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（英文）

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(p H 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210μm×180μm×150μm,X-射线衍射数据显示,该蛋白质晶体衍射分辨率为2.88,空间群为三方晶系,晶胞参数为a=b=118.71,c=150.51,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3/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.     

假坚强芽孢杆菌四氢嘧啶羟化酶的晶体制备及X-射线衍射研究

摘要:【目的】假坚强芽孢杆菌四氢嘧啶羟化酶蛋白纯化、晶体制备及X-射线衍射研究。【方法】通过PCR从假坚强芽孢杆菌OF4中克隆获得四氢嘧啶羟化酶基因,构建原核表达载体,经过原核表达,采用Ni-NTA亲和层析法和分子排阻色谱法纯化蛋白,289 K下采用座滴法进行晶体筛选和制备,在低温100 K下通过X-射线衍射仪(Rigaku MicroMax-007 HF)收集晶体衍射数据。【结果】通过原核表达及纯化成功获得了适合晶体生长的蛋白BpEctD。通过筛选最终在蛋白浓度为6.5 mg/mL及含有0.2 mol/L MgCl2·6H2O,0.1 mol/L Bis-Tris pH6.5,25% (W/V) 聚乙二醇3,350的缓冲液中获得了理想的蛋白晶体,其大小约为360 μm×240μm×60 μm,并在100K下成功收集了衍射数据,晶体衍射分辨率为2.40,空间群为三斜晶系P1,晶胞参数为a=45.18,b=58.87,c=68.81,α=77.48°,β=86.03°,γ=66.97°,每个不对称单位中含有2 个BpEctD单体,马修斯系数为2.44 3/Da,溶剂含量约为49.53%。【结论】衍射数据的成功收集为假坚强芽孢杆菌OF4四氢嘧啶羟化酶三维结构的解析奠定了前期基础,将有助于阐明四氢嘧啶羟化酶的催化机制。     

黄瓜27 kD LHC-Ⅱ复合物三维结构的电子晶体学初步研究

从黄瓜(Cucumis sativus L.)叶片中分离出只含有一种亚基(27 kD)的LHC-Ⅱ复合物.采用batch方法获得了其二维晶体,大小为0.7 μm×1.0μm,衍射能力达30 A.负染样品的二维投影结果表明,该晶体为p3对称性,晶胞参数为15.4 nm×15.4 nm,不同于以往报道的菠菜(Spinacia oleracea L.)或豌豆(Pisum satium L.)LHC-Ⅱ晶体,为另外一种晶型.采用tomography技术,收集了0°～-55°系列倾斜照片,进行三维重构.LHC-Ⅱ复合物是由6个单体组成的六元环,相邻2个单体分别从膜的两侧插膜,方向相反,在膜区靠疏水-疏水相互作用成二聚体,3个相同的二聚体相互连接成六元环.     

重组肺炎链球菌甘油脱氢酶活性及晶体培养的初步分析

甘油脱氢酶能够氧化胞内甘油为代谢活动提供能量,并参与调控与宿主粘附相关细菌蛋白质的表达,是潜在的抗菌药物靶点. 本文利用大肠杆菌BL21原核表达肺炎链球菌甘油脱氢酶,获得大量上清表达的目的蛋白. 再用Ni-NAT亲和层析、DEAE离子交换层析以及Superdex 200分子筛进行纯化. 经数据拟合,证实该酶在溶液中以同源二聚体形式存在. 光谱实验证实该重组表达甘油脱氢酶仍具有氧化甘油生成1, 3-二羟基丙酮的活性. 用Hampton试剂盒初筛晶体及棋盘法优化晶体,在0.1 mol/L Bicine PH 9.6,13% PEG MME 5000,0.2 mol/L KSCN,4% Dioxone条件下,得到了晶型好且有一定衍射能力的单晶,为解析肺炎链球菌甘油脱氢酶的三维结构及研究结构与酶活之间的关系奠定了基础.     

高粱NADP苹果酸脱氢酶的纯化及其分子特性

用Sephadex G-100柱和制备性等电聚焦电泳纯化了高梁叶片NADP苹果酸脱氢酶,得到电泳均一的酶制剂,酶比活力提高350倍。天然酶含有两个分子量为4万D的亚基。动力学研究表明Km(OAA)=31μmol/L:K_m(NADPH)=48 μmol/L;K_m(Mal)=11m mol/L:K_m(NADP)=45μmol/L。NADP为NADPH的竞争性抑制剂,抑制常数K_i=190 μmol/L。 在体外DTT为NADP苹果酸脱氢酶的激活所必需。DTT对该酶的激活受一小分子蛋白、NADP及离子强度所调节。     

富硒螺旋藻中含硒藻蓝蛋白的纯化、结晶及初步晶体学研究

从富硒螺旋藻中提取含硒藻蓝蛋白,经凝胶色谱和离子交换色谱纯化,应用悬滴气相扩散法,采用(NH4)2SO4和PEG4000作沉淀剂,获得了该蛋白质晶体的两种晶型.晶型Ⅰ属于单斜晶系,晶胞参数a=10.80 nm,b=11.70 nm,c=18.40 nm,β=90.2°,晶体空间群属于P21.单位晶胞中每个晶体学不对称单位含12个(αβ)单体,晶体衍射的最高分辨率达0.28 nm.晶型Ⅱ为六方晶系,晶胞参数为a=b=15.5 nm,c=4.03 nm,晶体空间群属于P63.晶体衍射的最高分辨率达0.28 nm.单位晶胞中每个晶体学不对称单位含1个(αβ)单体.对分子在晶体中的可能堆积方式进行了讨论.     

苏芸金杆菌伴孢晶体和芽孢孢衣的蛋白质组分及其与毒力的关系

研究了苏芸金杆菌(Bacillus thuringiensis)3个变种6个菌株的提纯伴孢晶体和芽孢对大蜡螟(Galleria mellonella)和大菜粉蝶(Pieris brassicae)的毒力、晶体的蛋白质和多肽成分及芽孢衣中的类晶体蛋白质成分.生物测定表明,晶体毒力高于芽孢,在总数量相同的情况下,晶体和芽孢近1:1的混合物的毒力高于单独的晶体或芽孢.芽孢衣中存在一种类似晶体蛋白质的成分,无晶体突变株及无效野生株的芽孢则无此种蛋白质且对两种昆虫无毒.变种wuhanensis和变种galleriae的晶体含MW138000的主要蛋白质和63000的次要蛋白质,经碱性缓冲液溶解后,上清液含MW138000的蛋白质,沉淀中含MW63000的蛋白质;变种aizawai的晶体中仅含138000的蛋白质.对大蜡螟无毒的HD-11(var.aizawai)晶体的蛋白质成分有别于上述晶体,其胰蛋白酶消化物的SDS凝胶电泳图型显示少2条多肽带,但对大菜粉蝶仍有效.结果表明,苏芸金杆菌的芽孢在昆虫病理中有重要作用,伴孢晶体的蛋白质和多肽成分与它们对昆虫的毒力特性之间有密切关系.     

利用巨大芽孢杆菌酶系合成L-丙氨酸和L-缬氨酸

本文研究了利用巨大芽孢杆菌ATCC_(39118)酶系合成氨基酸,同时也研究了丙氨酸脱氢酶、缬氨酸脱氢酶及葡萄糖脱氢酶的提纯工艺。所获得的AlaDH、ValDHc和GlcDH的比活性分别为11.2u/mg,7.8u/mg和23.0u/mg。为了进一步探讨由α-酮酸酶法转化成氨基酸的最适条件,我们对以上三种酶的主要性质,包括稳定性,最适pH、动力学常数、底物专一性及底物和产物对酶的抑制作用等进行了测定。同时用粗酶提取液和纯酶进行了由丙酮酸合成L-丙氨酸,由α-酮异戍酸合成L-缬氨酸的批量实验,在转化中葡萄糖脱氢酶作为NADH的再生酶。结果粗酶提取液催化L-丙氨酸产量的克分子转化率为80％,而纯酶催化的克分子转化率增加到92％。L-缬氨酸产量的克分子转化率也类似(93％)。     

Isolation and crystal ctructure of sulfur from Capparis spinosa fruits

从刺山柑果实中首次分离纯化得到S8单质.经X-射线单晶衍射分析,确认该化合物为S8晶体,M=256.48.晶体属斜方晶系,空间群Fddd.晶胞参数a=10.456(7)A°,b=12.908(9)A°,c=24.483(17)A°,V=3305(4)A°3,Z=16,F(000)=2048,R1=0.0915,wR2=0.1820.     

解淀粉芽孢杆菌产凝乳酶发酵优化

对实验室前期筛选到的一株解淀粉芽孢杆菌Bacillus amyloliquefaciens JNU002进行培养基和培养条件优化。单因素实验确定最佳碳源为乳糖,除麸皮外,其他氮源对产酶无明显促进作用,KH2PO4和Ca CO3等对产酶有促进作用。通过正交试验确定最佳培养基成分为麸皮16 g/L、乳糖10 g/L、KH2PO41 g/L、Ca CO31 g/L,其中麸皮对解淀粉芽孢杆菌产凝乳酶影响较大。在发酵温度37℃、种子液培养时间18 h、接种量0.5%、500 m L摇瓶装液量100m L时,较适宜解淀粉芽孢杆菌产凝乳酶。优化后解淀粉芽孢杆菌产凝乳酶活力达到12 766 SU/m L,比优化前提高了3.5倍。     

Complete genome sequence of the industrial strain Bacillus megaterium WSH-002

Bacillus megaterium, an industrial strain, has been widely used in protein production and the vitamin C industry. Here we reported a finished, annotated, and compared 4.14-Mbp high-quality genome sequence of B. megaterium WSH-002, which is the companion strain for Ketogulonicigenium vulgare in the vitamin C industry and is stocked in our laboratory.     

The determination of ascorbic acid and uric acid in human seminal plasma using an HPLC with UV detection

Oxidative stress has been proposed as one of the potential causes for infertility in men. Ascorbic acid and uric acid play important role in protection of spermatozoa against free radicals. A method for the simultaneous determination of ascorbic acid and uric acid in human seminal plasma using HPLC with UV detection and investigation their clinical significance as antioxidants protecting male germ cells against oxidative damage are described. Semen samples were obtained from consecutive male partners of couples presenting for a fertility evaluation. After liquefaction, the samples were centrifuged and the supernatants were diluted with dithiothreitol solution and after a filtration injected onto an analytical column. For the separation, a reverse-phase column MAG 1, 250 mm × 4.6 mm, Labiospher PSI 100 C18, 5 μm, was used. The mixture of ethanol and 25 mmol/L sodium dihydrogenphosphate (2.5:97.5, v/v), pH 4.70 was used as a mobile phase. Analytical performance of this method is satisfactory for both ascorbic acid and uric acid: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked seminal plasma were between 92.1 and 102.1%. We have found no significant differences in both ascorbic acid and uric acid concentration between the smokers and non-smokers (351.0 ± 237.9 μmol/L and 323.7 ± 99.5 μmol/L vs. 444.8 ± 245.5 μmol/L and 316.6 ± 108.9 μmol/L, p>0.05). This assay is a simple and reproducible HPLC method for the simultaneous measurement of ascorbic acid and uric acid in human seminal plasma.     

D-mannitol production by resting state whole cell biotrans-formation of D-fructose by heterologous mannitol and formate dehydrogenase gene expression in Bacillus megaterium

An in vivo system was developed for the biotransformation of D-fructose into D-mannitol by the expression of the gene mdh encoding mannitol dehydrogenase (MDH) from Leuconostoc pseudomesenteroides ATCC12291 in Bacillus megaterium. The NADH reduction equivalents necessary for MDH activity were regenerated via the oxidation of formate to carbon dioxide by coexpression of the gene fdh encoding Mycobacterium vaccae N10 formate dehydrogenase (FDH). High-level protein production of MDH in B. megaterium required the adaptation of the corresponding ribosome binding site. The fdh gene was adapted to B. megaterium codon usage via complete chemical gene synthesis. Recombinant B. megaterium produced up to 10.60 g/L D-mannitol at the shaking flask scale. Whole cell biotransformation in a fed-batch bioreactor increased D-mannitol concentration to 22.00 g/L at a specific productivity of 0.32 g D-mannitol (gram cell dry weight)(-1) h(-1) and a D-mannitol yield of 0.91 mol/mol. The nicotinamide adenine dinucleotide (NAD(H)) pool of the B. megaterium producing D-mannitol remained stable during biotransformation. Intra- and extracellular pH adjusted itself to a value of 6.5 and remained constant during the process. Data integration revealed that substrate uptake was the limiting factor of the overall biotransformation. The information obtained identified B. megaterium as a useful production host for D-mannitol using a resting cell biotransformation approach.     

排硫硫杆菌D6中硫化氢脱氢酶的纯化及性质

从烟气生物脱硫系统的好氧产硫磁性稳态流化床反应器中,经反复纯化分离出脱硫优势菌排硫硫杆菌菌株D6,采用四步工艺纯化出膜结合型硫化氢脱氢酶。SDS-PAGE测定显示其由α1β1亚基组成,光谱分析表明含有1 mol FAD/mol酶,血红素染色揭示小亚基上结合有1 mol血红素c/mol酶,该酶属于氧还蛋白家族。该酶的最适pH为8.6,对马心细胞色素c和硫化物的表观Km分别为2.5μmol/L和6.1μmol/L,反应计量实验表明其氧化产物为元素硫。硫化氢脱氢酶受到硫和亚硫酸盐的抑制,100μmol/L的氰化钾对该酶抑制率达72%。     

罗格列酮对海马神经元树突发育的影响

罗格列酮(rosiglitazone,Rosig.)是噻唑烷二酮类(thiazolidinediones,TZDs)过氧化物酶体增殖物激活受体γ(peroxisome proliferator-activated receptor gamma,PPARγ)的激动剂,近年来,临床研究发现其具有神经保护作用,但对其作用机制目前仍没有完全研究清楚．利用活细胞成像的方法,观察罗格列酮对大鼠海马神经元树突丝和树突树发育的影响及其机制．结果显示,罗格列酮浓度依赖的增高神经元树突丝密度,对树突丝长度、运动速度并没有影响．此外,罗格列酮也不影响树突树的总分支、总长度以及各级分支的数目和长度．PPARγ 特异性拮抗剂GW9662完全阻断了罗格列酮介导的树突丝密度增高．结果表明罗格列酮可能通过PPARγ途径影响神经元的早期发育,这可能是罗格列酮发挥神经保护作用的潜在机制．     

The resting metabolism of dodder seeds

Extracts of mature seeds of Cuscuta reflexa were examined for any deficiency in key enzymes. The activities of malate dehydrogenase, β-amylase and fructose 1,6-diphosphate aldolase exceeded 5.0 μmol substrate/min/g, while those of starch phosphorylase, α-amylase, acid phosphatase, phosphogluconate dehydrogenase (decarboxylating), aspartate aminotransferase, glucose 6-phosphate dehydrogenase, fructose 1,6-diphosphatase and alanine aminotransferase fell within the range 1 to 5 μmol/min/g and hexokinase, isocitrate dehydrogenase and alkaline phosphatase were below 1 μmol substrate/min/g seed powder. No activity of the following were found: acid invertase, alkaline invertase, phytase and glutamate dehydrogenase. Some of these observations were made also for seeds of Cuscuta campestris and Cuscuta indicora.     

Amino acids in cat fallopian tube and follicular fluids

Aminograms of tubal and follicular fluids were obtained using fluids collected by aspiratory puncture from six cats. The amino acids were separated and quantified by high-performance liquid chromatography analysis. The serum of the cats was used as control. The three most prevalent amino acids quantified in cat tubal fluid were glycine, glutamic acid, and taurine. Their mean concentrations were 840 μmol/l (μm), 808 μm and 596 μm, respectively. The three most prevalent amino acids quantified in cat follicular fluid were alanine, glutamine, and taurine. Their mean concentrations were 359 μm, 351 μm, and 258 μm, respectively. This result is consistent with aminograms of tubal fluid previously determined in other mammals. As previously observed in other species and humans, glycine was quantitatively the most abundant and most prevalent free amino acid in cat tubal fluid. The total quantity of amino acids in tubal fluid was similar in cats and other species. However, in contrast with other species studied, hypotaurine was not detected in tubal and follicular fluids of female cats.     

中国滑刃属一新种记述(滑刃目,滑刃科)

2001～2002年从山东省青州市韭菜Allium tuberosum的根际土壤中发现滑刃属Aphelenchoides1新种,定名为韭菜滑刃线虫Aphelenchoides alliussp.nov.。该虫具有以下特征:虫体大型;唇区缢缩,口针较长,无口针基部球;侧区具有6条侧线;生殖系统发育良好,卵巢具1～2个转折,卵母细胞单列;尾细锥形,末端收缩呈细指状,平截,刺状尾尖突生于其上;雄虫常见,尾钩状,末端具刺状尾尖突,交合刺典型,顶尖和喙突明显。已报道种中有8个种与新种相似,其中5个种虫体体长较短,小于500μm,易与新种分开,它们是:细尖滑刃线虫A.conimucronatus(L=280～440μm),高头滑刃线虫A.editocaputis(L=270～320μm),高山滑刃线虫A.montanus(L=390～400μm),稀少滑刃线虫A.rarus(L=300～320μm),沃恩滑刃线虫A.vaughani(L=360～430μm)。另3个种与新种相比较:中心滑刃线虫A.centralis侧线4条,尾巴较长,雄虫不常见;陶比恰滑刃线虫A.daubichaensis侧线4条,口针较短且有小的基部球,后阴子宫囊较短,卵巢无转折;一点红滑刃线虫A.emiliae口针基部球发达,中食道球椭圆形,更大19μm×15μm,后阴子宫囊较短。