauterine insemination for treating male factor infertility.     

Items from the Literature

Hazards in fixing small pieces of tissue for electron microscopy include damage, drying, or loss. Over the years, microstrainer tissue carriers have been developed to minimize these problems. Construction materials have included glass tubing, copper grids for electron microscopy, stainless steel screen, and bolting silk (Padawer 1951, Friend 1963, Bronskill 1970). Carriers made from plastic embedding molds (e.g., BEEM capsules) with either TEM grids attached to the conical tip (Buchanan 1965) or Nitex screen cloth held to one end by a retaining ring have proven to be inexpensive and popular, though the former has a very small filtration area and in the latter small tissues may be lost or crushed between the screen cloth and the bottom rim of the carrier. This note describes a carrier in which Nitex is permanently sealed to the bottom edee of a BEEM capsule cylinder.     

明胶—几丁糖膜的制备及其止血性能的实验研究

目的制备几丁糖膜以及研究其止血性能。方法 在透析后的几丁糖与明胶混合液中加入戊二醛交联后置入冻干机冻干制得几丁糖膜。取新西兰兔4只,在背部两侧对称性剪开直径1cm渗血伤口,分别用纱布和几丁糖膜止血,观察与创面的粘附情况,记录出血时间,以膜或纱布中Hb光度吸收值表示出血量。结果 几丁糖膜与创面粘附良好,几丁糖膜组、纱布组的出血时间分别为78.25±6.42秒、119.05±11.39秒,Hb光度吸收值为0.76±0.51、1.63±0.72,明显优于纱布对照组(P<0.01)。结论 几丁糖膜具有一定的止血性能。     

Removal of Stainable Cytoplasmic Substances from Cytogenetic Slide Preparations

A method to remove stainable cytoplasmic substances from cytogenetic preparation using RNase A treatment is reported. The preparations processed with this method are especially useful for the automated analysis of mi-cronuclei of cultured cells with cytochalasin B and of chromosome aberrations induced by radiation.     

紫草素的分离制备方法研究

采取CO2超临界萃取的方法从辽宁硬紫草中提取总紫草色素,用紫外分光光度法测定其含量。分别采用有机溶剂溶解碱液萃取法,直接碱水解法,以及Cu^2 络合法制备紫草素。通过三种紫草素制备方法的比较,确定先经Cu^2 络合纯化处理后再经水解法制备紫草素,收率较高,是实验室中简单、快速、高收率的由天然紫草分离制备紫草素的有效方法。     

Determination of alosetron in human plasma or serum by high-performance liquid chromatography with robotic sample preparation

A method of analysis for the determination of alosetron in human plasma or serum has been developed. The method was fully automated using a laboratory robot in order to improve analytical precision, efficiency and safety. The assay involved solid-phase extraction with reversed-phase HPLC separation and fluorescence detection. A validation exercise over the concentration range of 0.1 to 20 ng/ml demonstrated the selectivity, linearity, sensitivity, accuracy, precision, extraction efficiency, ruggedness and stability of the method. The method has been applied in support of numerous human pharmacokinetic/biopharmaceutic studies over the last five years.     

Suspension trapping (STrap) sample preparation method for bottom‐up proteomics analysis

Despite recent developments in bottom‐up proteomics, the need still exists in a fast, uncomplicated, and robust method for comprehensive sample processing especially when applied to low protein amounts. The suspension trapping method combines the advantage of efficient SDS‐based protein extraction with rapid detergent removal, reactor‐type protein digestion, and peptide cleanup. Proteins are solubilized in SDS. The sample is acidified and introduced into the suspension trapping tip incorporating the depth filter and hydrophobic compartments, filled with the neutral pH methanolic solution. The instantly formed fine protein suspension is trapped in the depth filter stack—this crucial step is aimed at separating the particulate matter in space. SDS and other contaminants are removed in the flow‐through, and a protease is introduced. Following the digestion, the peptides are cleaned up using the tip's hydrophobic part. The methodology allows processing of protein loads down to the low microgram/submicrogram levels. The detergent removal takes about 5 min, whereas the tryptic proteolysis of a cellular lysate is complete in as little as 30 min. We have successfully utilized the method for analysis of cellular lysates, enriched membrane preparations, and immunoprecipitates. We expect that due to its robustness and simplicity, the method will become an essential proteomics tool.     

S-腺苷蛋氨酸研究进展

S-腺苷蛋氨酸(SAM)是生物体内重要的中间代谢物质,参与多种生化反应．重点介绍了 S-腺苷蛋氨酸的制备和稳定性研究,同时综述了其生理功能、提取纯化和分析检测及临床应用．