地衣芽孢杆菌ws-6产纤维素酶条件优化

目的：通过优化该菌产酶条件,以期在饲料中的进一步应用建立基础。方法：纤维素酶活性用还原糖法测定,产酶条件采用单因素筛选与正交优化方法相结合。结果：培养基初始pH6.5、培养温度37℃、接种量2%2、50 mL三角瓶装液量30 mL、葡萄糖1.5%、羧甲基纤维素钠0.5%、酵母膏1.5%、KH2PO40.05%、NaCl 0.5%,培养48h后酶活性达到最高3.45U/mL;金属离子K＋、Mn2＋、Mg2＋、Zn2＋、Co2＋、Na＋对产酶有激活作用,Cu2＋则抑制酶活性。结论：酶活性比出发菌提高约1.4倍,对饲料制作中提高纤维降解具有一定的实用价值。     

外源NO对缺镁胁迫下玉米幼苗生长和离子平衡的影响

研究了在缺镁胁迫下,外源NO对缺镁玉米幼苗生长、根系活力和离子含量的影响。结果表明,缺镁胁迫使玉米幼苗株高、根长和干鲜重下降,根系活力降低,N元素在地上部和根部分配失调,新叶和老叶中Mg2＋、Cu2＋、Fe3＋、Mn2＋等离子含量下降,Ca2＋、K＋、Zn2＋等离子含量上升。根中Mg2＋离子含量下降,Ca2＋、K＋、Zn2＋、Cu2＋、Fe3＋、Mn2＋等离子含量上升。用100μmol·L-1一氧化氮供体硝普钠（SNP）处理后,玉米幼苗株高、根长、干重和鲜重均提高,根系活力增强,改善了N代谢,新叶中Ca2＋、K＋和Zn2＋等离子含量下降,Mg2＋、Cu2＋、Fe3＋和Mn2＋等离子含量提高,老叶中Mg2＋、Ca2＋、K＋和Zn2＋等离子含量下降,Cu2＋、Fe3＋和Mn2＋等离子含量提高,根中Mg2＋、Ca2＋、K＋、Cu2＋、Zn2＋、Fe3＋和Mn2＋离子含量均下降。实验结果表明,NO保护玉米幼苗免受缺镁胁迫的影响。     

重组N-乙酰鸟氨酸脱乙酰基酶的表达、纯化和复性研究

报道重组N-乙酰鸟氨酸脱乙酰基酶(NAOase)的研究进展。重组NAOase由大肠杆菌argE基因编码,在重组菌BL21(DE3)-pET22b-argE中的表达量为32.5%,大多以无活性的包涵体存在。低温诱导可增大有活性的可溶表达部分的比例。可溶性NAOase经Ni-NTA凝胶亲和纯化后得到SDS-PAGE电泳纯的酶,比酶活为1193.2u/mg蛋白。诱导条件影响整菌蛋白的成分及比例。37℃诱导生成的包涵体经尿素梯度洗涤后纯度较22℃高。低的蛋白浓度和合适的氧化还原体系是影响复性的关键因素。稀释法和透析法皆可使包涵体部分复性。在合适的条件下以稀释法复性时,约有17.78%包涵体可顺利复活。包涵体经尿素洗涤、溶解、Ni-NTA凝胶柱亲和纯化后,获得了高纯度的NAOase。     

极端耐热纤维素酶Cel74的表达、纯化与特性

从海栖热袍菌中克隆出编码热稳定性的纤维素酶基因,以热激载体pHsh为表达质粒,构建重组质粒phsh—Ceff4,并转化至大肠杆菌中进行表达。基因表达产物通过热处理和离子交换层析,重组酶纯度达电泳纯。对纯化的重组酶酶学性质研究表明,最适反应温度85℃,最适反应pH4．6,pH4．5—6．0之间酶的相对酶活在80％以上。Co^2＋对酶活性有促进作用,Ca^2＋、Mg^2＋、Zn^2＋不影响酶活性,而Cu^2＋、Ni^2＋、Mn^2＋对酶活性有抑制作用。     

西南岩溶地区黄荆叶片碳酸酐酶的稳定性

研究表明西南岩溶地区几种植物叶片中含有明显活性的碳酸酐酶(carbonic anhydrase, CA),从黄荆(Vitex negundo)叶片提取碳酸酐酶,该酶具有良好的热稳定性。阴离子及岩溶环境主要金属离子对碳酸酐酶活性有一定的影响,Ca2+、Mg2+低于10 mmol.L-1,Mn2+ 、Zn2+低于0.01 mmol.L-1,Co2+、Cu2+低于0.001 mmol.L-1,NO3-、Cl-、Br-、I-、NO2-低于0.01 mmol.L-1时,酶活性较稳定。0.001～0.01 mmol.L-1的Ca2+、Mg2+对该酶有激活作用。研究结果表明, 该酶比较适应岩溶土壤水体的离子环境从而保持酶活性的相对稳定。     

西南岩溶地区黄荆叶片碳酸酐酶的稳定性

研究表明西南岩溶地区几种植物叶片中含有明显活性的碳酸酐酶(carbonic anhydrase,CA),从黄荆(Vitex negundo)叶片提取碳酸酐酶,该酶具有良好的热稳定性.阴离子及岩溶环境主要金属离子对碳酸酐酶活性有一定的影响,Ca2 、Mg2 低于10 mmol·L-1,Mn2 、Zn2 低于0.01 mmol·L-1,Co2 、Cu2 低于0.001 mmol·L-1,NO3-、C1-、Br-、I-、NO2-低于0.01 mmol·L-1时,酶活性较稳定.0.001～0.01 mmol·L-1的Ca2 、Mg2 对该酶有激活作用.研究结果表明,该酶比较适应岩溶土壤水体的离子环境从而保持酶活性的相对稳定.     

一株假单胞菌(Pseudomonas sp．)产生的胞外脂酶

假单胞菌(Psendomonas sp．)生长在一定的培养条件中能产生胞外脂酶。 最适碳源为1．0％淀粉,氮源为1．0％蛋白胨。一些植物油,如橄榄油、糠油、菜油等能诱导脂酶的大量产生,诱导脂酶产生的橄榄油最适浓度为0．5％。无机离子在菌培养过程中对脂酶产率影响很大,K+、Na+、Mg2+、Ca2+等对脂酶产生有促进作用,而Mn2+、Ba2+、Zn2+、Fe3+、Co2+、Cu2+件等则抑制脂酶产生。非离子表面活性剂(tween、span及糖脂)能刺激胞外脂酶的产生。     

十和田/丽粳2号回交重组自交系糙米Zn含量与其他元素和农艺性状间相关性

用ICP-AES法测定了十和田/丽粳2号/十和田BC4F5回交重组自交系264个株系糙米Zn含量及其在株型间的遗传变异,分析了糙米Zn含量与16种元素(K、P、S、Mg、Ca、Mo、Ni、Fe、Cr、Na、Al、Cu、Sn、B、Mn和Sr)、9个农艺性状(结实率、实粒数、秕粒数、穗长、有效穗、花药长度、花药宽度、穗茎节间长和倒2叶长)、MDA(丙二醛)含量、3种酶(POD、SOD、CAT)活性间相关性,旨在揭示糙米Zn与其他元素、稻米产量、稻耐冷性等关联性。结果表明:回交重组自交群体糙米Zn含量为正态分布,有超亲优势株系;糙米Zn含量与8种元素(K、S、P、Mg、Ca、Fe、B、Mn)含量呈极显著相关,与5种元素(S、Mg、Ni、Cr、Al)含量呈显著偏相关;初步认为糙米Zn含量与稻米产量及耐冷性为负相关,与酶活性相关性不显著。     

海洋菌W11产中温淀粉酶的酶学特性

本研究从威海文登海域筛选获得一株产淀粉酶海洋菌W11,初步鉴定为弧菌,并探讨pH、温度、无机离子对淀粉酶活性和稳定性的影响及该酶底物浓度效应和Km值。结果表明:在pH7.5左右酶活性最高,pH在4.0～7.5范围内体现较强的稳定性。最适酶解温度为55℃,酶液在60℃以下有较好的热稳定性;Ba2+、Mn2+对淀粉酶有激活作用,而Cu2+、Mg2+、Zn2+则抑制淀粉酶活性,表观Km值为0.973mg/mL。海洋菌W11所产的中温淀粉酶保存温度范围较广、适应pH作用的范围广及稳定性较强,将有着广泛的应用潜力。     

斑点叉尾鮰源嗜麦芽寡养单胞菌胞外蛋白酶的特性研究

为了研究嗜麦芽寡养单胞菌胞外蛋白酶的酶学特性,本实验检测了不同的修饰剂、温度、pH、EDTA及Ba2+、Co2+、Cd2+、Ca2+、Mn2+、Hg2+、Cu2+及Mg2+等金属离子对酶活的影响,结果表明Ch-T、NBS和2-ME能显著抑制酶活性,而N-AI、PMSF和PCMB对酶活的影响不大,说明蛋氨酸残基、色氨酸残基和二硫键是酶活性的必需基团,而酪氨酸残基、丝氨酸残基和巯基与酶活性无直接关系。酶的最适温度为20℃,在pH为9.0时酶活最高,EDTA能显著影响酶活性,Ca2+、Hg2+、Cu2+能显著降低酶活性,而Co2+能使酶活增强,证实该酶为一种金属蛋白酶。同时检测了该酶的致病性,结果表明,该酶对小鼠及斑点叉尾鮰具有明显的致死作用,其LD50分别为4.33µg/g体重和3.49µg/g体重。     

Activation of sphingomyelinase from Bacillus cereus by Zn2+ hitherto accepted as a strong inhibitor

Sphingomyelinase (SMase) from Bacillus cereus has been known to be activated by Mg2+, Mn2+, and Co2+, but strongly inhibited by Zn2+. In the present study, we investigated the effects of several kinds of metal ions on the catalytic activity of B. cereus SMase, and found that the activity was inhibited by Zn2+ at its higher concentrations or at higher pH values, but unexpectedly activated at lower Zn2+ concentrations or at lower pH values. This result indicates that SMase possesses at least two different binding sites for Zn2+ and that the Zn2+ binding to the high-affinity site can activate the enzyme, whereas the Zn2+ binding to the low-affinity site can inactivate it. We also found that the binding of substrate to the enzyme was independent of the Zn2+ binding to the high-affinity site, but was competitively inhibited by the Zn2+ binding to the low-affinity site. The binding affinity of the metal ions to the site for activating the enzyme was determined to be in the rank-order of Mg2+ = Co2+ < Mn2+ < Zn2+. It was also demonstrated that these four metal ions competed with each other for the same binding site on the enzyme molecule.     

链霉菌壳聚糖酶的纯化及其酶学性质

分离纯化从烟台近海土壤筛选的链霉菌来源壳聚糖酶,并对其酶学性质进行研究。通过(NH4)2SO4分级沉淀分离得粗酶,透析后经Sephadex G-100柱纯化,得到2种壳聚糖酶(ChA和ChB)。SDS-聚丙烯酰胺凝胶电泳及Sephadex G-75凝胶过滤确定ChA的相对分子质量,研究ChA的最适底物水解条件、热稳定性、水解动力学及金属离子对酶活性影响。结果表明:ChA为单亚基蛋白,相对分子质量为4.16×104,在220和280 nm处呈现两个紫外吸收峰,催化水解壳聚糖的最适pH为5.0～5.5,最适温度为55℃。热稳定性实验表明:30℃温育1 h后酶活为初始酶活的33.3%,40℃温育1 h后酶活为初始酶活的22.2%。ChA的酶促反应初速率为6.2×10-3μmol/(mL.min),Vmax为0.318μmol/(mL.min),Km为1×10-2mg/mL,且对底物表现相对专一性。K+、Na+、Li+、Mg2+、Ca2+、Ba2+Zn2+、Cu2+和Co2+对ChA活力均表现为抑制作用,过渡金属离子Mn2+对酶有激活作用,重金属离子Hg2+、Ag+、Cd2+和Pb2+对酶均有较强的抑制作用。Mn2+和Zn2+的动力学研究表明,Mn2+对酶为混合型激活作用,Zn2+对酶为竞争性抑制作用。     

Effects of metal ions on sphingomyelinase activity of Bacillus cereus

Some divalent metal ions were examined for their effects on sphingomyelinase activity of Bacillus cereus. The enzyme activity toward mixed micelles of sphingomyelin and Triton X-100 proved to be stimulated by Co2+ and Mn2+, as well as by Mg2+. Km's for Co2+ and Mn2+ were 7.4 and 1.7 microM, respectively, being smaller than the Km for Mg2+ (38 microM). Sr2+ proved to be a competitive inhibitor against Mg2+, with a Ki value of 1 mM. Zn2+ completely abolished the enzyme activity at concentrations above 0.5 mM. The concentration of Zn2+ causing 50% inhibition of the enzyme activity was 2.5 microM. Inhibition by Zn2+ was not restored by increasing concentrations of Mg2+ when the concentration of Zn2+ was above 10 microM. Ba2+ was without effect. When sphingomyelinase was incubated with unsealed ghosts of bovine erythrocytes at 37 degrees C, the enzyme was significantly adsorbed onto the membrane in the presence of Mn2+, Co2+, Sr2+ or Ba2+. Incubation with intact or Pronase-treated erythrocytes caused enzyme adsorption only in the presence of Mn2+. In the course of incubation, the enzyme was first adsorbed on the membranes of intact bovine erythrocytes in the presence of Mn2+; then sphingomyelin breakdown proceeded with ensuing desorption of adsorbed enzyme. Hot-cold hemolysis occurred in parallel with sphingomyelin breakdown. In this case, the hydrolysis of membranous sphingomyelin as well as the initial enzyme adsorption took place in the following order: unsealed ghosts greater than Pronase-treated erythrocytes greater than intact erythrocytes.     

Metal ion requirement of bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase from rat liver

The metal ion requirement for both enzymatic activitiesof the bifunctional UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosaminekinase (E.C. 5.1.3.14/ 2.7.1.60), the key enzyme of N-acetylneuraminic acidbiosynthesis in ratliver, was investigated. UDP-N-acetylglucosamine 2-epimerase was active inimida-zole/HCl buffer in the complete absence of any metal ion. 200 mM Na + , K + , Rb + and Cs +activated enzymeactivity up to five-fold, whereas lower concentrations of thesemonovalent metal ions showed only a small effect on UDP-N-acetylglucosamine 2-epimeraseactivity. In sodium phosphate buffer the enzyme activitywas increased by 0.5 mM Mg , Sr , Ba and Mn , while in the presence of 200 mM NaCl UDP-N-acetyl-glucosamine2-epimerase activity showed astronger activation by these divalent metal ions. In imidazole/HClbuffer, UDP-N-acetylglucosamine2-epimerase activity was partially inhibited by 0.5 mM Be , Mg , Ba ,Mn , Sn and Fe , and completely inhibited by 0.5 mM Zn and Cd . Divalent metal ions were essen-tialforN-acetylmannosamine kinase activity, the most effective being Mg , followed byMn and Co .The optimal concentration of these metal ions was 3 mM. Less effective were Ni and Cd , whereas Ca ,Ba , Cu , Fe and Zn showed no effect on enzyme activity.     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

金属离子对地衣芽孢杆菌合成多聚γ-谷氨酸的影响

多聚γ 谷氨酸 [γ Ｐｏｌｙ(ｇｌｕｔａｍｉｃａｃｉｄ) ,γ ＰＧＡ]是由某些杆菌 (Ｂａｃｉｌｌｕｓ)合成的一种细胞外水溶性高分子氨基酸聚合物 ,是由Ｌ 谷氨酸、Ｄ 谷氨酸两种构型的单体通过γ 酰胺键聚合形成的[1 ] 。γ ＰＧＡ具有极佳的成膜性、成纤维性 ,阻氧性、可塑性、粘结性、保湿性和可生物降解等许多独特的理化和生物学特性[2 ,3] 。因此 ,γ ＰＧＡ可以被广泛用于医药制造 ,食品加工 ,蔬菜、水果、海产品防冻、保鲜 ,化妆品工业 ,烟草、皮革制造工业和植物种子保护等许多领域 ,是一种有极大开发价值和前景的多功能新型生物制…     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

耐酸性黑曲霉β-甘露聚糖酶的克隆及其在毕赤酵母中的表达分析

目的:克隆黑曲霉β-甘露聚糖酶基因,研究该基因在毕赤酵母中的表达情况。方法:运用RT-PCR从黑曲霉AN070902中克隆β-甘露聚糖酶cDNA片段,与载体pPIC9K相连,构建重组载体VMAN-pPIC9K,电转化毕赤酵母GS115,筛选产酶最高菌株进行5 L液体发酵,对该菌株所产重组酶进行酶学性质分析。结果:克隆获得1152 bpcDNA,编码由383个氨基酸残基组成的蛋白质,该蛋白质属于GH5家族,理论pI和相对分子质量分别为4.48和41.6×103;筛选获得的重组菌株VMAN-pPIC9K-GS115在5 L液体发酵中上清酶活达11 785 U/mL;表达的重组酶是一种酸性β-甘露聚糖酶,最适反应pH值为3.0,经pH2.0～9.0处理2 h后剩余酶活保持90%以上;该重组酶最适反应温度为65℃,70℃处理1 h后剩余酶活保持75%以上;该重组酶活性被1 mmol/L的Fe3+和Mn2+显著抑制,被1mmol/L的Co2+显著激活。结论:重组耐酸性β-甘露聚糖酶的特性,决定了其在工业生产中,特别是动物饲料和食品加工中具有应用价值。     

Physiologically relevant metal cofactor for methionine aminopeptidase-2 is manganese

The identity of the physiological metal cofactor for human methionine aminopeptidase-2 (MetAP2) has not been established. To examine this question, we first investigated the effect of eight divalent metal ions, including Ca(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+), on recombinant human methionine aminopeptidase apoenzymes in releasing N-terminal methionine from three peptide substrates: MAS, MGAQFSKT, and (3)H-MASK(biotin)G. The activity of MetAP2 on either MAS or MGAQFSKT was enhanced 15-25-fold by Co(2+) or Mn(2+) metal ions in a broad concentration range (1-1000 microM). In the presence of reduced glutathione to mimic the cellular environment, Co(2+) and Mn(2+) were also the best stimulators (approximately 30-fold) for MetAP2 enzyme activity. To determine which metal ion is physiologically relevant, we then tested inhibition of intracellular MetAP2 with synthetic inhibitors selective for MetAP2 with different metal cofactors. A-310840 below 10 microM did not inhibit the activity of MetAP2-Mn(2+) but was very potent against MetAP2 with other metal ions including Co(2+), Fe(2+), Ni(2+), and Zn(2+) in the in vitro enzyme assays. In contrast, A-311263 inhibited MetAP2 with Mn(2+), as well as Co(2+), Fe(2+), Ni(2+), and Zn(2+). In cell culture assays, A-310840 did not inhibit intracellular MetAP2 enzyme activity and did not inhibit cell proliferation despite its ability to permeate and accumulate in cytosol, while A-311263 inhibited both intracellular MetAP2 and proliferation in a similar concentration range, indicating cellular MetAP2 is functioning as a manganese enzyme but not as a cobalt, zinc, iron, or nickel enzyme. We conclude that MetAP2 is a manganese enzyme and that therapeutic MetAP2 inhibitors should inhibit MetAP2-Mn(2+).