透明颤菌血红蛋白的分离纯化与分析检测

透明颤菌血红蛋白(Vitreoscillahemoglobin,VHb)是惟一一种研究得较为透彻的原核生物氧结合蛋白血红蛋白。它支持细胞在微氧条件下进行好氧生长,克服发酵过程中的溶氧限制,因此在需氧微生物发酵工业中具有重要的应用价值。简述了VHb的分离纯化过程,综述了VHb的各种定性检测和定量分析方法,比较了各种检测分析方法的优缺点和适用性。提出利用改进的一氧化碳差光谱法以全细胞悬浮液为对象直接进行VHb的定量分析是发酵工业中应用VHb重组菌株的研究发展方向 。     

一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。     

耐有机溶剂极端微生物的耐受机制及应用

耐有机溶剂微生物是一类新颖的极端微生物,直到20世纪80年代才被系统地研究.它们通过各种耐受机制,有效抵御或降低有机溶剂对其细胞产生的毒害作用.因此,在全细胞催化、环境污染治理等领域,耐有机溶剂极端微生物具有广阔的工业应用前景.此外,深入透彻地了解耐有机溶剂极端微生物的各种耐受机制,有助于利用基因工程技术改造和优化现有耐有机溶剂极端微生物的各种性能,进一步拓展其工业应用领域.本文将从囊泡外排、改变细胞膜磷脂结构和组成等4个方面概述近年来耐有机溶剂极端微生物的耐受机制研究新进展,并介绍它们在全细胞催化等领域的应用.     

Heat Resistance of Salmonellae in Concentrated Milk

The heat resistance of Escherichia coli, Salmonella typhimurium, and Salmonella alachua in milk solutions containing 10, 30, 42, and 51% (w/w) skim milk for total solids was determined. Increased milk-solids level effected a significant increase in the heat resistance of each organism. Although E. coli was more heat-resistant than both strains of Salmonella in 10% milk, the situation was reversed in 42 and 51% milk. Prior growth temperature was found to exert a profound effect on the heat resistance of S. typhimurium. Growth of S. typhimurium in 42% milk solids for 24 hr did not greatly enhance the thermal resistance of the organism when heated in a fresh 42% solids concentrate. Application of a partial vaccum during heating greatly diminished the decimal reduction times of S. typhimurium and E. coli and, in addition, virtually eliminated the protective effect of increased solids level.     

Reactions of Peroxynitrite and Nitrite with Organic Molecules and Hemoglobin

In this work, the reactions of nitrite (NO2-) and peroxynitrite (ONOO-) with organic molecules as well as with hemoglobin (Hb) were examined and the potential interference with the detection of hydrogen peroxide and Hb was investigated. ONOO- at low concentrations (35-140 microM) induced a concentration-dependent oxidation of o-phenylenediamine and guaiacol, and this process can be improved by the addition of Hb in a concentration-dependent manner. This enhancing effect of Hb was possibly due to the formation of such highly reactive species as ferrylHb during the reaction of ONOO- and Hb. NO2- also oxidized the aromatic amine o-phenylenediamine, but its efficiency was much lower than that of ONOO-. A 300-fold excess of NO2- over hydrogen peroxide inhibited the oxidation of Pyrogallol Red mediated by hydrogen peroxide and Hb, which was due in part to the reaction of NO2- with Hb ferryl species compound I and compound II and the phenoxyl radical. These data suggest that ONOO- and NO2- can interfere with the detection of hydrogen peroxide. The overestimation or underestimation of the hydrogen peroxide detected is dependent upon the organic molecule utilized for detection and the relative rate of NO2-, superoxide, and ONOO- generation.     

Purification,Characterization, and Crystallization of Crocodylus siamensis Hemoglobin

Crocodylus siamensis hemoglobin was purified by a size exclusion chromatography, Sephacryl S-100 with buffer containing dithiothreitol. The purified Hb was dissociated to be two forms (α chain and β chain) which observed by SDS-PAGE, indicated that the C. siamensis Hb was an unpolymerized form. The unpolymerized Hb (composed of two α chains and two β chains) showed high oxygen affinity at 3.13 mmHg (P₅₀) and 1.96 (n value), and a small Bohr effect (δH⁺ = ?0.29) at a pH of 6.9–8.4. Adenosine triphosphate did not affect the oxygenation properties, whereas bicarbonate ions strongly depressed oxygen affinity. Crude C. siamensis Hb solutions were showed high O₂ affinity at P₅₀ of 2.5 mmHg which may assure efficient utilization of the lung O₂ reserve during breath holding and diving. The purified Hbs were changed to cyanmethemoglobin forms prior crystallization. Rod- and plate-shaped crystals were obtained by the sitting-drop vapor-diffusion method at 5 °C using equal volumes of protein solution (37 mg/ml) and reservoir [10–13 % (w/v) PEG 4000, with 0.1 M Tris buffer in present of 0.2 M MgCl₂·6H₂O] solution at a pH of 7.0–8.5.     

大孔树脂——溶剂萃取法精制高色价栀子黄色素的集成技术研究

以栀子为原料提取栀子黄色素,采用大孔吸附树脂--有机溶剂萃取相结合的集成技术,从栀子中分离纯化得到高色价的栀子黄色素.先采用大孔吸附树脂对栀子黄色素进行初步精制,以306型大孔吸附树脂为研究对象,探讨了大孔树脂对栀子黄色素的静态吸附率、吸附流速和洗脱剂浓度对吸附的影响,从而得到较为合适的工艺:吸附流速2.O mT/mi...     

有机溶剂对污泥厌氧发酵产酸的影响

有机酸是重要的化工原料,从市政污泥厌氧发酵过程提取有机酸、用于生产高附加值的产品可以实现废物资源化,本研究在确定最佳有机溶剂和萃取剂的基础上,考察了有机溶剂对城市污泥厌氧发酵生产有机酸的影响.结果表明,较合适的溶剂和萃取剂分别为磺化煤油和三烷基氧膦.少量磺化煤油对城市污泥发酵产生产有机酸有一定促进作用.     

Alleviation of Toxicity of Poisonous Organic Compounds on Hydrophilic Carrier/Hydrophobic Organic Solvent Interface

Growth tests of type culture strains were done on interfaces between nutrient agar and hydrophobic organic solvents. All strains examined were able to grow against n-paraffin (C₁₁-C₁₅), and many could grow against isooctane, and pentyl- and heptylbenzene. The interface cultivation method was also successful even when the solvent layer contained compounds toxic for microorganism.     

Purification and Characterization of Recombinant Polymeric Hemoglobin P1 of Glycera dibranchiata

The apoprotein of component P1 of the polymeric fraction of the intracellular hemoglobin of the marine polychaete Glycera dibranchiata has been expressed at a high level in Escherichia coli. The expressed globin was reconstituted with heme and purified. The N-terminal sequence of the recombinant P1 is identical to the cDNA-derived sequence of cloned P1 (Zafar et al., Biochem. Biophys. Acta, 1041, 117-123, 1990). Gel filtration, SDS-PAGE, optical spectra over the range 200-650 nm, and circular dichroism over the range 200-250 nm of the purified recombinant P1 were very similar to the polymeric fraction of native Glycera hemoglobin. The molar ellipticity at 222 nm provided an estimate of 77% for the α-helical content of the recombinant P1, in excellent agreement with that calculated from the crystal structure of Glycera monomeric component M-II. Although the oxygen binding affinity of the recombinant P1 is higher than that of the polymeric fraction of Glycera hemoglobin (3-4 torr vs 7-13 torr), which consists of at least six different single-chain hemoglobins, the Hill coefficient is lower (1.0-1.2 vs 1.2-1.4).     

Purification of Hemoglobin from the Actinorhizal Root Nodules of Myrica gale L

Hemoglobins are generally absent or present in low concentrations in the nodules of actinorhizal plants. An exception is Casuarina, where a hemoglobin occurs at relatively high concentration. However, this plant is unique in that Frankia, the microsymbiont, lacks the vesicles that are normally the site of nitrogen fixation. The present paper shows that a hemoglobin also occurs at high concentrations in Myrica gale L., an actinorhizal plant in which Frankia does form vesicles. Hemoglobin was extracted from root nodules under anaerobic conditions using a buffer containing CO, detergent, and a reducing agent. Carboxyhemoglobin was purified using gel filtration followed by aerobic ion-exchange chromatography. The optical absorption spectra of the oxy-, deoxy-, and carboxyhemoglobins were similar to those of other hemoglobins. The molecular mass of the native hemoglobin estimated by gel filtration was 38,500 D. The molecular mass of the subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 16,200 D, consistent with the mass of other hemoglobin subunits. Thus, the native hemoglobin is probably a dimer.     

有机相中脂肪酶催化不对称酯合成反应动力学的研究

有机相中脂肪酶催化不对称酯合成反应动力学的研究杨红,高修功,郭妮妮,曹淑桂,杨同书（吉林大学酶工程国家重点实验室,长春１３００２３）在环已烷中应用酵母脂肪酶催化外消旋２－辛醇和辛酸的不对称酯合成反应,研究了该反应的动力学机制,测定了表现动力学常数．利...     

有机溶剂中固定化脂肪酶催化硅醇的酯化反应

固定化Mucor miehei脂肪酶可催化有机硅醇和脂肪酸的酯化反应．对固定化酶用量、脂肪酸链长、不同有机硅醇底物、有机溶剂极性和水含量等影响因素进行了初步研究．     

Discovery of glpC, an Organic Solvent Tolerance-Related Gene in Escherichia coli, Using Gene Expression Profiles from DNA Microarrays

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.     

Purification of Saponin Compounds in Bupleurum falcatum by Solvent Partitioning and Preparative LC

Saponin compounds (saikosaponin c, a, and d) in Bupleurum falcatum were partially purified by solvent partitioning of the herbal extract using diethyl ether, distilled water, n-butanol, and acetone. After separation of the saponins by preparative LC, the purity of each saikosaponin was more than 94%. The identities of purified individual saikosaponins were confirmed by TLC, analytical LC, and fast-atom bombardment mass spectrometry.     

提高有机催化脂肪酶活性和稳定性的途径研究

随着非水酶学的发展,脂肪酶在有机催化反应中得到广泛应用,探寻提高有机催化脂肪酶的催化活性和操作稳定性的途径也随之成为研究热点.综述了近年来这一研究方向的成果及最新进展,着重阐述表面活性剂包衣、固定化以及二者结合的手段对脂肪酶在有机介质中的催化活性及操作稳定性的影响.     

Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta. 1. Purification and characterisation

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.     

Kinetics of Encapsulated Yeast Alcohol Dehydrogenase Dispersed in an Organic Solvent

Microcapsules dispersed in organic solvents provide a suitable environment for conducting enzyme reactions involving cofactors and hydrophobic substrates. Encapsulated YADH is active and stable in cyclohexane provided the pH is adjusted appropriately. Mass transfer does not influence batch reaction rates. Conversion in a fluidized-bed reactor containing encapsulated YADH/NAD⁺ and employing cyclohexane as the continuous phase depends strongly on residence time and inlet cinnamyl alcohol concentration. However, interpretation of these results is complicated by enzyme inactivation by the product, cinnamaldehyde, and interference from residual encapsulating agents.     

有机介质中脂肪酶催化外消旋苯甘氨酸甲酯的对映体选择性氨解反应

由水解酶催化的酯的对映体选择性水解和醇解反应 ,已在外消旋物质的拆分中得到广泛应用[1 ] 。近年来的一些研究表明 ,某些水解酶还可催化一些非天然酰基受体的转化 ,如过氧化氢、烷基胺、联胺和氨等。这些非天然酰基受体的转化反应在多肽的合成及手性化合物的拆分中显示出巨大的应用前景。其中 ,以氨为酰基受体的酶促氨解反应 ,是继酶促对映体选择性水解、酯化及转酯反应之后的另一制备光学纯化合物的新反应[2 ] 。目前国际上对这一新反应的研究尚属起步 ,国内未见有对该反应研究的报道。(Ｄ ,Ｌ) 苯甘氨酸是半合成 β 内酰胺类抗生素的重…