嗜热真菌纤维素酶的CBD与海栖热袍菌的纤维素酶融合表达

将嗜热真菌毛壳菌纤维素酶Cel7A的纤维素结合结构域编码区与极端嗜热厌氧菌海栖热袍菌的纤维素酶CelB基因进行融合, 构建重组质粒pHsh-CBD-CelB, 并在大肠杆菌中表达。对融合蛋白进行纯化, 通过热处理和离子交换层析, 纯化到的融合蛋白SDS-PAGE 电泳图谱显示为单一条带。对融合蛋白的特性研究, 结果表明融合蛋白降解CMC的最适反应温度为90°C, 结晶纤维素吸附实验表明该融合蛋白具有结合结晶纤维素的能力, 并且融合蛋白降解CMC与结晶纤维素的能力得到提高。     

微紫青霉CBHⅠ酶纤维素结合结构域在大肠杆菌中的分泌型表达及性质研究

为研究纤维素酶纤维素结合结构域的结构与功能 ,进而深入了解天然纤维素的生物降解机制和提高纤维素酶的生物工艺学价值 ,采用 PCR技术体外扩增了携带微紫青霉外切葡聚糖纤维二糖水解酶 ( CBH ) CBD编码区的 DNA片段 ,将 CBD编码区 DNA片段插入带有 Erwiniacarotovora pe1 B前导肽序列的大肠杆菌质粒 p KK- tac- new上进行了表达 .携带微紫青霉 CBDCBH 编码区的大肠杆菌重组菌株 DH5α( p KK- tac- new- 8)产生有活性的分泌型 CBDCBH 蛋白 .SDS-PAGE检测显示所产生的 CBDCBH 蛋白分子量约 1 0 .8k D.在 IPTG诱导下 ,该菌株所产生的CBDCBH 蛋白含量达 45.2 mg/L,且 90 %以上的 CBD蛋白分泌到培养物上清液中 .结晶纤维素 CF-1 1溶液经 CBDCBH 处理后 ,浊度比对照提高了 1 2 8.9% ,天然棉花纤维结构经 CBDCBH 处理后产生一定程度的非水解性降解作用 ,表明微紫青霉 CBDCBH 具有解聚天然结晶纤维素的作用 .     

半枝莲多糖的分离、纯化及其理化性质

唇形科植物半枝莲全草的热水提取液经乙醇沉淀、去蛋白、逆向流水透析、DEAE-纤维素柱层析和Sephadex G-100柱层析得纯品半枝莲多糖(S-BP)。凝胶过滤及聚丙烯酰胺凝胶电泳证明为单一均匀成分。SBP的比旋度为[α]_D~(20)=+26.2°,不含氮,分子量13000,具有890cm~(-1)红外吸收峰。经纸层析和气相色谱分析,SBP含鼠李糖、阿拉伯糖、木糖、甘露糖、半乳糖和葡萄糖,其克分子比为1.14:1.50:0.20:0.75:1.0:2.18。     

棕色固氮菌吡啶核苷酸转氢酶的分离纯化及性质的探讨

由棕色固氮菌菌体经超声波破碎处理的无细胞提取液,经50℃加热处理15分钟,硫酸铵区分,DEAE纤维素(DE—32)柱层析,SephadexG—100纯化,得到聚丙烯酰胺凝胶电泳均一的转氢酶制备物(胶浓度7.5％)。酶比活提高了     

明党参多糖的分离、纯化及其理化性质

伞形科植物明党参根的热水提取液经乙醇沉淀、去蛋白、逆向流水透析,DEAE-纤维素柱层析和 Sephadex G-100柱层析得纯品明党参多糖(CSP)。凝胶过滤及聚丙烯酰胺凝胶电泳证明为单一均匀成分。CSP 的比旋度为〔α〕_D~(20)=+170.9(C=0.4,H_2o),不含氮,分子量52000,具有845cm~(-1)红外吸收峰。经纸层析和气相色谱分析,CSP 含鼠李糖、阿拉伯糖、木糖、甘露糖、半乳糖和葡萄糖,其摩尔比为0.09:0.25:0.17:1.0:1.56:11.94。     

恶臭假单胞菌HspB的单晶培养及结晶条件优化

为了获得可用于X射线衍射的恶臭假单胞菌尼古丁代谢途径中关键单加氧酶Hsp B的单晶。定点突变PCR构建重组质粒,大肠杆菌中诱导表达,镍柱亲和层析、烟草蚀纹病毒(Tobacco etch virus,TEV)蛋白酶酶切和凝胶过滤层析纯化,悬滴扩散法进行结晶。成功构建重组质粒并获得高表达;比较了TEV蛋白酶柱上及透析酶切的效率,TEV蛋白酶透析酶切效率更高;确定了该纯化路线,获得电泳纯级的Hsp B蛋白。结晶条件初筛和正交优化后获得可培养Hsp B蛋白单晶的条件为22%PEG3350、0.1 mol/L Bis-Tris p H6.5、0.21 mol/L Mg Cl2、18℃、1?50比例加晶种。去除标签后的Hsp B蛋白获得了分辨率1.8?的单晶。     

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。     

鸡胚活性蛋白研究

鸡胚经加热除去杂蛋白,用水提取,活性蛋白含量为1mg/mL。测得SOD活性24μ/mL,25℃保存半年后活性仍保留75％.鸡胚活性蛋白对光照还原法产生的O(?)有较好的清除能力,对Fenton反应产生的·OH清除能力远高于甘露醇。对酪氨酸酶有强烈抑制。用上述提取液配制的霜剂经试用,均感皮肤柔软光滑,有祛斑、减轻面部皱纹的功效,无过敏反应。     

拟松材线虫纤维素酶活性与其致病性关系

【目的】研究拟松材线虫是否分泌纤维素酶以及纤维素酶与其致病力的关系。【方法】对拟松材线虫的不同致病性种群以及松材线虫的虫体蛋白提取液、分泌液的纤维素酶活性进行定量测定,比较群体间纤维素同工酶谱型差异。【结果】拟松材线虫也含有纤维素酶,并向体外分泌;且不同致病性种群的纤维素酶活性与其致病性有一定相关性,致病性越强纤维素酶活性越强。【结论】纤维素酶活性大小是导致拟松材线虫不同种群间致病性差异的重要原因,这对于全面认识松树线虫萎蔫病的致病机理有重要意义。     

糖苷水解酶7家族蛋白在纤维素降解中作用的研究进展

糖苷水解酶7家族（glycoside hydrolase family, GH7）是一类来源于真菌的水解酶,作用于纤维素结晶区或不定形区的β-1,4 键,可用于高效降解纤维素转化为可发酵的糖。GH7的成员具有高度保守序列以及相似三维结构,其催化结构域是由多个loop区围绕反向平行的β 折叠形成的β 三明治结构。目前已有17个GH7成员的结晶结构得到解析,明确了酶的结构与催化功能之间的关联,对GH7的来源及分类、蛋白序列、结构特征与催化纤维素降解功能关系的研究进展进行阐述。     

Algal heme oxygenase from Cyanidium caldarium. Partial purification and fractionation into three required protein components

Enzymatic heme oxygenase activity has been partially purified from extracts of the unicellular red alga Cyanidium caldarium, and the macromolecular components have been separated into three protein fractions, referred to as Fractions I, II, and III, by serial column chromatography through DEAE-cellulose and Reactive Blue 2-Sepharose. Fraction I is retained by DEAE-cellulose at low salt concentration and eluted by 1 M NaCl. Fraction II is retained by Blue Sepharose at low salt concentration and eluted by 1 M NaCl. Fraction III is retained on 2',5'-ADP-agarose and eluted by 1 mM NADPH, while Fraction II is not retained on ADP-agarose. Fractions I-III, have Mr values of 22,000, 38,000, and 37,000, respectively (all +/- 2,000), as determined by Sephadex gel filtration chromatography. In vitro heme oxygenase activity requires the presence of all three fractions, plus substrate, O2, reduced pyridine nucleotide, and another reductant. Ascorbate, isoascorbate, and phenylenediamine serve equally well as the second reductant, but hydroquinone can also be used, with lower activity resulting. Fractions I-III are heat sensitive and inactive by Pronase digestion. Fraction I has a visible absorption spectrum similar to that of ferredoxin and is bleached by dithionite reduction or incubation with p-hydroxymercuribenzoate. Fraction I can be replaced by commercially available ferredoxin derived from the red alga Porphyra umbilicalis, and to a smaller extent, by spinach ferredoxin. Fraction III contains ferredoxin-linked cytochrome c reductase activity and can be partially replaced by spinach ferredoxin-NADP+ oxidoreductase. Reconstituted heme oxygenase and ferredoxin-linked cytochrome c reductase activities are both abolished if Fraction I or III is preincubated with 0.1 mM p-hydroxymercuribenzoate, but heme oxygenase activity is only slightly affected if Fraction II is preincubated with p-hydroxymercuribenzoate. Preincubation of Fraction II with 0.5 mM diethylpyrocarbonate inactivates heme oxygenase in the reconstituted system, and 10 microM mesohemin partially protects this Fraction against diethylpyrocarbonate inactivation. Algal heme oxygenase is inhibited 80% by 2 microM Sn-protoporphyrin even in the presence of 20 microM mesohemin. Fraction II is rate limiting in unfractionated and reconstituted incubation mixtures. None of the three cell fractions could be replaced by bovine spleen microsomal heme oxygenase or NADPH-cytochrome P450 reductase.     

Identification of multiple cellular factors required for SV40 replication in vitro

The replication of simian virus 40 has been studied by using cell-free extracts derived from human 293 cells. Fractionation of this extract has led to the identification of three fractions that are required for efficient DNA synthesis. Initial fractionation of the crude extract by phosphocellulose chromatography has produced two fractions, I and II, neither of which is able to support replication separately, but when they are combined, efficient synthesis is restored. Both fractions are required, with SV40 T antigen, for the formation of a presynthesis complex at the SV40 origin. The major replication enzymes, DNA polymerase, DNA primase and the topoisomerases I and II all reside in fraction II. Fraction I has been subdivided into two subfractions (A and B) by DEAE-cellulose chromatography. Fraction A is essential for replication and is required for presynthesis complex formation. Fraction B stimulates DNA replication and is only required at the elongation stage. This multicomponent system has provided the foundation for identification of individual components that are required for DNA replication in vitro.     

Screening for specific calf thymus inhibitors (chalones) of T-lymphocyte proliferation.

Using agar colony assays with truly proliferating stimulated human T-lymphocytes and mouse granulocytes, two ultrafiltrate fractions were obtained from calf thymus which preferentially inhibited lymphocyte colony growth: Fraction I in the molecular range 1000-10,000 proved to be stable upon heating, prolonged storage and lyophilization, whereas Fraction II in the molecular range 10,000-30,000, was found to be unstable. Fraction I was also extracted with Tween 80 and cetyltrimethylammonium bromide. Chromatography of Fraction I on Biogel P6 and DEAE-cellulose further increased its specificity of inhibition for lymphocyte colony growth and revealed an estimated molecular weight of below 1400. Its inhibitory activity was found to be reversible and unlikely to result from spermine. Thus the properties of fraction I meet the requirements of a T-lymphocyte chalone as an endogenous non-cytotoxic and reversible inhibitor of T-lymphocyte proliferation.     

Comparative studies on fraction 1 protein from spinach and tobacco leaves

Fraction 1 protein of spinach and tobacco leaves was purifiedby Sephadex G-200 and DEAE-cellulose column chromatography.Immunological comparison of purified preparations of these proteinswas carried out with precipitin analysis using an antiserumof tobacco fraction 1 protein. Two different antigenic activitieswere found in tobacco fraction 1 protein, of which one was commonlyfound in the spinach protein and the other was specific to tobaccofraction 1 protein. The immunological results suggest that fraction 1 protein iscomposed of at least two different structures, one of whichis common to both spinach and tobacco proteins and the otherof which is specific to each of these proteins. This was confirmedfrom a chemical experiment. Fraction 1 protein was divided intolarge and small polypeptide components by sodium dodecyl sulfatetreatment and subsequent Sephadex G-100 column chromatography,then the amino acid compositions of each polypeptide of theseproteins were compared. The amino acid composition of the smallpolypeptide of spinach was different from that of tobacco, whileamino acid compositions of the large polypeptides of those proteinswere similar to each other. (Received July 1, 1968; )     

Multiple molecular forms of phosphoprotein phosphatase. III. Phosphorylase phosphatase and phosphohistone phosphatase of rabbit liver.

1. Phosphoprotein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) in the soluble fraction of rabbit liver which catalyzes the dephosphorylation of muscle phosphorylase a and phosphohistone (P-histone) was resolved into three active fractions by NaCl gradient elution from a DEAE-cellulose column (Fraction I, 11 and III in order of elution). They have different relative reaction rates for the two substrates and different degrees of stimulation by Mn-2+. Apparent Km values of Fraction I, II and III were 15, 20 and 16 muM for phosphorylase a, and 6.9, 5.3 and 4.4 muM for P-histone, respectively (with Mn-2+ in the assay mixture). 2. On sucrose density gradient centrifugation Fraction I and II were revealed to contain a major peak (7.0 S and 7.8 S, respectively) and a minor peak (4.0 S) of activity, while Fraction III contained only one peak (5.8 S). Freezing and thawing in the presence of 0.2 M mercaptoethanol dissociated all three fractions into subunits of similar molecular size (3.4 S), with concomitant enhancement of phosphorylase phosphatase activity. The Km values all became essentially the same (20 muM for phosphorylase a and 16 muM for P-histone). 3. The phosphorylase phosphatase and P-histone phosphatase activities could not be separated with any of the procedures described. Competition between the two phosphoprotein substrates was observed with some of the fractions.?     

Actin-activated ATPase from human erythrocytes.

A fibrillar protein complex, possessing ouabain-insensitive Ca2+-ATPase activity was isolated from human erythrocyte membranes by using a low ionic strength extraction procedure. Mg2+-ATPase activity was revealed upon addition of rabbit skeletal muscle actin, thus demonstrating the presence of a myosin-like protein in the crude extract of the erythrocyte membrane. Upon sodium dodecylsulfate gel electrophoresis, the extract showed mainly the doublet of subunit molecular weight bands of 230 000 and 210 000, and more than 10 faster moving bands. Gel filtration of the erythrocyte membrane extract on Sepharose 4B furnished 4 fractions. Fraction I, containing the doublet and 80 000, 60 000 and 46 000 subunit molecular weight bands was 5-fold purified with respect to Ca2+-ATPase activity, but was devoid of actin-activated Mg2+-ATPase activity. Fraction II, containing only the doublet, was devoid of Ca2+ and actin-activated Mg2+-ATPase activity. The 210 000 subunit molecular weight protein could be phosphorylated in the presence of Mg2+ in the crude extract and Fraction I but not in Fraction II.     

Extraction and purification of proteoglycans from various types of connective tissue

A new procedure for the isolation of proteoglycans has been described. Tissues are extracted with 4 M guanidinium chloride. the extracting solven is then exchanged for 7 M urea and the extract is chromatographed on a DEAE-cellulose column previously equilibrated with 7 M urea. Non-proteoglycan proteins were eluted with urea in weak salt solutions. Subsequently proteoglycans were eluted with strong salt solutions. By the procedure proteoglycans from tissues containing only small amounts of proteoglycans can be obtained virtually free from collagen in a form suitable for further fractionation.     

Metabolism of thyrotropin releasing hormone in brain extracts. Isolation and characterization of an imidopeptidase for histidylprolineamide.

An extract of porcine brain acetone powder incubated with thyrotropin-releasing hormone (TRH; pGlu-His-ProNH2) produces acid TRH (pGlu-His-Pro), histidine, and prolineamide. Fractionation of the brain extract by DEAE-cellulose chromatography produces three protein fractions which metabolize TRH. The activity of these fractions was characterized using TRH with a 3H-label on the histidine or proline as well as [His-3H]His-ProNH2. Fraction I contains pyroglutamate aminopeptidase and Fraction II contains TRH deamidase. Fraction III was found to contain a previously unrecognized enzyme which cleaves His-ProNH2 to histidine and proline. The histidylprolineamide imidopeptidase has been characterized. A competition study using a variety of compounds containing histidine or proline suggests that the best substrates for the imidopeptidase contain a free alpha-amino group on histidine and a blocked carboxyl group on proline, as is found in His-ProNH2. A survey of a variety of polypeptide hormones indicates that many of them inhibit the imidopeptidase activity. A kinetic study of the inhibition of the enzyme by adrenocorticotropic hormone (1-24) shows that the inhibition by polypeptide hormones is noncompetitive. We hypothesize that pituitary hormones may stimulate the production of (cyclo)-His-Pro by inhibiting alternate routes of TRH metabolism.     

Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.     

Intracellular cAMP binding proteins in Dictyostelium discoidium: differences in properties of the proteins from vegetative and differentiated cells.

Components responsible for intracellular cAMP binding from vegetative cells of Dictyostelium discoideum and from cells collected after 18 hr of differentiation were partially purified by DEAE-cellulose column chromatography. Their properties differ in a variety of aspects: The cAMP binding activity from vegetative cells is eluted at low ionic strength; it is inhibited by 5′-AMP; bound cAMP is not readily exchangeable, and finally, extensive dialysis of the crude extract enhances its cAMP binding activity. In contrast, the cAMP binding component from differentiated cells is not affected by dialysis and is not inhibited by 5′-AMP. It is eluted at higher ionic strength from DEAE and has a rapid dissociation rate for cAMP. The binding activity could be separated from protein kinase activity found in differentiated cells. A heat-stable dialyzable factor was found in both vegetative and differentiated cells which inhibited cAMP binding in vegetative cells but not in differentiated cells.