应用免疫和质谱法对亚心形扁藻氢酶蛋白进行亚细胞定位和分类

亚心形扁藻(Platymonas subcordiformis)是新发现的一株产氢海洋单细胞绿藻,经过胁迫调控可实现一定时间的持续产氢。氢酶是亚心形扁藻在胁迫条件下进行光合产氢的一个关键酶。但到目前为止,亚心形扁藻氢酶相关信息仍不清楚。利用蛋白合成抑制剂氯霉素和放线菌酮对亚心形扁藻氢酶活性进行考察,同时利用免疫印迹技术和免疫胶体金电镜对亚心形扁藻氢酶蛋白进行亚细胞定位分析。结果表明:亚心形扁藻氢酶蛋白可能由胞浆内合成,在叶绿体行使功能。采用免疫共沉淀技术富集亚心形扁藻细胞氢酶蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对免疫共沉淀复合物进行分离,从胶中切取目的蛋白条带,胶内酶解后进行基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析,得到相应的肽指纹图谱,通过搜索数据库检索初步断定亚心形扁藻氢酶蛋白为铁氢酶。     

木霉β-1,3-葡聚糖酶的分离纯化

目的:对木霉菌株LE02所产β-1,3-葡聚糖酶的分离纯化方法进行研究。方法:粗酶液分别用硫酸铵、乙醇和丙酮进行沉淀,再用DEAE-Sepharose CL-6B离子交换层析进一步分离纯化,并用SDS-PAGE法测其分子量。结果:硫酸铵分段盐析法沉淀酶蛋白的效果优于乙醇和丙酮沉淀;盐析得到的酶蛋白经透析浓缩后,再经DEAE-Sepharose CL-6B层析分离,可得到单一酶蛋白,总酶活回收率达78.71%,比酶活达到689.9U/mg,提高了53.74倍,经SDS-PAGE法测得该β-1,3-葡聚糖酶的分子量为80.137kDa。结论:采用硫酸铵分段盐析和离子交换层析法可获得电泳纯的β-1,3-葡聚糖酶,且酶活回收率高。     

亚心型四爿藻在CCCP作用下的光生物产氢的代谢途径

以添加CCCP(羰基氰化物间氯苯腙,Carbonyl cyanide m-chlorophenylhydrazone)的海洋绿藻亚心型四爿藻光生物制氢为研究体系,使用作用于光合系统不同位点的抑制剂研究该藻产氢过程不同时段的代谢途径。结果表明:四爿藻光生物产氢前期电子主要来自PS Ⅱ光解水以及胞内分解代谢,电子经由光合电子传递链传递至氢酶产生氢气;而后期释放的氢气则是通过不依赖光合电子传递链的发酵途径产生。产氢过程厌氧发酵代谢途径主要产物是乙酸、乙醇,其中乙醇代谢途径和氢酶竞争NAD(P)H,不利于氢气的积累。     

硫酸铵三步盐析对藻胆蛋白纯化的影响

主要研究了多次硫酸铵盐析对条斑紫菜藻胆蛋白提取纯化效果。对分离提取的对条斑紫菜藻胆蛋白溶液进行了3次硫酸氨溶液盐析,实验结果表明:55%饱和度可以将绝大部分藻胆蛋白盐析;采用不同组合(15%、20%、25%、30%、35%、40%、45%7个饱和度分别与50%、55%、60%3个饱和度两两组合)二步硫酸铵盐析沉淀藻胆蛋白,使R-藻红蛋白和C-藻蓝蛋白的盐析后纯度(A564/A280)分别达到了1.0和0.45以上,得率分别为1.4%和0.95%;第3次硫酸铵盐析使R-藻红蛋白、C-藻蓝蛋白的纯度分别达到了1.4和0.4以上,最终产率分别为1.3%和0.8%,而变藻蓝蛋白产率有所下降(从0.65%到0.49%),但纯度变化不大。实验证明了采用多次盐析方法可以很大程度提高藻胆蛋白纯度。     

简便厌氧透析装置

在一些对氧较敏感的酶(例如氢酶、固氮酶等)的分离纯化过程中,往往要采用硫酸铵分级沉淀除去杂蛋白,然后通过离子交换柱层析和凝胶过滤等方法,获得较均一的样品,进而研究它们的结构和     

厌氧真菌菌系乙酰酯酶的特性研究

利用厌氧培养技术,采用产酶培养基,培养课题组自行构建的一组厌氧真菌菌系,使之产乙酰酯酶。采用硫酸铵分级沉淀、透析袋透析、DEAE-纤维素离子交换柱层析、Sephadex G-75凝胶过滤柱层析,分离纯化所得到乙酰酯酶,研究其酶学性质。酶活力动态分析表明,乙酰酯酶在产酶培养基上,培养至第3天酶活力达到最高。乙酰酯酶最适温度为41℃,最适pH为9．0,Mg^2＋、K^＋、Ca^2＋对酶有一定的激活作用,Fe^3＋对酶有很强的抑制作用。该厌氧真菌菌系所产的乙酰酯酶,对于发酵木质纤维素类物质具有潜在应用价值。     

一步柱层析纯化螺旋藻藻蓝蛋白

采用硫酸铵盐析结合疏水层析技术分离纯化螺旋藻中的藻蓝蛋白.试验结果表明,在磷酸盐缓冲体系下藻蓝蛋白粗提液经1.25 mol/L硫酸铵盐析处理后离心脱气,只需采用一步Macro-Prep Methyl 疏水层析,藻蓝蛋白的纯度（A620/A280）可提高到4.017,回收率为19.38%.特征吸收峰和荧光光谱证实纯化后的产物符合藻蓝蛋白的性质,Native-PAGE电泳只出现单一染色带,表明纯化得到的藻蓝蛋白是均一的;SDS-PAGE电泳出现分子量为15.4 kDa、17.3 kDa的2条染色带,分别为藻蓝蛋白的α亚基与β亚基.     

洋葱伯克霍尔德氏菌产邻苯二酚2,3-双加氧酶的研究

对洋葱伯克霍尔德氏菌L 68生长及产邻苯二酚2,3 双加氧酶(C23D)的条件进行了研究,其最适产酶pH7.2;最适生长温度30～35℃;最适培养时间48h;苯酚浓度0.09%最有利于菌体产酶.对菌株L 68产生的C23D酶进行了纯化,超声波破碎后的细胞提取液经硫酸铵分级沉淀、DEAESepharoseFastFlow层析、Hydroxyapatite层析、SephadexG 150层析后,收率为20%,酶比活力提高了230倍.SDS PAGE检测得到了分子量为(34±1)kDa的蛋白.     

头孢菌素脱乙酰酶的重组表达及固定化

本文构建了利用trp启动子表达头孢菌素脱乙酰酶(CAH)的重组大肠杆菌DH5α-pCAH。重组菌在7L发酵罐(装液量2L)中发酵28 h,发酵液OD_(600)达到27,产酶313 kU/L发酵液,粗略估算重组蛋白占细胞总蛋白的70%。发酵生产的重组CAH粗酶液经过硫酸铵分级沉淀分离纯化和超滤除盐浓缩两步操作,纯化倍数为1.44,总酶活回收率56%,聚丙烯酰胺凝胶电泳检测纯化后蛋白没有明显杂蛋白条带出现。纯化后的CAH共价结合固定在环氧基载体LX-1000EP(c)上,通过对固定化条件的优化最终得到固定化酶比活443 U/g。该固定化酶重复催化50 mL 5%7-ACA底物100次后,酶活没有降低。     

基于激光共聚焦扫描显微技术分析激光对扁藻的影响

本文以亚心形扁藻为样品,波长1 341 nm的Nd∶YAP激光为光源,通过激光共聚焦扫描显微技术,研究Nd∶YAP激光辐照亚心形扁藻对亚心形扁藻叶绿体自体荧光强度和叶绿体面积大小的影响。Nd∶YAP激光辐照后的亚心形扁藻通过488 nm Ar^+激光激发获得亚心形扁藻自体荧光图像及其荧光光谱。结果表明,试验中除(10 W,60 s)辐照剂量组外,其余辐照剂量组均提高了亚心形扁藻的自体荧光强度,且所有的辐照剂量组均增大了亚心形扁藻的叶绿体面积。Nd∶YAP激光可刺激亚心形扁藻的叶绿体发育,促进藻细胞的生长,改善叶绿体光合作用的活性。     

Redox-dependent subunit dissociation of Azotobacter vinelandii hydrogenase in the presence of sodium dodecyl sulfate

Hydrogenases catalyze the reversible activation of dihydrogen. We have previously demonstrated that the purified hydrogenase from the nitrogen-fixing microorganism Azotobacter vinelandii is an alpha beta dimer (98,000 Da) with subunits of 67,000 (alpha) and 31,000 (beta) daltons and that this enzyme contains iron and nickel. The enzyme can be purified anaerobically in the presence of dithionite in a fully active state that is irreversibly inactivated by exposure to O2. Analysis of this hydrogenase by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) following boiling in SDS yields two protein staining bands corresponding to the alpha and beta subunits. However, when this enzyme was treated with SDS (25-65 degrees C) for up to 30 min under anaerobic/reductive conditions and then analyzed by anaerobic SDS-PAGE, a protein staining band corresponding to an apparent molecular mass of 58,000 Da was observed that stained for hydrogenase activity. Analysis of the 58,000-Da activity staining band by a Western immunoblot or a second aerobic SDS-polyacrylamide gel revealed that this protein actually consisted of both the alpha and beta subunits. Thus, the activity staining band (apparent 58,000 Da) represents the 98,000-Da dimer migrating abnormally on SDS-PAGE. Treatment of the anaerobically purified hydrogenase with SDS under aerobic conditions or under anaerobic conditions with electron acceptors prior to electrophoresis resulted in no activity staining band and the separated alpha and beta subunits. A. vinelandii hydrogenase was also purified under aerobic conditions in an inactive O2 stable form that can be activated by removal of oxygen followed by addition of reductant. This enzyme (as isolated), the activated form, and the reoxidized form were analyzed for their stability toward denaturation by SDS. We conclude that the dissociation of the A. vinelandii hydrogenase subunits in SDS is controlled by the redox state of the enzyme suggesting an important role of one or more redox sites in controlling the structure of this enzyme.     

Aerobic, inactive forms of Azotobacter vinelandii hydrogenase: activation kinetics and insensitivity to C2H2 inhibition

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H2 or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.     

A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test

A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mM), ammonium acetate (25 mM), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mM), L-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (5 mM), D-glucose (6 mM), glycerol (0.3%), imidazol (12.5 mM), DL-methionine (100 mM), mercaptoethanol (0.05%), sodium azide (19 mM, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mM), sodium nitrite (6.2 mM), sodium periodate (3.1 mM), sodium sulfite (12.5 mM), sodium thiosulfite (12.5 mM), sucrose (6 mM), and N,N,N',N'-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mM Tris buffer, urea, N,N'-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mM Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinctive colored products on addition of 5 microliter of test sample.     

The tryptophanase from Proteus rettgeri, improved purification and properties of crystalline holotryptophanase.

The inducible tryptophanase (L-tryptophan indole-lyase (deaminating) EC 4.1.99.1) was crystallized in holoenzyme from the cell extract of Proteus rettgeri. The purification procedure included ammonium sulfate fractionation, heat treatment at 60 degrees C, DEAE-Sephadex and hydroxylapatite column chromatographies. Crystallization was performed by the addition of ammonium sulfate to the purified enzyme solution containing 20% (v/v) glycerol, 0.1 mM pyridoxal phosphate and 10 mM mercaptoethanol. The crystallized enzyme was yellow and showed absorption maxima at 340 and 420 nm. The crystalline holotryptophanase preparation was homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The molecular weight of the enzyme was calculated as approx. 222 000. The amount of pyridoxal phosphate bound to the enzyme was determined to be 4 mol per mol of the enzyme. The enzyme is composed of four subunits of identical molecular size (mol. wt 55 000) and irreversibly dissociates into these subunits in the presence of a high concentration of sodium dodecylsulfate or guanidine hydrochloride. The NH2-terminal amino acid of the enzyme was identified as alanine.     

Isolation, purification and study of the stability of the soluble hydrogenase of Alcaligenes eutrophus Z-1]

Soluble hydrogenase was isolated from the hydrogen-oxidizing bacterium Alcaligenes eutrophus Z-1 and purified to electrophoretical homogeneity. The purification procedure included fractionation by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose and gelfiltration through Ultragel AcA-34. The resulting preparation had a specific activity of 25 mkmoles H2.min-1.mg of protein as measured by the rate of hydrogen evolution from sodium dithionite-reduced methyl viologen. The enzyme has a molecular weight of 200,000 and is made up of two subunits with mol. weights of 30,000 and two subunits with mol. weights of 65,000. The effects of pH, oxidants and reducers, as well as aerobic and anaerobic conditions on the hydrogenase preparations inactivation kinetics in intact cells and in a highly purified state were studied. The kinetic data suggest a possible existence of two enzyme forms differing in their activities and stabilities to denaturating influences.     

螺旋藻氢酶的纯化与生化特性

本研究用DE－52、SephadexG－75、SephadexG－100柱层析从螺旋藻分离纯化得到比活性提高200倍的氢酶,回收率为14％。凝胶柱层析和SDS－PAGE显示一条带,其分子量为56kd。氨基酸分析结果表明酸性氨基酸比例较大,等电聚焦测定结果证明其等电点为pH4．2。吸收光谱结果显示氢酶是铁硫蛋白。甲基紫晶（MV）是氢酶催化放氢的最佳电子供体,其Km（MV）为0．31mmol／L,最适pH值为7．5－8．0。     

Aerobic,inactive forms ofAzotobacter vinelandii hydrogenase: Activation kinetics and insensitivity to C2H2 inhibition

Azotobacter vinelandii hydrogenase (EC class 1.12), either purified or membrane-associated, was obtained aerobically in an inactive state. The kinetics of activation by treatment with a reductant (H₂ or dithionite) were determined. Three distinct phases of the activation were observed. Aerobically prepared, inactive hydrogenase was insensitive to acetylene inhibition, but could be rendered acetylene-sensitive by reduction with dithionite. These findings indicate that acetylene inhibition of hydrogenase requires catalytically active enzyme.     

Purification to homogeneity of Azotobacter vinelandii hydrogenase: a nickel and iron containing alpha beta dimer

Azotobacter vinelandii hydrogenase has been purified to homogeneity from membranes. The enzyme was solubilized with Triton X-100 followed by ammonium sulfate-hexane extractions to remove lipids and detergent. The enzyme was then purified by carboxymethyl-Sepharose and octyl-Sepharose column chromatography. All purification steps were performed under anaerobic conditions in the presence of dithionite and dithiothreitol. The enzyme was purified 143-fold from membranes to a specific activity of 124 mumol of H2 uptake . min-1 . mg protein-1. Nondenaturing polyacrylamide gel electrophoresis of the hydrogenase revealed a single band which stained for both activity and protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands corresponding to peptides of 67,000 and 31,000 daltons. Densitometric scans of the SDS-gel indicated a molar ratio of the two bands of 1.07 +/- 0.05. The molecular weight of the native enzyme was determined by three different methods. While gel permeation gave a molecular weight of 53,000, sucrose density gradient centrifugation and native polyacrylamide gel electrophoresis gave molecular weights of 98,600 +/- 10,000 and 98,600 +/- 2,000, respectively. We conclude that the A. vinelandii hydrogenase is an alpha beta dimer (98,000 daltons) with subunits of 67,000 and 31,000 daltons. Analyses for nickel and iron indicated 0.68 +/- 0.01 mol Ni/mol hydrogenase and 6.6 +/- 0.5 mol Fe/mol hydrogenase. The isoelectric point of the enzyme was 6.1 +/- 0.01. In addition, several catalytic properties of the enzyme have been examined. The Km for H2 was 0.86 microM, and H2 evolution was observed in the presence of reduced methyl viologen. The pH profile of enzyme activity with methylene blue as the electron acceptor has been determined, along with the Km and Vmax for various electron acceptors.     

Aerobically purified hydrogenase from Azotobacter vinelandii: activity, activation, and spectral properties

The hydrogenase from Azotobacter vinelandii is typically purified under anaerobic conditions. In this work, the hydrogenase was purified aerobically. The yields were low (about 2%) relative to those of the anaerobic purification (about 20%). The rate of enzyme activity depended upon the history of the enzyme. The enzyme preparations were active as isolated in H2 oxidation, and isotope exchange. The activity increased during the assay to a new maximal level (turnover activation). Treatment with reductants (e.g., H2, dithionite, dithiothreitol, indigo carmine) resulted in greater activation (reductant activation). Activation of the hydrogenase was accompanied by decrease in visible light absorption (300-600 nm) with maximal decreases at 450 and 345 nm which indicated the reduction of iron-sulfur clusters. The aerobically purified hydrogenase was susceptible to irreversible inactivation by cyanide. Pretreatment with acetylene did not influence activation of the hydrogenase. Once activated, the aerobically purified hydrogenase was indistinguishable from the anaerobically purified hydrogenase with respect to the catalytic properties tested.     

Purification and properties of hydrogenase from Megasphaera elsdenii

A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with Mr approximately 50 000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by O2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (epsilon 275 nm = 143 mM-1 cm-1) and a shoulder between 350 nm and 400 nm (epsilon 400 nm = 46 mM-1 cm-1). The enzyme catalyses hydrogen production from sodium dithionite at a low rate. The rate is greatly enhanced by addition of the electron donors flavodoxin, ferredoxin and methyl viologen. The kinetic data with these three electron donors suggest co-operativity, but no indication of self-association of the enzyme was obtained. Sodium chloride enhances the rate of hydrogen production with methyl viologen semiquinone and changes the kinetic behaviour of the enzyme with this electron donor, but causes inhibition of the reactions mediated by ferredoxin and flavodoxin. Two kinetic models were developed which are consistent with the kinetic data of the three electron donors tested. The apparent co-operativity for the hydrogen production can be fitted with the mathematical form of those models. The identical kinetic behaviour of the hydrogenase with the one-electron donors flavodoxin and methyl viologen semiquinone monomer and the two-electron donor ferredoxin indicates that the hydrogenase accepts two electrons in two separate, independent steps and further indicates that the two (4Fe-4S) clusters of the donor ferredoxin are independent. The interpretation of the kinetic data with methyl viologen semiquinone is complicated by the fact that the semiquinone dimerises, and that the formation of the dimer is enhanced by salt. Taking into account the association of this donor, the activity of the enzyme with methyl viologen semiquinone can be described by the sum of the activities of the enzyme with methyl viologen monomer and methyl viologen dimer. The enzyme catalyses the oxidation of hydrogen gas with methyl and benzyl viologen as electron acceptors to their semiquinone forms; both electron acceptors show Michaelis-Menten kinetics. The hydrogen oxidation activity with both electron acceptors is stimulated by addition of sodium chloride. The kinetic data of the oxidation of hydrogen with the two-electron acceptors used are consistent with the porposed models, if it is assumed that the pathway followed is compulsory. At this moment no choice can be made between the models proposed.