黄鳝碱性磷酸酶的分离纯化及其部分性质研究

经Tris-HCl缓冲液(pH8.6)抽提,正丁醇处理,30%-75%硫酸铵分级沉淀分离,DEAE-Sepharose离子交换柱层析,Sephacryl S-200凝胶过滤纯化,从黄鳝内脏组织中分离纯化出电泳纯的碱性磷酸酶。该酶提纯倍数为564倍,比活力达到3015U/mg。酶学性质和动力学性质研究表明,该酶催化磷酸苯二钠的水解反应,最适pH值为10.2,pH小于7和大于12均不稳定;最适温度为40℃,温度高于50℃不稳定;米氏常数Km值为1.17mmo1/L。金属离子对该酶的催化活力有不同的影响,K+对该酶活力无影响,Mg2+对该酶有激活作用,Zn2+对该酶有抑制作用。       

鲫鱼酸性磷酸酶酶学特性及不同效应物对酶活力的影响

经NaAc-HAc缓冲液(pH5.0)抽提,正丁醇处理,硫酸铵分级沉淀,DEAE-32离子交换层析,SephadexG-150凝胶过滤纯化,从鲫鱼内脏中分离纯化出电泳纯的酸性磷酸酶。该酶提纯倍数为30.82,比活力195.06U/mg。研究表明,该酶催化对硝基苯磷酸二钠水解反应,最适pH4.8,pH小于4和大于7时不稳定;最适温度45℃,温度高于50℃不稳定;米氏常数为0.23mmol/L,利用SDS-PAGE测定酶亚基分子量为33.3kD。化学修饰剂SUAN、PMSF、DTT、NBS对该酶活力影响不大,BrAc和IAc有明显抑制作用。金属离子对该酶催化活力有不同影响,Na+、K+、Ni2+、Co2+影响不显著,Mg2+、Ca2+、Ba2+、Mn2+有激活作用,Ag+、Cu2+、Pb2+、Cd2+有抑制作用,其中Mg2+、Ca2+、Pb2+、Cd2+对鲫鱼酸性磷酸酶荧光光谱的影响表明金属离子对酶活力的影响与酶构象改变有关。       

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

短小芽孢杆菌碱性蛋白酶BP的纯化和性质

短小芽孢杆菌产生的碱性蛋白酶BP经CM—Sephadex-C-50和Sephadex-G75两个柱层析,得到了聚丙烯酰胺凝胶电泳纯的酶组分,比活力从1307μ/mg提高到5538μ/mg.活力回收为21%,酶水解酪蛋白的最适反应温度为50℃,最适pH为9.5,Mn^2+、Ca^2+对酶有激活作用,Hg2+、Ag^+对酶有抑制作用.酶的热稳定性不高,但在Ca^2+保护下,热稳定性明显提高.酶的最适作用底物为酪蛋白,对血红蛋白、蛇毒蛋白、牛血清蛋白、卵蛋白、核糖核酸酶也有水解作用.对酪蛋白的Km为0.62%,V_max为50μg/min.DFP可完全抑制酶活性,PMSF和NBS也严重抑制酶活力,PCMB、_o-PTH和EDTA几乎不抑制酶活力.纯酶的分子量为25000Dal.该酶蛋白含有17种氨基酸,其中甘氨酸(Gly)和丙氨酸(Ala)为主要氨基酸.     

金属离子和脲对白蜡虫碱性磷酸酶的影响

各种金属离子及脲对白蜡虫Ericerus pela (Chavannes)碱性磷酸酶的活性有不同的影响。从白蜡虫雌成虫中分离纯化得到碱性磷酸酶,加入各种不同浓度的金属离子及脲测定酶的活力。一价金属离子Na⁺、K⁺、Li⁺对酶活力没有影响。碱土金属离子Ca²⁺、Mg²⁺、Ba²⁺对酶有激活作用,激活作用的大小顺序依次为Ca²⁺、Ba²⁺、Mg²⁺。第一过渡金属离子中,Mn²⁺、Co²⁺、Ni²⁺对酶有激活作用,而Zn²⁺、Cu²⁺有抑制作用。重金属离子Cd²⁺、Pb²⁺对酶有抑制作用。Ca²⁺激活作用表现为非竞争性激活效应。Cu²⁺抑制作用表现为非竞争性抑制效应。脲对碱性磷酸酶有变性失活作用,按脲浓度可分为低于3 mol/L和高于3 mol/L两种类型。低浓度的脲对白蜡虫碱性磷酸酶的活性抑制的动力学表现为混合型效应。     

鸡源和猪源乳杆菌胆盐水解酶的表达及酶学性质比较

【目的】随着抗生素生长促进剂(AGPs)在动物饲料中逐步禁止使用,AGPs替代物的研究成为热点。由于胆盐水解酶(BSH)在脂类代谢中的关键作用,成为AGPs替代物研究的一个重要方向。在原核表达和纯化的基础上鉴定鸡源和猪源乳杆菌BSH在酶学性质方面的差异性。【方法】分别对鸡源胆盐水解酶(BSHc)和猪源胆盐水解酶(BSHp)基因进行原核表达和蛋白纯化,通过测定对6种甘氨结合胆盐和牛磺结合胆盐的水解效率获得两种酶的酶学动力学性质,进而测定了温度、pH和金属离子对酶活力的影响。【结果】BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc对甘氨结合胆盐的水解效率较BSHp稍高;BSHc和BSHp的最适酶解温度分别为45°C和42°C;BSHc和BSHp的最适反应pH分别为6.0和5.4;含有Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)的金属盐对BSHc和BSHp的酶活力均具有不同程度的抑制作用,特别是Cu~(2+)和Fe~(3+)抑制作用比较强;含有Na~+、K~+、Mg~(2+)和Ca2+的金属盐对BSHc和BSHp酶活力的抑制作用相对较弱或无抑制作用,但KIO3对BSHc和BSHp酶活力具有强抑制作用,KI和CaCl_2对BSHp酶活力也具有较强的抑制作用。【结论】原核表达和纯化的BSHc和BSHp对甘氨结合胆盐的水解效率高于牛磺结合胆盐,BSHc的最适酶解温度和pH稍高于BSHp,Cu~(2+)、Fe~(3+)、Mn~(2+)和Zn~(2+)等金属离子对BSHc和BSHp酶活力具有明显抑制作用,试验结果为鉴定BSH抑制物进而研制AGPs替代物奠定了基础。     

额尔齐斯河野生丁(鐬)蛋白酶、淀粉酶活性的研究

目的:研究温度、pH、金属离子对新疆额尔齐斯河野生丁(鐬)消化道蛋白酶、淀粉酶活性的影响.方法:酶学和生化法.结果:丁(鐬)蛋白酶活性,后肠＞前肠＞中肠＞肝胰脏;淀粉酶活性,后肠＞中肠＞前肠＞肝胰脏.肠道、肝胰脏蛋白酶的最适温度分别为35℃、40℃,淀粉酶的最适温度均为40℃.消化道各部位蛋白酶、淀粉酶的体外最适pH为7.5.在试验的离子浓度范围,高浓度Ag 、Cu2 抑制酶活力;高浓度Fe3 、Mg2 、Ca2 促进酶活力;Pb 、Ba2 对消化道酶活力影响依浓度和部位而变.结论:消化酶最适温度均高于所处环境温度,最适pH值变化范围偏碱性;不同浓度金属离子对丁(鐬)消化道各部位蛋白酶、淀粉酶活力高低表现出不同的变化趋势.     

烟草头孢霉汞还原酶特性的研究

经检测,抗汞真菌烟草头孢霉(Cephalosporium tabacinum)F2菌株中具有汞五原酶活性。该酶是一种胞内酶,需NADH作为电子供体,催化Hg2+还原成为元素汞(Hgo)。该酶促反应还需硫氢基化合物,反应最适温度为30℃,最适pH范围为7．0-8.0。在25—30℃保温40分钟或在pH7．0保温2小时,酶活力均是稳定的。金属离子Ag+,Co2+,Cu2+,Zn2+,Mn2+和Ni2+在浓变为0．2-1．0mmol／L的范围内,对酶活力有不同程度的抑制作用,一定浓度的乙酸苯汞和铁氰化钾对酶活力也有部分抑制作用。     

金属离子对蜡蚧菌几丁质酶活力的影响

以蜡蚧菌(Ll)发酵液为材料,经分离纯化获得Ll几丁质酶(EC3.2.1.14)制剂.研究了金属离子对Ll几丁质酶活力的影响.结果表明,K+、Mg2+、Zn2+、Ca2+和Fe3+对几丁质酶活性有明显的促进作用,而Na+和Cu2+完全抑制几丁质酶的活性;Mn2+在低浓度时对酶有激活作用,随着浓度的升高表现出抑制作用;Fe2+和Ba2+的浓度低于0.5 mmol/L时对酶起抑制作用,而高于该浓度时则对酶有激活作用.     

重组磷脂酰丝氨酸合成酶的分离纯化及酶学性质研究

利对重组枯草芽孢杆菌(pBES-pss)表达的磷脂酰丝氨酸合成酶进行分离纯化及酶学性质研究.pBES-pss发酵后的粗酶液经硫酸铵盐析、中空纤维膜除盐浓缩、SP-Sepharose HP离子交换层析和Sephadex G-75凝胶层析,基本获得电泳纯的重组磷脂酰丝氨酸合成酶,比活力可达13.62 U/mg,分子量约为53 kD.酶学性质研究表明,该酶催化卵磷脂水解反应的最适pH8.0,最适温度为35℃.稳定性研究表明:该酶在pH 6.5～9.5 区间和低于45℃温度下稳定.表面活性剂及金属离子对该酶水解活性的影响结果表明,SDS、Tween20、Tween80对该酶有抑制作用,Triton X-100对该酶有增强作用;Mg2+、Zn2+、K+对该酶有抑制作用,Ca2+、Mn2+和EDTA对该酶有增强作用.     

蜗牛酶中一种β-葡萄糖苷酶的纯化及酶学性质的研究

通过DEAE-Sepharose离子交换层析和Sephadex G-100凝胶过滤层析的联用从中华白玉蜗牛消化酶中分离出1种具有人参皂苷Rb_1水解活性的β-葡萄糖苷酶.纯化后该酶在SDS-PAGE上呈单一蛋白质条带.反应最适pH为5.6,最适温度是80 ℃.pH稳定范围很广,在pH为4.0～11.0的溶液中和温度60 ℃以下保持长时间稳定状态,是一个耐碱和中等耐热的糖苷酶.Na~+、K~+、Li~+、Ca~(2+)、Mg~(2+)、EDTA、DTT和SDS不影响该酶活性,而Cu~(2+)、Ag~+和Fe~(3+)对该酶则具有明显的抑制作用.pNPG为底物的动力学参数Km和Vmax分别为0.182 mmol/L和0.189 μmol/(min·mg).     

Purification and characterization of murine protoporphyrinogen oxidase

The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4), has been purified to apparent homogeneity from mouse liver mitochondria. The purification involves solubilization from mitochondrial membranes with sodium cholate followed by ammonium sulfate fractionation and gel filtration on a Sepharose CL-6B column. The eluate is adjusted to 0.67 M (NH4)2SO4 and loaded onto a phenyl-Sepharose column. After salt washes, the enzyme is eluted with 0.5% sodium cholate and 0.5% Brij 35. The final step is high-pressure ion-exchange chromatography on a DEAE-5PW column. The purified protein has a molecular weight of approximately 65,000 by gel filtration chromatography on Sepharose CL-6B in the presence of 0.5% sodium cholate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows a single band corresponding to a molecular weight of 65,000. The absorption spectrum of the purified enzyme shows no evidence of a chromophoric cofactor. Purified protoporphyrinogen oxidase has a Km for protoporphyrinogen IX of 5.6 microM with a Vmax of 2300 nmol mg-1 h-1. It utilizes meso- and hematoporphyrinogen at about 10% the level of protoporphyrinogen. The pH optimum is broad with a maximum at 7.1. There is no stimulation or inhibition by any tested divalent cations, and sulfhydryl reagents have no inhibitory effect on the purified enzyme.     

Purification and properties of arylsulphatase A from chicken brain

1. Chicken brain arylsulphatase A was purified 2000-fold, with overall recovery 14%, by using ammonium sulphate fractionation, ethanol precipitation, Sephadex G-200 gel filtration and DEAE-Sephadex column chromatography. 2. The purified preparation was free from beta-glucuronidase, beta-galactosidase, acid phosphatase, inorganic pyrophosphatase and adenosine 3'-phosphate 5'-sulphatophosphate sulphohydrolase activities. 3. Polyacrylamide-gel electrophoresis indicated that the purified preparation was not homogeneous. 4. Chicken brain arylsulphatase was markedly inhibited by carbonyl reagents in the presence of traces of Cu(2+) in the system. Other metal ions such as Fe(2+) and Zn(2+), were inactive. 5. Ascorbic acid alone had no effect on enzyme activity but enhances the inhibition by Cu(2+). 6. Chicken brain arylsulphatase A resembled arylsulphatase A of other animal species in its kinetic properties such as K(m) value, anomalous time-activity relationship and the inhibitory effect of phosphate, sulphite and sulphate ions. However, its electrophoretic mobility, behaviour under zinc acetate fractionation and stimulation by Ag(+) were similar to arylsulphatase B of other animal species. Thus, this enzyme did not correspond to either arylsulphatase A or arylsulphatase B but properties of both. 7. The purified enzyme preparation can degrade cerebroside 3-sulphate.     

Purification and Characterization of a Maltotetraose-Forming Alkaline (alpha)-Amylase from an Alkalophilic Bacillus Strain, GM8901

An alkalophilic bacterium, Bacillus sp. strain GM8901, grown at pH 10.5 and 50(deg)C, produced five alkaline amylases in culture broth. At an early stage of the bacterial growth, amylase I (Amyl I) was produced initially and then, as cultivation progressed, four alkaline amylases, Amyl II, Amyl III, Amyl IV, and Amyl V, were produced from proteolytic degradation of Amyl I. A serine protease present in the culture medium was believed to be involved in Amyl I degradation. We purified Amyl I from the culture supernatant by ammonium sulfate precipitation, heparin-Sepharose CL-6B column chromatography, phenyl-Toyopearl column chromatography, and Mono Q HR5/5 high-performance liquid chromatography. The molecular weight of Amyl I was estimated to be about 97,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amyl I had an extremely high optimal pH of 11.0 to 12.0 and was stable in a broad pH range of 6.0 to 13.0. Amyl I had an optimal temperature of 60(deg)C and was stable up to 50(deg)C. Thermostability was increased in the presence of Ca(sup2+) and soluble starch. The enzyme required metal ions such as Ca(sup2+), Mg(sup2+), Cu(sup2+), Co(sup2+), Ag(sup+), Zn(sup2+), and Fe(sup2+) for its enzyme activity and was inhibited by 1 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. According to the mode of action of Amyl I on starch, Amyl I was classified as an (alpha)- and exo-amylase. Amyl I produced maltotetraose predominantly from starch via intermediates such as maltohexaose and maltopentaose.     

产碱性磷酸酶乳杆菌的筛选鉴定、酶的纯化及特性

【背景】碱性磷酸酶(alkaline phosphatase,ALP)是生物体内参与磷酸代谢的调控酶,不同物种的ALP性质与其生理功能有关,提纯后的ALP常用作工具酶,广泛应用于基因工程中,但目前关于乳酸菌中ALP的相关研究甚少。【目的】筛选出一株产ALP且具有潜在益生作用的乳杆菌,对该酶进行分离纯化,并对其性质进行探究,为今后益生菌的开发利用和ALP的工业化生产提供新的微生物资源。【方法】采集蒙古国4个地区的酸马奶样品,通过显色反应初筛和酶活检测复筛对产酶菌株进行筛选,经形态学观察、生理生化鉴定及16S rRNA基因序列同源性比较分析进行菌种鉴定。采用超声破碎法提取ALP,经硫酸铵沉淀、DEAE-52离子交换层析、Sephadex G-200凝胶过滤层析纯化该酶,SDS-PAGE电泳法检测其纯度。【结果】从78株乳酸菌中分离筛选出一株产ALP酶活性最高的乳杆菌(编号为Z23),16S rRNA基因序列长度为1 473 bp,鉴定结果表明为鼠李糖乳杆菌。纯化后的酶比活力为180.27 U/mg,纯化倍数为48.37,酶活回收率为17.05%,该酶亚基相对分子质量为46.7 kD。菌株所产ALP的最适温度为37℃,4℃时酶活最为稳定;最适pH为9.5,在pH 9.0-10.0之间,酶活稳定性可达90%以上;Mg2+和K+对ALP有明显激活作用,Ba2+和Cu2+在低浓度时对ALP有激活作用,高浓度时有抑制作用,Ca~(2+)、Zn~(2+)和EDTA对ALP有强烈的抑制作用。以不同浓度的p-NPP为底物,测得酶的Km值为3.42 mmol/L,Vmax值为1.24 mmol/(L·min)。【结论】本研究对蒙古国地区酸马奶中的益生菌资源有了更为明确的认知,为今后碱性磷酸酶产生菌的筛选和酶的应用开辟了新途径。     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

斜链拟青霉多糖活性组分的分离及其清除自由基活性

对斜链拟青霉胞外多糖进行体外清除自由基活性研究。利用醇沉法从斜链拟青霉发酵液中醇沉获得斜链拟青霉胞外多糖,经DEAE-纤维素-52柱层析后得到1个主峰P3和2个小峰P1与P2,对主峰P3进行Sephadex G-100柱层析,表明P3为纯化物; 应用DPPH-酶标法和化学发光法分别对P3进行清除DPPH·、·OH和O-2·能力测试。结果表明：P3对DPPH·、·OH和O-2·均具有明显的清除能力,且与浓度呈量效关系,IC50分别为0.083、0.121和0.214 mg/mL。     

酸性α-淀粉酶的分离纯化与酶学性质研究

纯化了枯草芽胞杆菌xm-1菌株酸性α-淀粉酶,并对其酶学性质进行了研究。通过硫酸铵沉淀和Sephadex G-75凝胶层析将酸性α-淀粉酶粗酶液纯化了32.5倍,活力回收率为10.0%。酶性质测定结果表明,该酸性α-淀粉酶分子量约为60kD,最适反应温度为45℃、最适作用pH5.0,该酶在pH3.4-6.0下稳定,高温耐受性差。Cu2+、Zn2+、EDTA对酶有不同程度的抑制作用,Ca2+和Mn2+对酶具有较强的激活作用。     

Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli

The SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6x His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54kDa. The optimal temperature and pH of the purified rSAP6 were 40 degrees C and 3.4, respectively. The enzyme was stable below 45 degrees C and between pH 2.6 and 5.0. The results show that Mn(2+) had an activating effect on the enzyme, while Cu(2+), Mg(2+), Zn(2+) and Ag(+) acted as inhibitors of the enzyme. However, Ca(2+) had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.     

Biochemical and immunochemical characterisation of human diadenosine triphosphatase provides evidence for its identification with the tumour suppressor Fhit protein

We describe here the purification and characterisation of the human enzyme diadenosine triphosphatase isolated from human platelets and leukocytes, offering biochemical and immunochemical evidence to identify this enzyme with the novel tumour suppressor Fhit protein, a homodimer composed of approximately 17 kDa monomers. It catalyses the Mg(2+)-dependent hydrolysis of diadenosine triphosphate, Ap(3)A, to AMP+ADP. The fluorogenic substrate di-ethenoadenosine triphosphate, epsilon-(Ap(3)A), and Fhit antibodies were used for enzymatic and immunochemical characterisations, respectively. Human Ap(3)Aase presents a native molecular mass of approximately 32 kDa and no significant differences were found in K(m) values (2 microM), activating effects by Mg(2+), Ca(2+), and Mn(2+), optimum pH (7.0-7.2) or inhibition by Zn(2+) and diethyl pyrocarbonate between the human enzyme and the recombinant Fhit protein. Suramin is a very potent competitive inhibitor of both human Ap(3)Aase and Fhit protein with K(i) values in the range 20-30 nM. Both human and rat Ap(3)Aase activity co-purifies with Fhit immunoreactivity under gel filtration, ion-exchange and affinity chromatography. Homogeneous human Ap(3)Aase preparations analysed by SDS-PAGE and Western blot analysis with Fhit antibodies elicit immunochemical responses corresponding to a approximately 17 kDa polypeptide, indicating a dimeric structure for the enzyme Ap(3)Aase. The strong inhibition of Fhit enzyme by the drug suramin, supports the need to investigate the therapeutic potential of Fhit-Ap(3)Aase mediated by its interaction with suramin or related drugs.