Synthesis,Characterization and Antibacterial Properties of Rare Earth (Ce^(3+) ,Pr^(3+) ,Nd^(3+) ,Sm^(3+) ,Er^(3+) ) Complexes with L-Aspartic Acid and o-Phenanthroline

Five novel ternary complexes of rare earth ions with L-Aspartic acid (Asp) and o-phenanthroline (Phen) were synthesized in ethanol aqueous solution. Their compositions were characterized by elemental analysis, molar conductance, FT-IR, Raman, UV-VIS and TG-DTA. The compositions of the complexes were confirmed to be: RE( Asp)3PhenCl3·3H2O (RE: Ce3+ , Pr3+ , Nd3+ , Sm3+ , Er3+ ). The antibacterial activity test shows that all these complexes exhibit excellent antibacterial ability against Escherichia coli, Staphylococcus aureus and Candida albicans. The antimicrobial spectrum of the complexes are broad.

Investigating the mechanisms of isochorismate synthase:An approach to improve salicylic acid synthesis and increase resistance to Fusarium head blight in wheat

Salicylic acid(SA),a vital endogenous hormone,plays a crucial role in plant growth and the response to abiotic and biotic stress.Isochorismate synthase(ICS)and phenylalanine ammonia lyase(PAL)are critical rate-limiting enzymes for SA synthesis.Fusarium head blight(FHB)seriously threatens the safety of wheat production,but increasing the content of SA can enhance FHB resistance.However,the pathway of SA synthesis and regulation in wheat remains unknown.In this study,three wheat ICS(TaICSA,TaICSB,and TaICSD)were identified,and their functions were validated in vitro for isomerizing chorismate to isochorismate.The mutation of one or two homoeoalleles of TaICSA,TaICSB,and TaICSD in the wheat variety‘Cadenza’reduced SA levels under ultraviolet treatment and Fusarium graminearum infection,further enhancing sensitivity to FHB.Overexpression of TaICSA can significantly enhance SA levels and resistance to FHB.To further study SA synthesis pathways in wheat and avoid interference with pathogenicity related genes,the leaves of wild-type Cadenza and different TaICS mutant lines were subjected to ultraviolet treatment for transcriptomic analysis.The results showed that 37 PALs might be involved in endogenous SA synthesis,and 82 WRKY and MYB family transcription factors may regulate the expression of ICS and PAL.These results were further confirmed by RT-PCR.In conclusion,this study expands our knowledge of SA biosynthesis and identifies TaICSA,as well as several additional candidate genes that encode transcription factors for regulating endogenous SA levels,as part of an efficient strategy for enhancing FHB resistance in wheat.

Electrocatalytic oxidation and determination of dopamine at a carbon ionic liquid electrode modified with nafion-L-aspartic acid composite film

The electrocatalytic oxidation of dopamine(DA)was studied by electrochemical approaches at a carbon ionic liquid electrode(CILE)modified with the composite film of nafion and L-aspartic acid(NL-CILE).The CILE was fabricated by replacing non-conductive organic binders with a room-temperature hydrophobic ionic liquid,1-butyl-3-methyl-imidazolium hexafluorophosphate.The composite film of NL was used as matrix to adsorb DA and catalyze the oxidation of DA in phosphate buffer solution(PBS).The electrochemical response of DA was investigated at the NL-CILE,the traditional carbon paste electrode(TCPE),CILE and the nafion modified CILE(N-CILE)in 0.1M PBS(pH 7.4),respectively.The results showed the superiority of NL-CILE to N-CILE,CILE and TCPE in terms of provision of higher sensitivity,faster electron transfer and better reversibility.Under optimum condition,the oxidation peak current was rectilinear with DA concentration range from 0.1μM to 0.1mM,with a detection limit of 0.03μM(S/N=3)by differential pulse voltammetry.The proposed method was applied to determine DA in samples successfully.

Synthesis and Crystal Structure of a New Ternary Complex of Nickel(II) with L-Aspartic Acid and 1,10-Phenanthroline

A new ternary complex of nickel(II) with L-aspartate (L-Asp) and 1, 10-phenan- throline(Phen), [Ni3(L-Asp)(Phen)5(H2O)3](ClO4)4?4.75H2O, has been synthesized in a mixed sol- vent of H2OC2H5OH at the pH value of 3. It crystallizes in triclinic, space group P1 with a = 11.861(2), b = 18.384(4), c = 19.746(3) ?, α = 107.68(1), β = 105.94(1), γ = 103.41(2)°, V = 3703.6(11) ?3, Mr = 1745.66, Z = 2, Dc =1.565 g/cm3, μ = 0.989 mm-1, F(000) = 1791, R = 0.0643 and wR = 0.1611. The complex contains two cations, binuclear [Ni2(L-Asp)(Phen)3(H2O)]2+ and mononu- clear [Ni(Phen)2(H2O)2]2+. All the nickel ions in the complex are six-coordinated with a distorted octahedral geometry, but the coordination environments for them are different. There exist hydrogen bonds and π-π stacking interactions in the complex.

Selective Recognition and Detection of L-Aspartic Acid by Molecularly Imprinted Polymer in Aqueous Solution

Molecularly imprinted polymers selective for L-aspartic acid (LAA) have been prepared using the carboxy-betaine polymer bearing zwitterionic centres along the backbone. LAA is well known to promote good me-tabolism, treat fatigue and depression along with its significance in accurate age estimation in the field of forensic science and is an important constituent of ‘aspartame’, the low calorie sweetener. In order to study the intermolecular interactions in the prepolymerization mixture between the monomer and the template (LAA)/non-template (DAA), a computational approach was developed. It was based on the binding energy of the complex between the template and functional monomer. The results demonstrate that electrostatic in-teractions primarily guide the imprinting protocol. The MIP was able to selectively and specifically take up LAA from aqueous solution, human blood serum and certain pharmaceutical samples quantitatively. Hence, a facile, specific and selective technique to detect the amino acid, LAA in the presence of various interfer-rants, in different kinds of matrices is presented.

Synthesis of L-Aspartic Acid and Lactone Copolymers

Biodegradable poly(alc-alt-Asp) was synthesized by ring-opening polymerization of the monomer 3-(S)-[(benzyloxycarbonyl)methyl] -morpholine-2, 5-dione and subsequent catalytic hydrogenation. Copolymers of the monomer with glycolide, D,L-lactide and L-lactide were also prepared.

Pectate lyase is a factor in the adaptability for Heterodera glycines infecting tobacco

The soybean cyst nematode, Heterodeara glycines, is a serious pathogen of soybean, and reported to be the host of a wide range of Fabaceae. In the present study, the host specificity and reproductivity of two populations of H. glycines collected from soybean and tobacco were identified and characterized. The comparative identity between β-1,4-endoglucanase, pectate lyase and chorismate mutase of H. glycines parasitizing on soybean and tobacco were 99, 97 and 98%, respectively. The qR T-PCR analysis indicated that the expression of pectate lyase 2 gene was significantly higher in second-stage juveniles of H. glycines Henan population parasitizing on tobacco than that of H. glycines Shanxi population parasitizing on soybean. In addition, the pectic acid content of cell wall was significantly higher(45%) in the roots of tobacco than the roots of soybean. Our results indicate that the changes in transcript parasitism genes may be a result of long-term evolution illustrating how a plant-parasitic nematode adapts to the host environment for optimal infestation and survival.

Cloning and characterization of a new endo-type polyG-specific alginate lyase from bacteria Vibrio sp. QD-5

Seven bacterial clones with alginate-utilizing activity were isolated from rotten kelp. By activity test, the Vibrio sp. QD-5 with the potential alginate-degrading capability was chosen to carry out the draft genome sequencing, and the result showed that the Vibrio sp. QD-5 containing an alginate lyase gene cluster. One of these genes, aly-IV, was cloned and characterized for the first time. After overexpression, Aly-IV, with a molecular mass of about 62 kDa and a theoretical isoelectric point (pI) of 5.12, was purified to a specific activity of 1 256.78 U/mg and showed highest activity at 35°C in the Tris-HCl buffer at pH of 8.9. Moreover, the enzyme activity was enhanced by the metal ions of Na+, K+ and Mg2+ under certain concentration. Aly-IV degraded favorably polyG blocks in an endo-type, yielding monomer and dimer as the main products. Due to its high substrate specificity, Aly-IV could be used as a potential tool for production of polyG oligosaccharides with low degree of polymerization (DP) and for determining the fine structure of alginate.

代谢工程改造酿酒酵母发酵生产β-胡萝卜素

β-胡萝卜素属于多烯烃类,是水果和蔬菜中含量最高的类胡萝卜素,能够预防并减轻心脏病,白内障和癌症等疾病,市场需求量较大,利用生物合成法生产β-胡萝卜素具有较好的应用前景。胞质乙酰辅酶A是β-胡萝卜素合成的重要前体,如何提高胞质乙酰辅酶A的含量是高效合成β-胡萝卜素的关键。该研究利用米曲霉(Aspergillus oryzae)来源的ATP-柠檬酸裂解酶基因(ATP-citrate lyase,ACL)转化出发菌株ZS20,同时过表达酿酒酵母(Saccharomyces cerevisiae)自身CAB 1基因,从而增加胞质乙酰辅酶A含量,β-胡萝卜素产量显著提高,菌株L5的摇瓶发酵产量达到333.3 mg/L,是出发菌株的2.1倍。在此基础上,菌株L5回补缺陷的腺嘌呤、赖氨酸、亮氨酸和色氨酸合成关键基因得到菌株L11,β-胡萝卜素摇瓶发酵产量为378.1 mg/L。经发酵罐发酵优化后,产量提高到1152.7 mg/L。

微泡菌QZHA1褐藻胶裂解酶MAAL1的酶学性质研究

为探究Microbulbifer sp.QZHA1褐藻胶裂解(Escherichia coli)酶MAAL1的酶学性质,将褐藻胶裂解酶基因maal1构建至pET-28a表达载体并利用大肠杆菌BL21(DE3)进行异源表达。研究发现:重组酶MAAL1与来源于Microbulbifer sp.ALW1菌株的褐藻胶裂解酶(WP_23625014.1)同源性最高,为93.69%,且与PL7家族蛋白聚为一支;重组酶MAAL1最适温度为35℃,最适pH为7.5,在pH为5.5~10.5范围内保存24 h仍能保持60%以上的酶活力;MAAL1具备良好的耐有机溶剂特性,在测试的9种有机溶剂中,除异丙醇外,其他有机溶剂在添加量达到30%(体积分数)后,酶活力依然保持在59%以上;重组酶MAAL1最适条件下酶活力为4.3 U/mg,米氏常数(K_(m))值为1.08 mg/mL,最大反应速率(V_(max))为4.75 mg/(mL·min),催化常数(Kcat)值为4.52 s^(-1);重组酶MAAL1对聚β-D-甘露糖醛酸(polymannuronic acid,PolyM)具有底物特异性,对聚α-L-古罗糖醛酸(polyguluronic acid,PolyG)无降解能力;薄层层析分析显示,MAAL1降解海藻酸钠的主要终产物是单糖、二糖和三糖,降解PolyM的主要终产物是二糖和三糖,降解MG杂合片段(heteropolymeric MG blocks,PolyMG)的主要终产物为单糖和二糖,故MAAL1是一种内切型聚甘露糖醛酸裂解酶。重组酶MAAL1具有良好的pH和有机溶剂耐受性,对PolyM具有底物特异性且不降解PolyG,该特性褐藻胶裂解酶首次在微泡菌属中被发现,该酶可为制备甘露糖醛酸寡糖(mannuronate oligosaccharides,MAOS)提供新的候选用酶。

弧菌DS32褐藻胶裂解酶基因vralg1的异源表达和酶学性质研究

对从福建省东山湾沉积物样品中筛选到的菌株Vibrio sp. DS32的褐藻胶裂解酶基因vralg1进行克隆和异源表达,并对其酶学性质进行评估。以DS32基因组为模板,克隆褐藻胶裂解酶基因vralg1,构建了pET-vralg1重组表达载体,并在大肠杆菌中实现了异源表达,对重组酶VRALG1的酶学性质、底物特异性和完全降解产物等进行了测定。结果表明:重组酶VRALG1最适温度为35℃,在5~50℃范围内相对酶活力达到80%以上,最适pH为6.5~7.5,在pH为6.0~9.0范围内保温1 h后相对酶活力在90%以上;重组酶VRALG1最大反应速率为5.919 mmol/(L·min),米氏常数为3.712 mmol/L,最适条件下比活力为5.874 U/mg;K^(+)、Cs^(+)、Na^(+)、咪唑和乙醇对酶活性影响较小,5 mmol/L或50 mg/mL浓度下相对酶活力保持90%以上,EDTA对酶的抑制作用明显,1 mmol/L浓度下可使酶完全失活;重组酶VRALG1对海藻酸钠和聚古罗糖醛酸具有较高的降解活性,TLC分析显示产物主要为单糖、二糖和三糖混合物,结合底物特异性分析,推测重组酶VRALG1是具有明显聚古罗糖醛酸偏好性的内切型双功能褐藻胶裂解酶。本研究成功克隆了弧菌DS32中褐藻胶裂解酶基因并实现了其在大肠杆菌中的异源表达,所得重组酶VRALG1具有优良的海藻酸钠降解活性和明显的聚古罗糖醛酸偏好性,可以用于制备低聚合度的褐藻寡糖。

Key genes expression of reductive tricarboxylic acid cycle from deep-sea hydrothermal chemolithoautotrophic Caminibacter profundus in response to salinity, pH and O_2

CO2 fixation pathway of Caminibacter profundus, a chemolithoautotrophic e-Proteobacteria from deep-sea hydrothermal vent, was determined and characterized by genetic and enzymatic analyses. Gene expression of key enzymes for CO2 fixation in response to salinity, pH and O2 in Medium 829 were also investigated. The results demonstrate that C. profundus contained aclB, porA and oorA, the genes encoding key enzymes of reductive tricarboxylic acid （rTCA） cycle. However, genes fragments of cbbL and cbbMencoding key enzyme of Calvin cycle were not recovered. Key enzymatic activities of ATP citrate lyase （ACL）, pyruvate： ferredoxin oxidoreductase （POR） and 2-oxoglutarate： ferredoxin oxidoreductase （OOR） were also present in C. profun- dus. The combination of genetic and enzymatic analyses confirm that C. profundus adopted rTCA cycle for carbon assimilation. The results of aclB and oorA relative expressions of C. profundus demonstrate that the ranges of environmental factors for high genes expression were sea salt 3.0%-5.0% （optimum 3.0%）, pH 5.0-6.5（optimum pH 6.5）, anaerobic to microaerobic conditions （optimum 1.0% 02）. Gene expression pat- terns under different conditions show similar patterns with bacterial growth, revealing that key rTCA cycle genes provided molecular basis for bacterial growth and propagation. Our results suggest that C. profun- dus could regulate key genes of rTCA cycle for carbon assimilation and energy metabolism in response to environmental fluctuations in hydrothermal vent.

竹醋液对常温贮藏砂糖橘的保鲜效果

以不同浓度(50、150、250 mL/L)竹醋液(bamboo pyroligneous acid,BPA)和蒸馏水(对照)处理健康、机械造伤及接种指状青霉的砂糖橘,测定常温(25±1)℃贮藏过程中果实腐烂率、果实侵染率、病斑指数、果皮总类黄酮、总酚含量和苯丙氨酸解氨酶(phenylalanine ammonia lyase,PAL)、过氧化物酶(peroxidase,POD)活性等指标评价贮藏效果。结果显示,50、150 mL/L BPA处理能降低常温贮藏期间果实腐烂率(36 d时处理组比对照组低15%),提高果皮总类黄酮含量、PAL和POD活性(贮藏0~20 d),此外还降低了机械伤果实腐烂率。250 mL/L BPA处理能减缓指状青霉接种后砂糖橘的腐烂和病斑扩展,显著提高果皮总酚、总类黄酮含量和PAL活性(P<0.05)。综上,150 mL/L BPA可显著提高砂糖橘的常温贮藏性(P<0.05),而250 mL/L BPA抑制指状青霉的效果较好。

氨基酸序列分析揭示苯丙氨酸/酪氨酸解氨酶在陆生植物中的演化

苯丙烷途径在陆生植物木质素和黄酮类等次生代谢产物的生物合成中至关重要,其中一步必需的反应为苯丙氨酸解氨酶(PAL)将底物苯丙氨酸脱氨催化为下游产物。最新研究发现了能够同时催化苯丙氨酸和酪氨酸脱氨的双功能酶—苯丙氨酸/酪氨酸解氨酶(PTAL),但是对诸多问题,例如怎样精准鉴定PAL和PTAL、植物PAL演化为PTAL其氨基酸序列发生了什么变异、哪些植物类群包含PTAL等,仍属未知。为了探索PAL基因家族在陆生植物中的演化及关键变异,本研究利用PAL氨基酸全长序列构建了陆生植物不同类群系统发育树,比较和分析了PAL和PTAL氨基酸序列并模拟了其蛋白质三维构象。结果表明,以PAL氨基酸序列构建的系统发育树,能够反映已知陆生植物的系统关系,PAL和PTAL的氨基酸序列中存在着8个差异位点,其中121和123这两个关键位点非常稳定,可以联合用于准确鉴定包含PAL和PTAL的植物,并且I121L和F123H的关键变异,导致只能与苯丙氨酸结合的PAL演化为也能同时结合酪氨酸的PTAL。

Cis- and Trans-Cinnamic Acids Have Different Effects on the Catalytic Properties of Arabidopsis Phenylalanine Ammonia Lyases PAL1, PAL2, and PAL4

Abstract: Cis-cinnamic acid (CA) is a naturally occurring compound, presumably converted from trans-CA in higher plants. To investigate the effect of cis-CA on the activity of Arabidopsis phenylalanine ammonia lyase (PAL), AtPAL1, AtPAL2, and AtPAL4 genes were isolated using reverse transcription poly-merase chain reaction. These genes were fused to a glutathione S-transferase gene and overexpressed in a heterologous prokaryotic system of Escherichia coli. The purified PAL1, PAL2 and PAL4 enzymes were characterized biochemically to determine the effects of cis-CA on the kinetic parameter Km. The results showed that cis-CA is a competitive inhibitor for PAL1, but not PAL2 and PAL4, whereas trans-CA acts as a competitive inhibitor for all three PAL isomers, suggesting that cis- and trans-CA have different effects on the catalytic activity of PAL.

双酶法制备L-天冬氨酸的循环生产工艺

目前,工业上采用化学法将马来酸转化为富马酸,然后用L-天冬氨酸裂解酶菌株全细胞催化富马酸和氨水生产L-天冬氨酸,并采用硫酸酸化方法使L-天冬氨酸结晶。两步催化反应步骤繁琐,并且全细胞催化富马酸为L-天冬氨酸的菌体消耗量大,硫酸结晶法会产生废品硫酸铵。为了克服以上不足,将两步反应合并,利用实验室构建的马来酸异构酶和L-天冬氨酸裂解酶共表达的大肠杆菌(Escherichia coli BL21(DE3)Bptm-Easp)细胞裂解液催化马来酸铵生产L-天冬氨酸;对酶催化反应条件进行了优化,得到最佳条件:采用pH8.0的200 g/L马来酸铵溶液作为底物,加入马来酸异构酶总活力为2000 U/L的E.coli BL21(DE3)Bptm-Easp细胞裂解液,于40℃反应24 h,马来酸可转化完毕;反应液经活性炭脱色后,采用马来酸代替硫酸使L-天冬氨酸结晶,经洗涤、干燥后得到L-天冬氨酸晶体,结晶收率达到81.80%;采用碳酸氢铵和少量氨水中和结晶后的马来酸滤液,作为下一批次反应的底物溶液;在此基础上进行了4批次马来酸滤液转化反应,马来酸转化率均在99%以上,L-天冬氨酸结晶收率均在80%以上,L-天冬氨酸纯度达到98%以上。

黑曲霉酸性果胶裂解酶在大肠杆菌中的表达和酶学性质研究

酸性果胶裂解酶能以β-反式消去作用断裂果胶中的α-1,4糖苷键形成不饱和寡聚半乳糖醛酸,对食品等工业中高酯化果胶降解具有重要意义。该研究将黑曲霉Aspergillus niger AG11来源的酸性果胶裂解酶(pelA)在大肠杆菌Escherichia coli BL21(DE3)中进行表达,并进行了蛋白标签优化和酶学性质研究。最终确定在N端融合碳水化合物结合结构域(carbohydrate-binding structural domain,CBM)时,pelA的胞内酶活力最高,达到3.25 U/mL,比初始条件下提高4.12倍。对CBM切除前后pelA的酶学性质进行测定,结果表明pelA的比酶活力、最适反应温度及最适反应pH分别为15.45 U/mg、40℃和5.0,pelA在30~50℃孵育2 h保持80%以上活性,这与pelA+CBM相同,在40℃、pH为4.0~5.0时孵育2 h,相对酶活力保持在60%以上,此时pelA+CBM具有80%以上的活性。以柑橘果胶为底物,pelA的K m和k cat分别为4.44 mmol/L和31.44 s^(-1)。该研究结果为pelA的分子改造和在工业生产中的应用奠定了基础。

苯丙氨酸解氨酶和绿原酸与小麦抗赤霉病性的关系

对小麦赤霉病不同抗性的苏麦三号(R)、扬麦四号(MR)和南农824(S)三个品种的苯丙氨酸解氨酶(PAL)比活力(u·hr^(-1)·mg^(-1)protein),在拔节期的顶叶中分别测得6.26,12.28和25.12;在孕穗期的麦穗中分别测得140.1,143.3和167.7。滴注接种后8天,苏麦三号、扬麦四号和南农824的病穗中 PAL 比活力与健康穗中的比值分别为0.53,0.67和1.34。用 HPLC 法测定麦穗中绿原酸含量(μg·g^(-1)dry WT),在接种4、8和12天后的苏麦三号穗中分别为32.4,52.4和27.7;扬麦四号穗中分别为60.0,68.0和94.4;南农824穗中分别为123.8,142.2和158.9。绿原酸含量在苏麦三号接种穗中均最低,在南农824接种穗中均最高。禾谷镰刀菌分生孢子在0.05%、0.1%和0.2%的绿原酸溶液中萌发数比对照分别增加214%,614%和970%。上述结果表明,小麦麦穗接种后的 PAL 比活力变化以及绿原酸含量与其抗赤霉病程度呈负相关。

蝴蝶兰组培褐变与酚酸类物质及相关酶活性的关系

【目的】寻找与蝴蝶兰褐变相关的主要酚酸,并了解与总酚含量及相关酶活性之间的关系。【方法】以3个褐变程度不同的蝴蝶兰品种P.‘China Best Girl’(A1)、P.amabilis BL.‘Jude Butterfly’(B3)和Dtps.King Shiang’sRose×Jetgreen Firbird(R4)为材料,利用高效液相色谱对其进行了9种酚酸的定性定量分析,并在以叶片为外植体的初代培养过程中,研究了总酚含量、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)和过氧化物酶(POD)的动态变化。【结果】初步证明绿原酸、邻苯二酚、儿茶酚、咖啡酸及没食子酸、对羟基苯甲酸、香豆酸可能与蝴蝶兰褐变相关,苯甲酸对蝴蝶兰褐变影响很小;在褐变过程中,褐变程度高的品种,总酚含量也高;PAL和PPO活性与褐变程度呈正相关,POD与褐变有很大关系;总酚含量与PAL活性呈正相关,与PPO和POD活性呈负相关。【结论】初步阐明了与蝴蝶兰褐变相关的酚酸种类,以及褐变程度与总酚含量及相关酶活性的关系。