人脾DNA拓扑异构酶Ⅰ的提纯及其性质的研究

 经三个步骤从人脾提纯了拓扑酶Ⅰ。SDS-PAGE和等电聚焦均显示单一蛋白带。分子量77kD,pI7.3。新生霉素和香豆霉素A_1不抑制酶活性。本实验表明,反应最适K~(+)或Na~(+)浓度为180mmol/L。反应活性不要求Mg~(2+),但5mmol/L存在时,活性增加90％。在pH5.0～9.0均有活性,但在7.5～8.0时活性较大。30℃放置24小时或50℃处理30分钟活性基本丧失。最适反应温度为37℃。对-氯汞苯甲酸强烈抑制酶活性。     

Purification and properties of deoxyribonuclease from human urine.

The DNAase in human urine was purified about 30-fold with a recovery of 28%. This involved DEAE-cellulose and phosphocellulose chromatography steps and gel filtration on Sephadex G-75. The enzyme required divalent cations such as Co2+, Mg2+, Mn2+ and Zn2+ for activity, but Ca2+, Cu2+ and Fe2+ were ineffective. EDTA and G-actin inhibited the reaction. The maximum activity was observed at pH 5.5 in acetate buffer plus Co2+ or Mg2+ and Ca2+. It had a molecular weight of approximately 38 000, estimated by gel filtration on Sephadex G-75 and isoelectric point of around pH 3.9. The enzyme is an endonuclease which hydrolyzes native, double-stranded DNA about 3 to 4 times faster than thermally denatured DNA to produce 5'-phosphoryl- and 3'-hydroxyl-terminated oligonucleotides. The final preparation was free of non-specific acid and alkaline phosphatases, phosphodiesterase and ribonuclease activities.     

镰刀菌Q7-31T内切葡聚糖酶Egn21的分离纯化及酶学性质

【目的】为进一步研究镰刀菌Q7-31T产生的植物细胞壁降解酶的酶系信息。【方法】以1%(W/V)蛋白胨为氮源,0.5%(W/V)燕麦秸秆为碳源,20°C、120 r/min振荡培养3 d,诱导发酵培养菌株,获得的粗酶液经过Sephacry S-100凝胶柱层析和DEAE琼脂糖弱阴离子交换柱层析,最终得到纯化的内切葡聚糖酶,并对其进行酶学性质分析及串联质谱鉴定。【结果】研究表明:Egn21的分子质量为44.25 kDa,等电点为4.91;酶学特性研究显示:Egn21降解羧甲基纤维素的最适反应温度为40°C,在45°C以下比较稳定。该酶最适pH为6.0,在pH为5.0–8.0条件之间比较稳定。Co~(2+)、Zn~(2+)和Mg~(2+)对其没有明显作用,而Fe~(2+)、Ca~(2+)、K~(+)、Na~(+)和Mn~(2+)对酶活性有抑制作用,Hg~(2+)会使酶失去活性。【结论】从Q7-31T中分离纯化得到的内切葡聚糖酶Egn21,经过酶学特性与串联质谱鉴定结果显示其属于GH5家族。     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

Purification and characterization of the FokI restriction endonuclease

The restriction endonuclease FokI from Flavobacterium okeanokoites was purified to homogeneity. Based on gel filtration, sedimentation and sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, the following properties of the enzyme were determined: FokI exists in one active monomeric form, and has an Mr of 64-65.4 x 10(3).FokI is a strongly basic protein with an isoelectric point of 9.4. The enzyme exhibits restriction activity in the pH range 5.0 to 10.5 (maximum level at pH 7.0-8.5) and its divalent cation requirement is satisfied not only by Mg2+, but also by Co2+, Mn2+, Ni2+, Cd2+, Zn2+ and Fe2+.     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.     

黑曲霉(Aspergillus niger)β-D-葡萄糖苷酶的纯化及其性质

利用垂直板凝胶制备电泳从黑曲霉(Aspergillus niger,AS 3.316)中分离提纯了β-D-葡萄糖苷酶(EC3.2.1.21),经凝胶电泳鉴定为单一带。酶作用的最适pH为4.4,在pH4.0—6.2稳定;最适温度65℃,热稳定性较好,于60℃保温4小时,活力保留80％。此酶作用于纤维二糖的Km值为6.09mM。聚丙烯酰胺薄层等电聚焦测得其pI值为5.5;用SDS凝胶电泳测得其分子量为77000。此酶不仅能水解纤维二糖和对硝基苯-β-D-葡萄糖苷,还能微弱地水解对硝基苯β-D-半乳糖苷和β-D-木糖苷。金属离子Fe~(2+)、Hg~(2+)、Cu~(2+)、Al~(3+)、Hg~+和Ag~+等对此酶有不同程度的抑制作用,蛋白质侧链修饰剂N-溴代琥珀酰亚胺对此酶有较强的抑制作用,2-羟基-5-硝基溴苯对酶也有一定的抑制作用,推测色氨酸残基对β-D-葡萄糖苷酶的活力是非常必要的。     

Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.     

Studies on phospholipases from Streptomyces. II. Purification and properties of Streptomyces hachijoensis phospholipase D.

1. Phospholipase D [EC 3.1.4.4] from Streptomyces hachijoensis was purified about 570-fold by column chromatography on DEAE-cellulose and Sephadex G-50 followed by isoelectric focusing. 2. The purified preparation was found to be homogeneous both by immunodiffusion and polyacrylamide disc gel electrophoresis. 3. The isoelectric point was found to be around pH 8.6 and the molecular weight was about 16,000. 4. The enzyme has maximal activity at pH 7.5 at 37 degrees. The optimal temperature is around 50 degrees at pH 7.5, using 20 min incubation. 5. The enzyme was stable at 50 degrees for 90 min. At neutral pH, between 6 and 8, the enzyme retained more than 95% of its activity on 24 hr incubation at 25 degrees. However, the enzyme lost 80% of its activity under the same conditions at pH 4.0. 6. The enzyme was stimulated slightly by Ca2+, Mn2+, and Co2+, and significantly by Triton X-100 and ethyl ether. It was inhibited by Sn2+, Fe2+, Fe3+, Al3+, EDTA, sodium dodecyl sulfate, sodium cholate, and cetylpyridinium chloride. 7. This phospholipase D hydrolyzes phosphatidylethanolamine, phosphatidylcholine, cardiolipin, sphingomyelin, phosphatidylserine, and lysophosphatidylcholine, liberating the corresponding bases. 8. The Km value was 4mM, determined with phosphatidylethanolamine as a substrate.     

一种新型具有纤溶活性的沙蚕金属蛋白酶的分离纯化及鉴定

通过硫酸铵分级盐析、DEAE-Sepharose FF阴离子交换色谱、CM-Sepharose FF阳离子交换色谱和Sephacryl S-100 HR凝胶过滤色谱,从沙蚕体内分离纯化出一种新型的具有纤溶活性的金属蛋白酶,命名为NVMP.采用SDS-PAGE和MALDI-TOF MS 质谱检测,该酶是一种分子质量为28~32 kD的单链蛋白,等电聚焦电泳显示其等电点为8.0. NVMP酶活性被EGTA完全性抑制,表明其是一种典型的金属蛋白酶,最适温度为40 ℃,最适pH为6,Cu2+、Co2+和Zn2+可阻断其酶活性,而Ca2+ 和Mg2+可增强蛋白酶活性.经肽指纹图谱分析发现,NVMP是一种未知的新蛋白. NVMP可直接水解纤维蛋白,也可通过激活纤溶酶原转变成纤溶酶的方式,间接水解纤维蛋白.因此,NVMP对预防和治疗血栓性疾病具有一定的药用价值.     

Purification and properties of polyvinyl alcohol oxidase with broad substrate range obtained from Pseudomonas vesicularis var. povalolyticus PH

Extracellular PVA oxidase produced by Pseudomonas vesicularis var. povalolyticus PH was purified to homogeneity by ammonium sulphate fractionation followed by successive column chromatography, and a study made of its characteristics. The molecular weight of the purified enzyme was estimated to be 75,000 by gel filtration and 85,000 by SDS-PAGE, suggesting that it consists of monomeric protein. Its isoelectric point was 5.7. The purified enzyme was colourless, and contained one atom of iron per molecule. It exhibited a broad pH activity profile with maximum activity at pH 10.0, and was stable between pH 6.0 and 10.0. The optimum temperature for enzyme activity was 40°C, with stability up to 45°C. The enzyme activity was inhibited strongly by Fe2+, Hg2+ and Sn2+, and weakly by Cu2+, EDTA, thiourea and IAA. The enzyme exhibited activity toward several secondary alcohols, suggesting that it was a secondary alcohol oxidase. In particular, the enzyme exhibited strong activity towards the larger secondary alcohols such as 2-octanol and 4-decanol, and relatively strong activity towards cyclohexanol and benzyl alcohol.     

Purification and some properties of uridine diphosphate N-acetylglucosamine pyrophosphorylase from Neurospora crassa.

Uridine diphosphate N-acetylglucosamine pyrophosphorylase (EC. 2.7.7.23) of Neurospora crassa has been purified approximately 210-fold with dithiothreitol as the stabilizing agent by use of chromatographic techniques. The enzyme preparation appeared to be homogeneous when subjected to electrophoresis. The molecular weight was estimated as approximately 37 000 by gel filtration. The enzyme had an isoelectric point around pH 4.4. Maximum activity of the enzyme was observed at pH 7.5. The enzyme required Mg2+, which may be replaced by other divalent cations such as Mn2+ and Co2+ for lesser degrees of effectiveness. The enzyme was strictly specific for UDP-N-acetylglucosamine as the substrate. The estimated values of Km were 2.2 mM for UDP-N-acetylglucosamine and 5.4 mM for inorganic pyrophosphate. The enzyme activity was highly stimulated by the addition of dithiothreitol or dithioerythritol but was lost by sulfhydryl inhibitory reagents.     

β-葡聚糖酶的分离纯化和特性研究

对里氏木霉所产β-葡聚糖酶粗酶液通过饱和硫酸铵沉淀、Sephadex G-100 柱层析和DEAE-Sephadex A-50 柱层析进行纯化,比活提高14.60倍,活力回收6.62%。酶特性研究表明,最适温度和pH分别为60℃和5.0,在pH低于5.0时酶较稳定,酶的热稳定性在60℃以下。 Cu～2+、 Mn～2+ 、Mg～2+ 、Fe～3+ 和K+对酶有抑制作用, Zn～2+、Ca～2+、 Co2+和 Fe～2+ 有激活作用。     

短小芽孢杆菌A-30耐碱性木聚糖酶的纯化及性质研究

木聚糖广泛存在于自然界 ,通常占高等植物干重的 1 5%～ 30 % ,由木糖经β- 1 ,4-糖苷键连接起来形成主链 ,并由阿拉伯糖、乙酰基甘露糖、葡萄糖醛酸等复杂侧链共同组成 .在众多可降解木聚糖的酶中 ,β-内切木聚糖酶 ( E.C3.2 .1 .8,β- 1 ,4- xylanxylanohydrolase)起主要作用 .在纺织、制浆造纸、饲料及食品等工业中具有潜在的应用价值 .近年来 ,欧美等国已将其应用在造纸制浆工业 ,降低了漂白时氯的用量 ,改善了纸张性能 ,并且减少了环境污染 .对木聚糖酶的研究成为生物技术领域研究的热点之一 .国内外对来源于不同菌种的木聚糖酶的分离…     

粗毛栓菌诱变菌株SAH-12漆酶的分离纯化及酶学性质研究

粗毛栓菌Trametes gallica诱变菌株SAH-12是通过紫外诱变选育所得的漆酶高产菌株,Active-PAGE分析表明SAH-12在高氮低碳无机盐培养液(LM3)中至少分泌3种漆酶同工酶(Lac1、Lac2、Lac3)。采用硫酸铵盐析、透析和Sephadex-G75分子筛层析从其培养液中分离纯化得到电泳纯的Lac1,纯化倍数6.54,酶活性回收59.7%。Lac1经SDS-PAGE验证为一条带,其表观分子量为61.5kDa。Lac1为一种糖蛋白,含糖量11.6%,等电点pI4.40,催化氧化底物ABTS的最适反应温度为60℃,最适pH为2.6,Km值为25μmol/L。Lac1在40℃(pH4.0)以下和pH1.5～5.0(28℃)范围内稳定。金属离子Fe2+、Ag+、Hg2+和Cr3+与抑制剂DTT、SDS、EDTA和DMSO对Lac1有抑制作用,其中Fe2+和DTT完全抑制酶活,而Cu2+对酶有明显激活作用,Mn2+、Zn2+对酶活影响不大。Lac1不仅可使一些合成染料明显脱色,而且对苹果汁多酚祛除也有较好效果。40℃用该酶(1U/mL)处理苹果汁5h,其多酚含量可降低40%。     

米氏凯伦藻溶血毒素的溶血反应特征

探讨了温度、pH值、二价阳离子等对米氏凯伦藻(Karenia mikimotoi Hasen)溶血毒素溶血活性的影响,分析了米氏凯伦藻溶血毒素的溶血反应特征.结果表明,实验室培养米氏凯伦藻的溶血活性约为64.69±6.43 HU L-1,单个藻细胞的溶血活性为6.17±0.61×10-6 HU;在实验温度(0～37℃)下,溶血活性随温度的增加而增加;pH6.0时的溶血活性最高;Cu2+、Mg2+、Mn2+、Ca2+、Co2+、Zn2+和Hg2+等对米氏凯伦藻的溶血活性的影响不同.离子浓度为5 mmol/L时,Hg2+的抑制作用最强.高浓度Hg2+对红细胞的集合效应不但阻止了Hg2+进入血细胞诱导的溶血作用,而且阻止了毒素对细胞膜的破坏,但这种抑制作用可被EDTA消除.     

β-葡聚糖酶的分离纯化和特性研究*

对里氏木霉所产β-葡聚糖酶粗酶液通过饱和硫酸铵沉淀、Sephadex G-100 柱层析和DEAE-Sephadex A-50 柱层析进行纯化,比活提高14.60倍,活力回收6.62%。酶特性研究表明,最适温度和pH分别为60℃和5.0,在pH低于5.0时酶较稳定,酶的热稳定性在60℃以下。 Cu～2+、 Mn～2+ 、Mg～2+ 、Fe～3+ 和K+对酶有抑制作用, Zn～2+、Ca～2+、 Co2+和 Fe～2+ 有激活作用。     

Studies on nitrate reductase of Clostridium perfringens. Purification, some properties, and effect of tungstate on its formation.

Nitrate reductase (NaR) linked to reduced methyl viologen from Clostridium perfringens was purified by ammonium sulfate precipitation. DEAE-cellulose chromatography, disc electrophoresis on polyacrylamide gel, and triple DEAE-Sephadex chromatography. The specific activity was increased 1,200-fold with a yield of 9%. The purified preparation was nearly homogeneous in disc electrophoresis. It was brown, and its spectrum showed a slight shoulder near 420 nm as well as a peak at 280 nm. The molecular weight was found to be 90,000 based on s020,w (5.8S) and 80,000 by Sephadex G-100 gel filtration. In SDS-polyacrylamide electrophoresis, it showed only a single band with a molecular weight of 90,000; it had no subunit structure. The isoelectric point was pH 5.5, and the optimum pH was 9. Mn2+, Fe2+, Mg2+, and Ca2+ stimulated the activity. Km for nitrate was 0.10 mM, and nitrate was stoichiometrically reduced to nitrite in the presence of 2 mM Mn2+. Ferredoxin fraction obtained from extracts of the bacterium was utilizable as an electron donor at pH 8. Cyanide and azide inhibited the enzyme. The formation of NaR was induced by nitrate and inhibited by 0.5 mM tungstate, but recovered in the presence of 0.1 mM molybdate; NaR of C. perfringens appears to be a molybdo-iron-sulfur protein.