巨大芽孢杆菌WSH-002丙氨酸脱氢酶066晶体制备及X-射线衍射分析

丙氨酸脱氢酶可逆催化丙氨酸脱氨生成丙酮酸,在氨基酸和酮酸的合成及代谢中至关重要.本研究通过PCR从巨大芽孢杆菌WSH-002中克隆并构建了丙氨酸脱氢酶基因(aldBM066)的原核表达载体,经原核表达后,采用Ni-NTA亲和层析法和阴离子交换色谱法纯化获得蛋白AldBM066,在289 K下以座滴法进行晶体生长条件筛选和制备.通过对蛋白质结晶条件的筛选,最终在蛋白质浓度为15 mg/mL及含有0.1 mol/L 乙酸钠（pH 5.0）和2.4 mol/L甲酸钠的缓冲液中获得了理想的蛋白质晶体,晶体大小约为210 μm×180 μm×150 μm,X-射线衍射数据显示,该蛋白质晶体衍射分辨率为2.88 A,空间群为三方晶系,晶胞参数为a=b=118.71 A,c=150.51 A,α=β=90°,γ=120°,每个不对称单位中含有1个AldBM066单体,马修斯系数为2.62 A3/Da,溶剂含量约为53.02%.衍射数据的成功收集为解析巨大芽孢杆菌WSH-002中丙氨酸脱氢酶的三维结构奠定了前期基础,将有助于阐明以单体存在的丙氨酸脱氢酶的催化机制.     

四氢嘧啶羟化酶的研究进展

作为相容性物质,5-羟化四氢嘧啶不仅可以调节渗透压,还可以稳定蛋白结构,在医药、生物制造和化工行业具有广阔的发展前景。四氢嘧啶羟化酶属于Fe2+与2-酮戊二酸依赖型双加氧酶超家族,主要催化四氢嘧啶生成5-羟化四氢嘧啶。我们简要介绍了四氢嘧啶羟化酶的基因来源、活性检测、蛋白结构、催化机理及活性中心等方面的研究进展。     

四氢嘧啶羟化酶的克隆表达、酶学性质及其在羟基四氢嘧啶合成中的研究

四氢嘧啶羟化酶(EctD)是双加氧酶超家族的重要成员,其底物四氢嘧啶和产物羟基四氢嘧啶的应用广泛,因此EctD在生物制造领域具有重要的应用价值。从来源于新疆盐湖的需盐色盐杆菌(Chromohalobacter salexigens)中获得四氢嘧啶羟化酶基因(ectD),并在E.coli BL21(DE3)中进行表达,对异源表达的EctD酶学性质进行研究。结果表明：EctD的最适温度是30℃、最适pH是7.5,在30℃、pH 6.5～8.0条件下具有较好的稳定性;Fe²⁺、Ba²⁺、Mn²⁺、Mg²⁺、Fe³⁺和Ca²⁺离子可增强EctD的酶活,而Co²⁺、Zn²⁺和Cu²⁺离子明显抑制EctD的酶活;动力学参数为K_m=7.63 mmol/L、k_cat/K_m =1.01 1 L/(mmol·s)、V_max ...     

利用X-衍射法测定农药助剂对苏云金杆菌LB-2菌株晶体蛋白质立体结构的影响

运用X-射线衍射法研究几种化学物质及保存温度对苏云金杆菌LB-2菌株伴孢晶体的影响,测得晶体结构变化与经生物测定得到的毒力之间有较好的相关性。     

一株可用于四氢嘧啶制备的中度嗜盐菌

<正>四氢嘧啶(1,4,5,6-四氢-2-甲基-4-嘧啶羧酸)是中度嗜盐细菌的一种渗透压补偿性溶质[1],可以平衡细胞的渗透压,并且在高温、冷冻和干燥等逆境下,对酶、DNA、细胞膜和整个细胞提供保护作用,因此四氢嘧     

Cobetia marina CICC10367渗透压冲击下羟基四氢嘧啶的合成及释放

摘要:【目的】筛选获得耐受渗透压冲击的羟基四氢嘧啶合成菌株,利用“细菌挤奶”工艺提高羟基四氢嘧啶的产率。【方法】从盐池中分离羟基四氢嘧啶合成菌株,并对其进行形态、生理生化及16S rDNA鉴定。考察了培养基及其NaCl浓度对羟基四氢嘧啶合成的影响,在优化的条件下利用“细菌挤奶”工艺制备羟基四氢嘧啶。【结果】筛选获得的一株羟基四氢嘧啶合成菌株,鉴定为Cobetia marina CICC10367（C. marina CICC10367）。NaCl浓度为90 g/L的、谷氨酸单钠为唯一碳氮源的培养基有利于羟     

X射线衍射晶体法解析脱卤酶DehDIV-R结构的研究

R-2-卤代酸脱卤酶能立体选择性水解R-2-卤代酸。解析酶的单晶结构对提高酶的选择性和活性提供了直接的结构指导,是目前酶结构领域研究的前沿。以实验室前期得到的来自假单胞菌ZJU26的R-2-氯丙酸脱卤酶(Deh DIV-R)为研究对象,采用X射线衍射晶体法进行结构解析。采用pp SUMO载体融合表达Deh DIV-R蛋白,依次通过Ni-NTA亲和层析、透析酶切、二次NiNTA亲和层析以及凝胶过滤层析纯化得到单一条带,且均一性好的蛋白。接着对结晶条件进行初筛与优化,得到的最佳结晶条件为0.1mol/L HEPES p H 7,12%PEG 6 000,0.2mol/L Mg Cl2,8mmol/L CHAPS。晶体在上海同步辐射光源BL18U1线站上收集衍射数据,采用分子置换法成功解析获得了分辨率为2.35的Deh DIV-R的晶体结构。Ramachandran图表明98.02%的氨基酸位于最适区,证明了该结构的合理性。Deh DIV-R的纯化、结晶以及结构解析为进一步深入了解其结构和功能奠定了基础。     

产高活性抗凝溶栓双活性蛋白的短小芽孢杆菌的筛选及鉴定

摘要:【目的】筛选可产生抗血栓活性物质的细菌。【方法】利用VY/4平板、酪蛋白平板从水样、土样、兔粪、羊粪、朽木等20多个样品中筛选目的菌株;利用纤维蛋白平板和纤维蛋白试管检测抗血栓活性;利用形态学特征、理化性质、16S rRNA序列同源性鉴定目的菌株。【结果】得到5株可产生抗血栓活性物质的细菌,重点研究了菌株LDS33,发现其分泌的胞外蛋白在纤维蛋白平板上和纤维蛋白试管中均显示出强烈的溶栓活性,通过试管法发现此蛋白质同时具有较强的抗凝活性。结合形态学、理化性质、16S rDNA序列及进化树分析,发现该菌株属于硬壁菌门芽孢杆菌目芽孢杆菌科芽孢杆菌属的短小芽孢杆菌,将其命名为Bacillus pumilus LDS.33。【结论】短小芽孢杆菌LDS33可产生高活性的抗凝溶栓双活性蛋白。     

假坚强芽胞杆菌中乙醇降解相关酶的克隆、表达及酶学特性

【目的】研究假坚强芽胞杆菌OF4中乙醇脱氢酶和乙醛脱氢酶的酶学特性。【方法】通过引物设计,采用PCR技术从嗜碱芽胞杆菌OF4的基因组DNA中扩增获得乙醇脱氢酶(adh)基因和乙醛脱氢酶(aldh)基因,构建表达载体,通过异源原核表达,Ni-NTA柱层析纯化酶蛋白,分析其酶学特性。【结果】乙醛脱氢酶的最适反应温度为35℃,最适反应pH值为8.0,酶蛋白的活力为979.6 U/mg,其稳定性在25℃和35℃下比45℃稍好;尽管由于乙醇脱氢酶的表达量低而未能纯化获得酶蛋白,但通过双基因共表达及乙醇耐受性实验发现乙醇脱氢酶也具备较高的催化活性。【结论】成功地从假坚强芽胞杆菌OF4中克隆获得了乙醇脱氢酶和乙醛脱氢酶基因,二者共同作用能够较大提高宿主对乙醇的耐受性。     

极端耐盐放线菌白色普氏菌YIM 90005T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆

摘要：【目的】 为了研究耐盐放线菌对高盐环境的适应机理。【方法】 用HPLC定量检测了极端耐盐、丝状产孢放线菌——白色普氏菌（Prauserella alba） YIM 90005T在不同盐浓度下胞内相容性溶质的种类和含量。【结果】 结果发现,四氢嘧啶和5-羟基四氢嘧啶是其主要的相容性溶质。在培养基NaCl浓度为10%时,四氢嘧啶在胞内累积浓度最大,为18.77 μg/mg干菌体重。之后随NaCl浓度的升高,胞内的四氢嘧啶含量逐渐减少,而5-羟基四氢嘧啶的含量逐渐增加,在该菌耐受的最高NaCl浓度下（24% w/v）,胞内5-羟基四氢嘧啶含量达到最大值,为22.98 μg/mg干菌体重。设计兼并引物,利用染色体步移,克隆得到四氢嘧啶及5-羟基四氢嘧啶合成相关基因ectABCD。序列分析表明,ectABCD位于一个操纵子中。进一步对不同NaCl浓度培养条件下ectB,D的表达量进行定量分析,结果表明该基因簇表达量随着培养基中NaCl浓度的增加而增大。【结论】 研究结果证实5-羟基四氢嘧啶是P. alba YIM 90005T在极高盐浓度条件下起渗透调节及保护的相容性溶质。     

Reidentification of facultatively alkaliphilic Bacillus firmus OF4 as Bacillus pseudofirmus OF4

With a view toward verifying the original classification of alkaliphilic Bacillus firmus OF4, physiological and biochemical characteristics were more extensively catalogued than in original studies, and this catalog was supplemented with 16S rDNA sequence homology and more extensive DNA–DNA hybridization analyses. Phylogenetic analysis of this alkaliphile based on the comparison of multiple 16S rDNA sequences from Bacillus species indicated that this strain is most closely related to Bacillus pseudofirmus. Consistently, in the DNA–DNA hybridization analysis of the alkaliphile and Bacillus reference strains, the highest level of DNA–DNA relatedness (96%) was found between the alkaliphile and the B. pseudofirmus type strain (DSM 8715^T). The findings support the conclusion that this alkaliphile strain is more closely related to B. pseudofirmus than to B. firmus, and we propose the future use of the designation B. pseudofirmus OF4. Received: April 20, 1999 / Accepted: August 31, 1999     

Superoxide dismutase from Bacillus stearothermophilus: Crystallization and preliminary X-ray diffraction studies

Superoxide dismutase from Bacillus stearothermophilus is a dimeric manganese-containing enzyme with a molecular weight of 40,000. It has been crystallised in the monoclinic space group P2₁ with unit cell dimensions of $\text{a = 50}\text{A}\text{?}\text{, b = 70}\text{A}\text{?}\text{, c = 69}\text{A}\text{?}$ and β = 111 ° 10′. The asymmetric unit appears to be the dimer.     

Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum

Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized. The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A. They are suitable for a high-resolution X-ray diffraction analysis.     

Crystallization and preliminary X-ray diffraction studies of two mutants of lactate dehydrogenase from Bacillus stearothermophilus.

Bacillus stearothermophilus lactate dehydrogenase, one of the most thermostable bacterial enzymes known, has had its three-dimensional structure solved, the gene coding for it has been cloned, and the protein can be readily overexpressed. Two mutants of the enzyme have been prepared. In one, Arg171 was changed to Trp (R171W) and Gln102 was changed to Arg (Q102R). In the other, the mutation Q102R was maintained, but Arg171 was changed to Tyr (R171Y). In addition, an inadvertent C97G mutant was present. Both mutants have been crystallized by the hanging drop vapor diffusion method at room temperature. Bipyrimidal crystals have been obtained against (NH4)2SO4 in 50 mM piperazine HCl buffer. The crystals belong to space group P6(2)22 (P6(4)22) (whereas the native enzyme, the structure of which has been solved by Piontek et al., Proteins 7:74-92, 1990) crystallized in the space group P6(1)) with a = 102.3 A, c = 168.6 A for the R171W, Q102R, C97G triple mutant, and a = 98.2 A; c = 162.1 A for the R171Y, Q102R, C97G mutant. These crystal forms appear to contain one-quarter of a tetramer (M(r) 135,000) in the asymmetric unit and have VM values of 3.8 and 3.3 A3/dalton, respectively). The R171W mutant diffracts to 2.5 A and the R171 Y mutant to approximately 3.5 A.     

Crystallization and preliminary X-ray diffraction studies of a toxic crystal protein from a subspecies of Bacillus thuringiensis

The toxic crystal protein (Mr 64,000) from a subspecies of the bacterium Bacillus thuringiensis has been solubilized and recrystallized yielding diffraction quality crystals. Crystals are obtained by a change in pH and ionic strength using Na2CO3. They can also be obtained by a change in ionic strength only using NaBr as the precipitant. The space group of both forms is C222(1) with a = 133, b = 116, c = 104 A and one molecule/asymmetric unit. Still photographs show reflections to 3.0-A resolution.     

Crystallization and preliminary X-ray diffraction analysis of BcOMT2 from Bacillus cereus: a family of O-methyltransferase

O-Methyltransferases (OMTs), one of the ubiquitous enzymes in plants, bacteria, and humans, catalyze a methyl-transfer reaction using S-adenosylmethionine and a wide range of phenolics as a methyl donor and acceptor, respectively. Substrates for most bacterial OMTs have largely remained elusive, but recent investigation using BcOMT2, an OMT from Bacillus cereus, suggested that ortho-dihydroxyflavonoids could serve as substrates. To elucidate the functional and structural features of BcOMT2, we expressed, and purified BcOMT2, and crystallized an apoenzyme and its ternary complex in the presence of a flavonoid and S-adenosylhomocysteine. Each crystal diffracted to 1.8 angstroms with its space group of C2 and P2(1)2(1)2(1), respectively. Structural analysis of apo-BcOMT2 and its ternary complex will provide the structural basis of methyltransfer onto (iso)flavonoids in a regiospecific manner.     

Crystallization and preliminary X-ray analysis of phenylalanine hydroxylase from rat liver

Phenylalanine hydroxylase from rat liver has been crystallized from polyethylene glycol 4500 with sodium formate. The crystals are tetragonal rods and belong to space group P4(1)22 or P4(3)22 with unit cell dimensions a = b = 57.6 A and c = 304.1 A. They diffract to at least 2.4 A resolution and have one molecule per asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of mandelonitrile lyase from almonds

Single crystals of three different isoenzymes of (R)?(+) mandelonitrile lyase (hydroxynitrile lyase) from almonds (Prunus amygdalus) have been obtained by hanging drop vapor diffusion using polyethylene glycol 4000 and isopropanol as co-precipitants. The crystals belong to the monoclinic space group P2_l with unit cell parameters a = 69.9, b = 95.1, c = 95.6 Å, and β = 118.5°. A complete set of diffraction data has been collected to 2.6 Å resolution on native crystals of isoenzyme III. © 1994 Wiley-Liss, Inc.