海洋微生物有机磷降解酶的纯化与性质研究

从长期受有机磷农药污染的海水中分离得到1株能高效降解农药的芽胞杆菌M-1,通过离子交换层析、凝胶过滤层析等方法从发酵液中分离纯化了有机磷农药降解酶,SDS-PAGE测得该酶的分子质量约为45 kD。酶反应最适pH为7.5,最适反应温度为30℃,30℃下保温30 min,酶活力基本不变,高于30℃酶活力则迅速下降;K 、Na 、Ca2 、Mn2 对酶活性有促进作用,Hg2 、Zn2 和Cu2 等对酶有抑制作用。     

Purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures

The present study is focused on purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures--hormidium sp. A27 DM, Lyngbya sp. A09 DM and Halomicronema sp. A32 DM. 'Phycoerythrin' was successfully purified and characterized. On SDS-PAGE, the PE purified from all three young cultures showed four bands--corresponding to α and β subunits of each of PE-I and PE-II. However, phycoerythrin purified after prolonged growth of Phormidium sp. A27 DM and Halomicronema sp. A32DM showed only one band corresponding to 14 kDa whereas Lyngbya sp. A09 DM continued to produce uncleaved phycoerythrin. The absorption spectra of purified PEs from all the three young and old cultures showed variations however the fluorescence studies of the purified PEs in all cases gave the emission spectra at around 580 nm. The described work is of great importance to understand the role of phycoerythrin in adapting cyanobacteria to stress conditions.     

发菜藻蓝蛋白基因克隆及原核表达

发菜（Nostoc flagelliforme）是一种陆生固氮蓝藻,具有重要的经济和生态价值。运用双向电泳技术、MALDI-TOF-TOF/MS鉴定和数据库检索,获得藻蓝蛋白部分氨基酸序列并设计简并性引物,克隆藻蓝蛋白基因并研究其表达。结果表明,发菜藻蓝蛋白α和β亚基两个基因的编码序列及两者之间的间隔序列全长为1097bp,编码β亚基和α亚基的基因序列全长分别为519bp和489bp,β亚基基因序列位于α亚基基因序列上游,两者之间通过89bp的基因片段连接,GenBank登录号为GU549478,并对推译的α和β亚基三维结构进行了预测。将藻蓝蛋白的α和β亚基基因在大肠杆菌中表达,获得了符合预期的外源重组蛋白。研究结果为进一步研究发菜藻蓝蛋白的分子结构及生物学功能奠定了基础。     

Thermal stability improvement of blue colorant C-Phycocyanin from Spirulina platensis for food industry applications

C-Phycocyanin (C-PC) is a blue pigment in cyanobacteria, rhodophytes and cryptophytes with potential use as a value-added food colorant. Its stability was studied by examining the thermal degradation reactions in a range of temperature (25-80 °C) before and after the addition of selected edible preservatives. The natural protein crosslinker methylglyoxal does not stabilize significantly C-PC whereas addition of honey or high concentration of sugars greatly diminish the blue color degradation occurring when C-PC is exposed to high temperature.Data show that the sugar preservative effect on the C-PC blue color is related to the final concentration of sugar added rather than the type of sugar. For this reason the best preservative was found to be fructose, which is the most soluble sugar among those tested, at saturation concentration. Exploratory sterilization studies have been carried out with six blue/green fructose syrups made by mixing C-PC with the natural yellow pigment Carthamus tinctorius. Both after a “low temperature” and a “high temperature” sterilization procedure the syrups remain clear and maintain their bright color with only partial blue color degradation. After the sterilization process, the syrups were monitored for two months, in such observation period the loss of blue color is minimal.     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Purification and characterization of two thermostable protease fractions from Bacillus megaterium

Protease enzyme from Bacillus megaterium was successively purified by ammonium sulfate precipitation, ion exchange chromatography on DEAE-cellulose and gel filtration chromatography on Sephadex G-200. The purification steps of protease resulted in the production of two protease fractions namely protease P1 and P2 with specific activities of 561.27 and 317.23 U mg^?1 of protein, respectively. The molecular weights of B. megaterium P1 and P2 were 28 and 25 KDa, respectively. The purified fractions P1 and P2 were rich in aspartic acid and serine. Relatively higher amounts of alanine, leucine, glycine, valine, thereonine valine and glutamic acid were also present. The maximum protease activities for both enzyme fractions were attained at 50 °C, pH 7.5, 1% of gelatine concentration and 0.5 enzyme concentrations. P1 and P2 fractions were more stable over pH 7.0–8.5 and able to prolong their thermal stability up to 80 °C. The effect of different inhibitors on the protease activity of both enzyme fractions was also studied. The enzyme was found to be serine active as it had been affected by lower concentrations of phenylmethylsulfonyl fluoride (PMSF). Complete dehairing of the enzyme-treated skin was achieved in 12 h, at room temperature.     

Purification and characterization of protein methylase II from Helicobacter pylori

Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS-PAGE. On non-denaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4x(78+29)=428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a K(m) value of 5.0x10(-6) M for S-adenosyl-L-methionine and a V(max) of 205 pmol methyl-(14)C transferred min(-1) mg(-1) protein.     

Purification and characterization of a lysine aminopeptidase from Kluyveromyces marxianus

A lysine aminopeptidase was purified from the yeast Kluyveromyces marxianus. This enzyme was purified 100-fold from a soluble extract obtained at 100,000g. The purification procedure consisted in fractionated precipitation with ammonium sulfate and five chromatography steps. The native enzyme had a molecular mass of 46 kDa assessed through gel filtration. This aminopeptidase depicted an optimal pH of 7.0 and was stable at a pH range of 4-8, its optimal temperature was 45 degrees C and the enzyme became unstable at temperatures above 55 degrees C. The isoelectric point of the purified enzyme was 4.4. Michaelis constant and Vmax for L-lysine-p-nitroanilide were 0.33 mM and 2.2 mM min(-1) per milligram of protein, respectively. The enzyme was strongly inhibited by bestatin, o-phenanthroline and, to a lesser extent, by EDTA, suggesting that this enzyme is a metalloprotease. Our results suggest that the lysine aminopeptidase from Kluyveromyces marxianus might be of biotechnological relevance.     

雅致放射毛霉AS3.2778碱性蛋白酶的纯化及性质研究

利用盐析,离子交换,疏水层析及凝胶过滤的方法从雅致放射毛霉AS3.2778的发酵麸曲中分离纯化出一碱性蛋白酶,其纯化提高了22.7倍,酶活回收率16.1%,最终比酶活可达到6094u/mg。电泳分析发现,该蛋白酶是一单体蛋白,其分子量大约在32KDa。性质分析表明：该蛋白酶在60℃、pH8.5～10.5具有最大催化活性;在40℃以下,pH6.0～9.0的范围有很好的稳定性;1mM的PMSF可以完全抑制其活性,显示该蛋白酶属于丝氨酸蛋白酶家族。底物专一性的研究发现,该蛋白酶有相当广泛的肽键选择性,对绝大多数由疏水性氨基酸（尤其是亮氨酸）构成的肽键有很强的水解能力。     

Purification and characterization of aminopeptidase (pumAPE) from Ustilago maydis

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.     

Purification and characterization of xylanase from Aspergillus ficuum AF-98

The purification and characterization of xylanase from Aspergillus ficuum AF-98 were investigated in this work. The extracellular xylanase from this fungal was purified 32.6-fold to homogeneity throughout the precipitation with 50–80% (NH₄)₂SO₄, DEAE-Sephadex A-50 ion exchange chromatography and Sephadex G-100 chromatography. The purified xylanase (specific activity at 288.7 U/ mg protein) was a monomeric protein with a molecular mass of 35.0 kDa as determined by SDS-PAGE. The optimal temperature and pH for the action of the enzyme were at 45 °C and 5.0, respectively. The xylanase was activated by Cu²⁺ up to 115.8% of activity, and was strongly inhibited by Hg²⁺, Pb²⁺ up to 52.8% and 89%, respectively. The xylanase exhibited K_m and V_max values of 3.267 mg/mL, 18.38 M/min/mg for beechwood xylan and 3.747 mg/mL, 11.1 M/min/mg for birchwood xylan, respectively.     

Purification and characterization of tubulin from ginkgo pollen

 Tubulin was purified by a combination of acetone powder preparation, DEAE Sephadex A-50 chromatography, Sephacryl S-300 gel filtration, and Mono Q anion exchange chromatography from the pollen of ginkgo (Ginkgo biloba L.), a typical gymnosperm. The average yield of tubulin is 2 mg per 100 g of pollen grain. The purified tubulin is electrophoretically homogeneous. It seems to be composed of two subunits on SDS-PAGE and is resolved as two major spots on two-dimensional electrophoresis, preliminarily indicating that there are no obvious tubulin isotypes in ginkgo pollen. The apparent molecular weights of the two subunits are about 54 kDa and 52 kDa respectively, estimated from the SDS-PAGE. It was also demonstrated that tubulin from ginkgo pollen is immunochemically related to animal brain tubulin, and the purified tubulin was polymerized to microtubular aggregates in the presence of taxol and GTP in vitro. Received: 13 April 1996 / Revision accepted: 24 March 1997     

蛹虫草无性型深层培养液中纤溶酶的分离纯化和酶学性质研究

【目的】确立蛹拟青霉深层培养液中高纯度、高纤溶活性纤溶酶的分离纯化方法并测定其酶学性质。【方法】采用硫酸铵盐析、Sephadex G-25凝胶色谱、Phenyl-Sepharose HP疏水相互作用色谱、CM-Sepharose FF弱阳离子交换色谱和Superdex 75凝胶色谱对蛹拟青霉纤溶酶进行分离。用Lowry法测定蛋白质浓度,纤维蛋白平板法测定其纤溶活性,SDS-PAGE鉴定其纯度并确定其分子量,IEF法测定其等电点。【结果】研究发现,以蔗糖和豆饼为培养基主要基质时,蛹拟青霉深层培养可以产生至少两种纤溶酶。提纯后的纤溶酶Ⅱ比活力达到800.46 U/mg,总纯化倍数为30.07倍。纤溶酶Ⅱ的相对分子量和等电点分别为32 kD和9.3±0.2。纤溶酶Ⅱ是一种糖蛋白,总含糖量为0.98%(W/V)。该酶可以顺次降解人血纤维蛋白(原)的α、β和γ链。其最适作用pH及温度分别为7.4和41°C。Aprotinine与PMSF对该纤溶酶的活性完全抑制,推测此纤溶酶可能是一种丝氨酸蛋白酶。【结论】单一的高纤溶活性纤溶酶的获得和酶学性质的确定,为该酶开发成为新型溶栓药物提供了理论依据。     

卵孢金孢藻(Chrysosporum ovalisporum)——中国水华蓝藻新记录

卵孢金孢藻(Chrysosporum ovalisporum(Forti)Zapomelováet al.)是一种在欧洲地中海地区和澳大利亚较常见的水华蓝藻,但我国目前还未有该物种分布的报道。本文对采自上海市滴水湖中的藻种进行分离培养,获得了一株纯化藻株CFWA01007,经光学显微镜观察,初步判定其为卵孢金孢藻,对其形态特征进行了详细描述并测定了其16S rRNA基因序列,结果与来自欧洲和北美的Chrysosporum ovalisporum(Forti)Zapomelováet al.相似度极高,故判断其即为卵孢金孢藻,这是该种在中国的首次发现。对该种的分类学和生态学特征进行了讨论。     

Fatty acid composition of six freshwater wild cyanobacterial species

Hydroxy, n-saturated, branched, dioic, and unsaturated fatty acids in six freshwater wild cyanobacteria (Chroococcus minutus, Lyngbya ceylanica, Merismopedia glauca, Nodularia sphaerocarpa, Nostoc linckia, and Synechococcus aeruginosus) collected from different lakes and springs of Israel have been identified by gas chromatography-mass spectrometry (GC-MS).     

Purification and characterization of xylanase from alkali-tolerant Aspergillus fischeri Fxn1

Abstract Alkali-tolerant Aspergillus fischeri Fxn1 produced two extracellular xylanases. The major xylanase ( M _r 31000) was purified to electrophoretic homogeneity by ammonium sulfate precipitation, anion exchange chromatography and preparatory PAGE. Xylose was the major hydrolysis product from oat spelt and birch wood xylans. It was completely free of cellulolytic activities. The optimum pH and temperature were 6.0 and 60 °C, respectively. pH stability ranged from 5 to 9.5 and the t_{1 / 2} at 50 °C was 490 min. It had a K _m of 4.88 mg ml⁻¹and a V _max of 588 μmol min⁻¹ mg⁻¹. The activity was inhibited (95%) by AlCl₃ (10 mM). This enzyme appears to be novel and will be useful for studies on the mechanism of hydrolysis of xylan by xylanolytic enzymes.     

Purification of a kappa-carrageenase from marine Cytophaga species

A mixture of extracellular carrageenases was isolated from the cell-free medium of a culture of marine Cytophaga sp. 1k-C783 grown on ZoBell 2216 E broth with 0.1% commercial carrageenan. A single active peak of kappa-carrageenase was separated and purified from the mixture by ammonium sulfate precipitation, ion-exchange chromatography, and Sephadex G-200 gel filtration chromatography. Molecular weight of the purified kappa-carrageenase was estimated as 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The purified kappa-carrageenase had pH optimum 7.6 and temperature optimum 25 C.     

Purification and physicochemical characterization of a cotyledonary lectin from Luetzelburgia auriculata

A lectin was purified from the cotyledons of Luetzelburgia auriculata (Fr. All) Ducke by affinity chromatography on agarose-N-acetyl-D-galactosamine. The lectin is a potent agglutinin for rabbit erythrocytes, reacts with human red cells, but is inactive against cow, sheep, and goat erythrocytes. Hemagglutination of rabbit erythrocytes was inhibited by either 0.39 mM N-acetyl-neuraminic acid or N-acetyl-D-galactosamin, 12.5 mM D-lactose or D-melibiose, 50 mM D-galactose or raffinose. Its hemagglutinating activity was lost at 80 degrees C, 5 min, and the activation energy required for denaturation was 104.75 kJ mol(-1). Chromatography on Sephadex G-100, at pH 7.6, showed that at this hydrogenic ionic concentration the native lectin was a homotetramer (123.5 kDa). By denaturing SDS-PAGE, LAA seemed to be composed of a mixture of 29 and 15 kDa polypeptide subunits. At acidic and basic pHs it assumed different conformations, as demonstrated by exclusion chromatography on Superdex 200 HR 10/30. The N-terminal sequence of the 29 kDa band was SEVVSFSFTKFNPNQKDII and the 15 kDa band contained a mixture of SEVVSFSFTKFNPNQKDII and KFNQIVAVEEDTDXESQPQ sequences, indicating that these bands may represent full-length and its endogenous fragments, respectively. The lectin is a glycoprotein having 3.2% neutral carbohydrate, with a pI of 5.8, containing high levels of Asp+Asn and Glu+Gln and hydroxy amino acids, and low amount or absence of sulfur amino acids. Its absorption spectrum showed a maximum at 280 nm and a epsilon (1%) x (1cm) of 5.2. Its CD spectrum was characterized by minima near 228 nm, maxima near 196 nm and a negative to positive crossover at 210 nm. The secondary structure content was 6% alpha-helix, 8% parallel beta-sheet, 38% antiparallel beta-sheet, 17% beta-turn, 31% unordered and others contribution, and 1% RMS (root mean square). In the fluorescence spectroscopy, excitation of the lectin solution at 280 nm gave an emission spectrum in the 285-445 nm range. The wavelength maximum emission was in 334.5 nm, typical for tryptophan residues buried inside the protein.     

Purification and characterization of two agarases from Agarivorans albus OAY02

Two extracellular β-agarases were purified from Agarivorans albus OAY2, which was isolated from seaweed collected in Qingdao, Shandong, China. The fermentation process was optimized with enzyme activity improving from 1.06 U/ml to 2.65 U/ml. Agarases were purified by ammonium sulfate fractionation and ion-exchange chromatography. Purification resulted in a 107-fold increase in agarase-a activity and a 52-fold increase in case of agarase-b, with high specific activities of 2715 and 1338 U/mg, respectively. The molecular masses of the agarases were estimated to be 50 kDa and 107 kDa, respectively. The optimum temperatures for the activities of agarase-a and agarase-b were 40 °C and 50 °C, respectively. Agarase-a was stable at 30 °C, while agarase-b was stable at 50 °C. The optimum pH values for agarase-a and agarase-b were both 9.0. Agarase-a was inhibited by Cu²⁺, Mn²⁺, Co²⁺ and EDTA, while agarase-b was inhibited by Cu²⁺, Mn²⁺ and Fe²⁺. The results of ¹³C NMR and TLC showed that hydrolysis of agarose by agarase-a produced neoagarobiose (NA2), neoagarotetraose (NA4) and neoagarohexaose (NA6). Agarase-b hydrolyzed agarose to yield neoagarobiose (NA2) and neoagarotetraose (NA4). Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) and the enzymatic product results indicated that agarase-a and agarase-b might be novel agarases.     

玉米矮花叶病毒提纯及特性的研究

改进病毒纯化方法,获得了较高纯度、较强侵染活性的提纯病毒制品。提纯病毒的产量为0.7—1.2mg/100g病叶,病毒粒体大小为720—750×14nm,SDS-聚丙烯酰胺凝胶电泳测得的外壳蛋白分子量为36000道尔顿,用提纯病毒免疫家兔得到较高特异性的抗血清,微量沉淀反应的效价为1/2048,间接ELISA法测出的感病叶汁液的最高稀释倍数为3200—6400倍。