l-Sorbose Oxidase from Trametes sanguinea

A crude inhibitor for pancreatic lipase was extracted from soybean seeds. The lipase activity decreased curvilinearly with an increase in inhibitor concentration. At a low inhibitor concentration, enhanced inhibition was observed by the co-existence of protein such as bovine serum albumin in the reaction mixture. The lipase activity was inhibited immediately after the addition of inhibitor which did not cause the significant destraction of substrate emulsion. The lipase activities of Aspergillus niger, Rhizopus delemar and castor bean seeds were also inhibited. The inhibition was observed when various oil substrates such as soybean oil, linseed oil, olive oil emulsions and Ediol were used, and the extent of inhibition varied among them. Column chromatography of inhibitor on Sephadex G–100 showed that the molecular weight of a main peak of inhibitor was estimated as about 80,000.     

Acid Protease Produced by Trametes sanguinea,a Wood-destroying Fungus

A wood-destroying fungus, Trametes sanguinea, produced a potent acid protease in a submerged culture. Maximum proteolytic activity of the culture was attained after 140-hours cultivation in a medium containing dextrin and corn steep liquor. The acid protease was obtained in crystalline form from the mycelium-free culture filtrate by the following successive treatments: acetone precipitation, ionexchange column chromatography, ammonium sulfate fractionation, dialysis, and crystallization by acetone. Throughout the over-all process, the acid protease was purified approximately 30-fold with about 8% recovery of the original activity.     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

Purification and Some Properties of Tetanolysin

Tetanolysin was purified from the culture fluid of a strain of Clostridium tetani by ammonium sulfate fractionation, acetone precipitation and repeated gel filtration. Two hemolysins with different molecular weights were separated by gel filtration, and the smaller one, tetanolysin, was further purified. The purification raised the specific activity of tetanolysin 1,050-fold to 500 HU/μg of protein. The purified preparation gave a single, relatively broad band on polyacrylamide gel electrophoresis, in which the activity was roughly parallel with the protein concentration. However, on sodium dodecylsulfate-gel electrophoresis it gave two bands with nearly equal amounts of proteins, showing molecular weights of 53,000 and 48,000±3,000. Furthermore, isoelectric focusing revealed four peaks of the activity whose isoelectric pHs were 6.1, 5.6, 5.3, and 6.6 in decreasing order of the activity. These findings suggest that the preparation contains four hemolysins with different pis, which are classifiable into two groups by molecular size. The preparation was completely free of tetanus neurotoxin and proteases. Tetanolysin was more strongly inhibited by cholesterol and more rapidly adsorbed onto erythrocytes than θ-toxin of Cl. perfringens.     

N-氨甲酰基-D-氨基酸酰胺水解酶的快速纯化及性质

通过硫酸铵分级沉淀、疏水层析及阴离子交换层析等三步 ,有效地从一菌株NO .2 2 6 2中纯化了N 氨甲酰基 D 氨基酸酰胺水解酶。结果表明 ,酶活性回收约 2 0 %,纯化了 8 4倍。天然PAGE与SDS PAGE分析表明 ,该酶分子为同源四聚体 ,单体分子量约为 3 5kD。酶催化反应的最适pH为 7 7～ 8 0 ,最适温度为 45℃。以N 氨甲酰 DL 丙氨酸为底物时 ,Km =1 3×1 0 - 3 mol L ,Vmax=0 .3 3mol min。二价金属离子Ni2 + 有激活作用 ,Zn2 + 有明显的抑制作用 ,而Co2 + 对酶活无影响。该酶N 末端 8个氨基酸残基依次为TRQKILAF。     

Purification and Some Properties of Milk Protease

Separation of acetic acid from palm oil mill effluent (POME) to increase its concentration by an anion exchange resin was examined as a preliminary study for its recovery from POME that had been anaerobically treated by sludge from a palm oil mill. This paper concerns the acetic acid thus separated for producing bacterial polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus. It was found that sludge particles in POME strongly inhibited the adsorption of acetic acid on the anion exchange resin. Removing the sludge particles from the POME facilitated the separation of acetic acid from the POME efficiently. The concentrated acetic acid thus obtained from anaerobically treated POME could be used as a substrate in the fed-batch production of polyhydroxyalkanoate by Alcaligenes eutrophus.     

Purification and Some Properties of Two Boticins

Two bacteriocins (boticins) were elaborated without induction by strain S5, a nontoxigenic variant of Clostridium botulinum type E. After separation of the two active entities by gel filtration on Sephadex G-50, a large particle with boticin activity was isolated by density gradient ultracentrifugation, and a small soluble boticin was purified by continuous curtain electrophoresis and chromatography on sulfoethyl-Sephadex. Large and small boticins were purified 200- and 3,000-fold, respectively, with yields of 50% or more. The small boticin, a basic substance with molecular weight under 30,000, was the predominant species; the large boticin, a negatively charged particle with particle weight greater than 40 x 10(6), represented less than 20% of the total activity. Both purified boticins were resistant to heat and were attacked by proteolytic enzymes, but the large boticin was less thermostable and less sensitive to proteolytic enzymes than was the smaller variety. The activity of the large boticin was not reduced by treatment with urea or deoxyribonuclease. Both boticins exhibited sporostatic and bactericidal activities for C. botulinum type E, strain 070. A suspension of type E strain 070 vegetative cells was rendered nonviable within 9 min by the small boticin. The lethal action of this bacteriocin was not reversed by trypsin.     

嗜酸氧化亚铁硫杆菌硫氰酸酶的表达、纯化及其性质鉴定

目的:嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)硫氰酸酶是硫代谢中极其重要的酶,其作用主要包括氰化物的解毒,铁-硫蛋白的合成,以及硫胺、硫尿苷或烟碱乙酸胆碱的生物合成,硫氰酸酶的研究对揭示生物冶金机理具有重要的推动作用.方法:以A.ferrooxidans ATCC23270基因组为模板设计引物,通过PCR扩增得到编码硫氰酸酶的基因,目的基因片段与原核表达载体PLM1构建重组体,然后转入大肠杆菌(Eschcrichia coli,E.coli)DH5a感受态中,基因测序正确后,重组质粒再转入E.coli BL21感受态中,加IPTG诱导蛋白表达,用一步亲和层析法纯化出浓度和纯度都较高的硫氰酸酶.结果:SDS-PAGE分析证实蛋白分子量为21kD,紫外可见光分析,确定硫氰酸酶中含有铁硫簇,酶活测定发现重组硫氰酸酶在体外不具有酶活性,可能与酶反应条件及信号肽的切断有关.结论:体外成功克隆.表达,纯化出重组体硫氰酸酶,其基本性质也得到阐述.     

Purification and Some Properties of Plant Serine Acetyltransferase

Serine acetyltransferase (SATase) that catalyzes the conversionof L-serine to O-acetyl-L-serine (OAS) in the presence of acetyl-CoAwas highly purified from rape leaf extract, using a coupledassay system in which OAS is converted to cysteine by enzymaticaction of exogenous cysteine synthase. Through purificationprocedures including heat treatment, ammonium sulfate fractionationand successive column chromatographies on DEAE-Toypearl, Blue-Toyopearland Toyopearl HW-55, the specific activity was raised to about4,800-fold over that in the crude extract. The molecular weightof rape enzyme was estimated to be about 350,000 by gel filtrationcolumn chromatography. The cysteine-forming activity of thefinal preparation was completely dependent on L-serine, acetyl-CoAand sulfide. However, this preparation had low activity forL-cysteine synthesis from L-serine even in the absence of exogenouscysteine synthase, suggesting that plant SATase exists as ahigh-molecular weight enzyme complexed with cysteine synthase. (Received November 6, 1987; Accepted March 25, 1988)     

Purification and Some Properties of Bacteriophage ST-1

Bacteriophage ST-1 is shown to be a small, isometric, single-stranded deoxyribonucleic acid (SS-DNA) virus with a diameter of about 260 nm. Standard methods for growth, assay, preparation of high-titer lysates, and purification of the phage are suggested. ST-1 infects K-12 and not C strains of Escherichia coli and requires a divalent cation to adsorb to susceptible bacteria. Adsorption also requires an activation of the particle brought on by incubation at 37 C. The latent and eclipse periods are essentially identical (9 to 11 min) in ST-1 infections, with an average burst size about 250 phages per cell. Multiple densities of ST-1 infectivity are observed during purification in CsCl gradients. The virus recovered from different densities has the same sedimentation coefficient and, therefore, all phage containing fractions are pooled during purification. The purified ST-1 particle has a sedimentation coefficient of 121S relative to phiX-174 (114S) in a sucrose gradient and a molecular weight of 6.8 x 10(6) (as estimated from its relative sedimentation). The nucleic acid is assumed to be SS-DNA on the basis of (i) the specific incorporation of (3)H-thymine, (ii) the dependence of its UV absorption on temperature, and (iii) its reaction with formaldehyde. ST-1 SS-DNA sediments at 24.4S relative to phiX-174 SS-DNA (23.8S).     

变色栓菌锰过氧化物酶同工酶的纯化及其性质研究

变色栓菌(Trametes versicolor)胞外产酶培养液经硫酸铵沉淀、DEAE-cellulose DE52离子交换柱层析后,获得两个活性组分D1和D2,其中活性组分D2经Phenyl SepharoseTM6Fast Flow疏水层析后,所得样品MnP1经SDS-PAGE检测已达到电泳纯。活性组分D1经Phenyl SepharoseTM6Fast Flow疏水层析、Sephacryl S-200HR凝胶过滤层析后,所得样品MnP2经SDS-PAGE检测已达到电泳纯。两种同工酶MnP1及MnP2,各自的比活力为579.09、425.00U/mg;纯化倍数为17.51、12.85;活力回收率为6.17%、2.47%。由SDS-PAGE法测得MnP1及MnP2的表观分子量分别为46.3kD、43.0kD。两种同工酶催化DMP(2,6-二甲氧基酚)氧化反应的最适pH值及最适反应温度有所不同,最适pH值分别为pH5.8、pH6.2,最适反应温度分别为60℃、65℃。在45℃以下,pH4.0~7.0之间,MnP1及MnP2的稳定性好。DMP为最佳酶促反应底物,以DMP为底物的Km分别为13.43μmol/L、12.45μmol/L。在无Mn2 存在的条件下,酶促反应几乎不发生。EDTA在较高浓度时抑制酶的活性,DTT在所试浓度下都完全抑制酶的活性。     

Purification and Some Properties of Two Aminopeptidases from Sardines

Two fish aminopeptidases designated as aminopeptidases I and II were purified by DEAE-cellulose chromatography, gel filtration on Sephadex G-200, and isoelectric focusing. The final preparations of enzymes I and II were judged nearly homogenous by polyacrylamide gel I, electrophoresis. The molecular weights of enzymes I and II were determined by gel filtration to be 370,000 and 320,000, respectively. The isoelectric points were 4.1 (I) and 4.8 (II), Both enzymes were inhibited by EDTA and activated by Co⁺⁺. Bestatin could inhibit enzyme I but not enzyme II. Enzymes I and II rapidly hydrolyzed not only synthetic substrates containing alanine or leucine but also di-, tri-, and tetra-alanine. Judged from all of these properties, sardine aminopeptidases resemble human alanine aminopeptidase. Enzyme I retained more than 70% of its original activity in 15% NaCl, suggesting the enzyme participates in hydrolyzing fish proteins and peptides during fish sauce production.     

Purification and Some Properties of Candida albicans DNA Polymerases

Abstract

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained bv DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.     

双胸蚓纤溶酶的纯化及性质

 用硫酸铵分段盐析、超滤膜分级分离及DEAE-纤维素、Sephadex A-25和Sephadex G-50三种柱层析方法从双胸蚓组织的粗提取液中分离纯化出一种纤溶酶,分子量为29kD,由一条肽链组成。此晦具有强烈的溶解纤维蛋白的作用,对家兎实验性血凝块也具有明显的溶解作用。此酶的最适pH为8.0,在pH7.6～8.4之间活力相差不到2％;酶在PH4.7—11.0范围内稳定;酶作用的最适温度为57℃;此酶热稳定性较好,于25～50℃保温3小时,酶活力基本不变,60℃时,活力保留65％。金属离子Na~(+)、K~(+)、Mg~(2+)等可提高此酶的活力,而Hg~(2+)、Ca~(2+)等金属离子对此酶有不同程度的抑制作用。     

Purification and Some Properties of Chitinase from Momordica charantia

Chitinase was purified from Momordica charantia L. by affinity chromatography. The purified enzyme showed single band on sodium dodecyl sulfate polyacrylamicle gel electrophoresis and the molecular weight was estimated as 35 kD. The enzyme was stable at temperatures up to 50℃ or less than 10 % loss of activity in 1 h. Its optimum temperature was about 45 ℃. Its suitable pH had a rather wide range from pH 4.4 to pH 6.8 and the optimum pH was about 6.2. The activity of the enzyme was similar in root and stem. In the lower leaves,the activity was higher than that of the upper.     

蟾蜍细胞溶素的纯化及其生化特性研究

利用阴离子交换和凝胶过滤柱层析等方法对蟾蜍卵黄外被细胞溶素进行了分离纯化,获得了高纯度的样品．该酶的质量为３２ｋＤ,其特异性ＭＣＡ－人工合成底物为Ｂｏｃ－Ｇｌｎ－Ａｒｇ－Ａｒｇ－ＭＣＡ,能被ＤＦＰ、ＳＢＴＩ、ｌｅｕｐｅｐｔｉｎ和ｐ－ＡＭＰＳＦ等蛋白酶抑制剂所强烈抑制,但不受ｃｈｙｍｏｓｔａｔｉｎ、ｂｅｓｔａｔｉｎ、Ｅ－６４和ＥＤＴＡ等的影响,表明该酶是一种丝氨酸类型的蛋白酶     

Purification and Some Properties of Pheophorbidase in Chenopodium album

A novel enzyme, pheophorbidase, which catalyzes the conversionof pheophorbide a to C-13²-carboxylpyropheophorbide a, was purifiedfrom Chenopodium album leaves. The purified enzyme showed twobands of 28 kDa and 29 kDa on SDS-PAGE. The molecular mass ofthe native pheophorbidase was 105 kDa. The N-terminal aminoacid sequence for the 28-kDa protein could be determined, whereasthe N-terminus of the 29-kDa protein was blocked. Immunochemicaland enzyme activity analyses revealed that pheophorbidase islocated in an extra-plastidic part of the cell. (Received September 7, 1998; Accepted October 26, 1998)