Preliminary report of NAD+-dependent amino acid dehydrogenase producing bacteria isolated from soil

BACKGROUND: Amino acid dehydrogenases (L-amino acid: oxidoreductase deaminating; EC 1.4.1.X) are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD+, NADP+ or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria (PKU), maple syrup urine disease (MSUD), homocystinuria (HCY) and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteria. METHODS: The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed. RESULTS: In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD+-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat/Km (s(-1).mM(-1)) values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83. CONCLUSION: This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria.     

Partitioning of glutenin flour of special wheat using aqueous two-phase systems

The partitioning behavior of the glutenin proteins was evaluated in aqueous two-phase systems (ATPSs) formed by sulfate salts (lithium or sodium) and poly(ethylene glycol) (PEG) with average molar mass of 1500 g mol⁻¹ or 4000 g mol⁻¹. The partition coefficients for the glutenin proteins in each ATPS were investigated as a function of the temperature (278.2 K–318.2 K), tie line length (TLL) and electrolyte nature. In all ATPS, the majority of glutenin proteins spontaneously concentrate in the polymer-rich phase (K_p > 1). The partition coefficient is very dependent on the salt nature and the ATPS formed by PEG + lithium sulfate presents higher K_p values as compared with the ATPS formed PEG + sodium sulfate. An increase of molar mass of polymer promotes a decrease of K_p. Thermodynamic parameters of transfer (Δ_trG, Δ_trH and Δ_trS) were obtained by the application of the Van’t Hoff equation (VHE). The values obtained by VHE indicate that the transfer of glutenin proteins to the polymer-rich phase has an enthalpic origin.     

大豆凝集素、脲酶和胰蛋白酶抑制剂的分离纯化

采用硫酸铵盐析、羟基磷灰石柱层析以及Sephadex凝胶过滤等方法,同时从大豆中分离纯化大豆凝集素、脲酶和胰蛋白酶抑制剂。三者的蛋白质回收率分别为0.39％、0.23％和0.13％。本方法与DEAE—Sephadex A_(50)柱层析及酸化—Sepharose 4 B、胰蛋白酶—Sepharose 4 B、卵类粘蛋白—Sepharose 4 B亲和层析分离纯化方法作了比较。本法简便易行,效果较好。     

应用~(15)N标记氮肥研究肥料氮素在橡胶树幼苗叶片中的动态变化

本文以热研7-33-97芽接橡胶幼苗为试材,应用15N示踪技术研究相同施氮量情况下,肥料氮素在橡胶树幼苗叶片中的动态变化规律。氮肥品种为15N标记的CO(NH2)2、15N标记(NH4)2SO4、15N标记Ca(NO3)2和15N双标记NH4NO3。结果表明,橡胶树幼苗叶片对四种15N标记氮肥吸收分配势(Ndff值)总体上均随施肥时间的延长表现为逐渐增加的趋势;但各氮肥处理间叶片Ndff值存在显著差异,同一生长期橡胶树幼苗叶片对肥料氮的征调能力总体上表现为:NH4NO3-15NCa(NO3)2-15N(NH4)2SO4-15NCO(NH2)2-15N。通过分析不同生长时期叶片叶绿素相对含量(SPAD值)和Ndff值,发现两者之间总体呈极显著的正相关关系。     

Isolation,partial purification and characterization of a potato peel ∞-solanine cleaving glycosidase

An enzyme which hydrolyzes the rhamnose from ∞-solanine was isolated and partially purified by ammonium sulfate precipitation from the peels of Kennebec and Wauseon potatoes. Enzyme activity was found to be present in the 60 to 80% ammonium sulfate fraction. Enzyme activity was increased by the use of non-ionic detergents with a mixture of Triton X-100:Triton CF-54 (2:1, w/w) providing the greatest increase in activity. The pH optimum for the rhamnosidase for both varieties was found to be 6.0 while of the temperatures evaluated 42 °C was the optimum temperature. The effect of substrate concentration on enzyme activity was determined for both varieties and the Vmax and Km were calculated from Lineweaver-Burke double reciprocal plots. The Vmax and Km for the Kennebec and Wauseon rhamnosidases were found to be 0.27 mg/ml and 5.4 x 10^-4 M and 0.48 mg/ml and 8.2 x 10^-4 M, respectively.     

玉米叶片谷氨酸合酶(GOGAT)的纯化和特性研究

玉米叶片的谷氨酸合酶(GOGAT)经硫酸铵分级沉淀处理后,与粗酶液相比较被纯化了1.71倍。在缓冲溶液pH值为7、酶溶液体积为6.25 mL、反应时间为12 min的条件下,谷氨酸合酶(GOGAT)的活性最高,该酶对底物谷氨酰胺(L-GIn)、α-酮戊二酸(α-KG)和电子供体(NADH)专一,其最适浓度分别为55、60、0.3 mmol/L,Km值分别为4.13、7.78、0.001 4 μmol/L。     

Efficient Purification of R-phycoerythrin from Marine Algae (Porphyra yezoensis) Based on a Deep Eutectic Solvents Aqueous Two-Phase System

R-phycoerythrin (R-PE), a marine bioactive protein, is abundant in Porphyra yezoensis with high protein content. In this study, R-PE was purified using a deep eutectic solvents aqueous two-phase system (DES-ATPS), combined with ammonium sulphate precipitation, and characterized by certain techniques. Firstly, choline chloride-urea (ChCl-U) was selected as the suitable DES to form ATPS for R-PE extraction. Then, single-factor experiments were conducted: the purity (A₅₆₅/A₂₈₀) of R-PE was 3.825, and the yield was 69.99% (w/w) under optimal conditions (adding 0.040 mg R-PE to ChCl-U (0.35 g)/K₂HPO₄ (0.8 g/mL, 0.5 mL) and extracting for 20 min). The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results revealed that the purified R-PE contained three main bands. One band was presented after purification in native-PAGE. The UV-vis spectra showed characteristic absorption peaks at 495, 540, and 565 nm. R-PE displayed an emission wavelength at 570 nm when excited at 495 nm. All spectra results illustrated that the structure of R-PE remained unchanged throughout the process, proving the effectiveness of this method. Transmission electron microscope (TEM) showed that aggregation and surrounding phenomena were the driving forces for R-PE extraction. This study could provide a green and simple purification method of R-PE in drug development.     

茉莉花芳樟醇β-D吡喃葡萄糖苷酶的分离与纯化

茉莉花芳樟醇 β_D吡喃葡萄糖苷酶 (LGA)丙酮粉经过pH6 0柠檬酸缓冲液萃取、 3 0 %和 80 %硫酸铵分步沉淀 ,以及CM_Toyopearl65 0M柱层析 ,用 0 - 0 5MNaCl柠檬酸缓冲液梯度洗脱酶蛋白 ,最后用快速蛋白液相色谱 (FPLC)的SephacrylS_2 0 0柱分离系统纯化出了LGA ,LGA蛋白的SDS_PAGE图谱在分子量 1 4 4万道尔顿处显示了一条谱带。它可以催化芳樟基 β_D_吡喃葡萄糖苷分解释放芳樟醇     

蒸汽爆破处理对剑麻纤维组分分离的影响

采用高温、高压水蒸汽瞬间爆破处理剑麻纤维。通过处理前后剑麻纤维的水溶物、碱溶物、木质素含量、纤维素含量的比较,分析研究了蒸汽爆破处理条件对剑麻纤维组分分离效果的影响。试验结果表明:爆破前预处理、处理温度(压力)、维压时间是影响组分分离的重要因素:随着处理强度的增大,纤维中木质素含量降低,纤维素含量增大;爆破前的不同试剂预浸泡处理中,质量分数为17.5％的NaOH和质量分数为0.1％的H2SO4预浸泡处理效果较优,水和质量分数为1％的NaOH浸泡处理效果较差。      

Extraction of oil from Jatropha curcas L. seed kernels by enzyme assisted three phase partitioning

Three phase partitioning has been evaluated for extraction of oil from Jatropha curcas L. seeds. This process consisted of simultaneous addition of t-butanol (1:1, v/v) and 30% (w/v) ammonium sulphate to the slurry prepared from Jatropha seed kernels. Combination of sonication and enzyme treatment with a commercial preparation of fungal proteases at pH 9, led to 97% oil yield within 2 h.     

茶叶黄酮类物质的双水相系统纯化及抗氧化研究

采用PEG/盐的双水相系统纯化茶叶黄酮,探究萃取机理及其抗氧化活性。以茶叶为原料,茶叶黄酮得率为指标;采用单因素和响应面法试验设计,研究PEG的种类和用量、盐的种类和用量、温度、pH对茶叶黄酮提取率的影响,根据Box-Behnken中心组合方法进行3因素（Na₂SO₄的质量分数、PEG600、MgCl₂添加量）3水平的试验设计,进行响应面（RSM）分析。通过分析还原力和DPPH体系,评估不同浓度黄酮的抗氧化活性,并与参照抗氧化剂BHT和Vc进行比较。结果显示最佳条件：pH为5,萃取温度30℃,5mL 15% Na₂SO₄,6g PEG600,MgCl₂的添加量为2.9%,萃取率为96.31%。本方法所形成双水相体系,操作简便,萃取率高,方法重现性好;茶叶黄酮有较强的铁还原能力和清除DPPH自由基能力,茶叶黄酮是一个潜在的天然安全的有效的抗氧化剂。     

硫酸铝和硫酸酸化拟建茶园土壤的初步研究

用Ａｌ２（ＳＯ４）３和Ｈ２ＳＯ４作为酸化剂对拟植茶园的中偏碱性土壤进行土壤酸化试验，结果表明，两种酸化剂均能迅速酸化土壤，Ａｌ２（ＳＯ４）３的酸化效果高于Ｈ２ＳＯ４，前者能使土壤ｐＨ值明显下降，交换性酸总量显著上升。经淋洗后，交换性钙、镁含量明显下降，交换性钾、锰含量变化不大     

水稻种子脂氧合酶基因OsLOX1的原核表达、纯化及鉴定

脂氧合酶是动植物体内催化脂质降解的关键酶,也是茉莉酸合成途径的第一个关键酶。以先前克隆到的水稻种子脂氧合酶基因OsLOX1全长cDNA为模板,用含有特异酶切位点的P1、P2为引物,通过PCR方法将它构建到大肠杆菌表达载体pET30a(+)上并转化到大肠杆菌菌株BL21（DE3）中,获得相应的重组工程菌。经过20℃条件下的IPTG诱导,OsLOX1重组蛋白在BL21（DE3）菌株中得到表达,经生化特性分析,发现该重组蛋白具有LOX催化活性,其最适pH和温度分别为4.8和30℃。该重组子可进一步应用于体外生产茉莉酸和研究植物种子LOX结构与功能的关系。     

海南省7县市美人蕉瘟病菌的生物学特性

测试了近年来在我国海南引起美人蕉瘟病大流行的PyriculariacannaecolaHashioka菌丝生长的温度、pH、营养要求等生物学特性。结果表明:该菌菌丝生长的温度范围为15~35℃,最适温度30℃;该菌对pH值有较大的适应性,在pH3~9内均可生长,最适pH值为5.5;能利用甘油、淀粉、葡萄糖和蔗糖作碳源,甘油是最适的碳源;能利用KNO3、谷氨酸、硫酸铵、磷酸二氢铵和硝酸铵作氮源进行生长,但KNO3优于L-谷氨酸,硫酸铵优于磷酸二氢铵和硝酸铵。来自不同地区的7个菌株生物学特性基本相似。     

不同氮源条件下橡胶树小苗NH4+和NO3-的吸收特征

采用改进常规耗竭法,研究(NH)SO4,KNO3,NH4NO3三种氮源条件下橡胶树小苗对N4+和NO3-的吸收特性.结果表明:不论是单一氨源(NH4+-N或NO3--N)还是NH4+-N和NO3--N的混合氮源,橡胶树小苗对NH4+的吸收量均大于NO3-的吸收量,其差异在浓度大于0.5 mmol/L的混合氮源下达显著水平.混合氮源条件下,不论是NH4+还是NO3-的吸收量,均比单一氮源条件下的大,且其差异分别在浓度小于1.25 mnlol,L(NH4-)和0.25 mmol/L(NO3-)时达显著水平.吸收动力学特征表现为,单一氮源条件下,橡胶树小苗对NH4+的最大吸收速率和离子亲和力大于NO3-,表明小苗更喜好NH4+-N.而在NH4+-和NO3-N的混合氮源条件下,橡胶树小苗吸收NH4+的Vma值和离子亲和力都较单一氮源时高;吸收NO3-的Vma和KmI值都有所下降,但Km值的下降幅度比Vma值的大,说明混合氮源条件能同时促进橡胶树小苗对NH4+和NO3-的吸收.与单一NH4++N营养相比,橡胶树小苗可能更喜好NH4+-N和NO3--N混合营养.用LB法和H法2种方法处理数据,橡胶树小苗吸收NH4+和NO3-的动力学特性基本相同,不同之处仅在于H法所得的参数值Vma和Km普遍略大于LB法的.     

根际溶氧量与氮素形态对水稻根系特征及氮素积累的影响

 为研究根际溶氧量和氮素形态对水稻根系生长及对氮素利用的影响,设计了两个试验：1)分别以铵硝混合营养（NH4NO3）和全铵营养[(NH4)2SO4]为氮源,在营养液中将杂交籼稻国稻1号和常规粳稻秀水09培养6周,在第4周和第6周时取样测定植株的生物量和氮素含量;2) 将上述铵硝混合营养下培养4周的国稻1号水稻植株,以分根培养的方式进行不同溶氧量处理,测定水稻生物量、根系形态和氮积累量的差异。结果表明: 1）根际溶氧量较低时(溶氧量0~1.0 mg/L),铵硝混合营养比单一的铵态氮营养显著提高植株的生物量,国稻1号生物量增加69%,秀水09增加41%;铵硝混合营养显著提高了水稻的根系数量、最长根长、根干质量和根系活力以及植株的氮积累量,国稻1号的地下部和地上部氮积累量分别提高60%和52%,秀水09则分别提高了41%和33%。2) 分根实验中,铵硝混合培养的国稻1号,其增氧(溶氧量8.0~9.0 mg/L)处理的根系生物量增加21.6%, 根系数量、最长根长和根体积分别增加27%、14%和10%,而氮积累量提高了11%。增氧和铵硝混合营养均对促进水稻根系生长和氮素积累具有正互作效应。     

茶树叶绿素酶提取、分离纯化与性质的初步研究

在优化茶树叶绿素酶提取及活性分析条件的基础上,对茶树叶绿素酶的分离纯化及其性质进行了研究,结果表明:加入与鲜叶等量PVP,用pH7.5的磷酸二氢钠-磷酸氢二钠缓冲液提取的叶绿素酶活性最高,叶绿素酶的活性分析最佳条件为在45℃条件下反应15min;经过分离纯化后,叶绿素酶的纯度提高了55.96倍,酶的得率为9.45%,SDS-PAGE结果显示有一条蛋白质的条带,酶的亚基分子量为37.2kD;对纯化后的叶绿素酶的性质研究表明,该酶最适pH为7.5,45℃时候反应活性最高,在4℃保存条件下,随着存放时间的延长,酶活性逐渐下降,保存一个月后叶绿素酶活性剩余45%,用2mmol/L和7mmol/L的金属离子(Ca2+、Mg2+、Zn2+、Fe3+)、EDTA处理,都对叶绿素酶活性有不同程度的促进作用。     

Effects of Rhizosphere Dissolved Oxygen Content and Nitrogen Form on Root Traits and Nitrogen Accumulation in Rice

Dissolved oxygen and nitrogen form have important effects on rice root growth and nitrogen availability.An indica hybrid rice,Guodao 1,and a conventional japonica rice,Xiushui 09,were cultured in hypoxic nutrient solution with NH4NO3 or(NH4)2SO4 as the nitrogen source for six weeks in pools.A portion of the Guodao 1 seedlings after treatment in the pools for four weeks were transferred to a split-root system at different dissolved oxygen contents and cultured for an additional two weeks.Biomass,root morphol...     

X-ray photoelectron spectroscopy characterization of gold nanoparticles functionalized with amine-terminated alkanethiols

Gold nanoparticles (AuNPs) functionalized with a short chain amine-terminated alkanethiol (HS-(CH(2))(2)NH(2) or C2 NH(2)-thiol) are prepared via a direct synthesis method and then ligand-exchanged with a long chain amine-terminated alkanethiol (HS-(CH(2))(11)NH(2) or C11 NH(2)-thiol). Transmission electron microscopy analysis showed the AuNPs were relatively spherical with a median diameter of 24.2?±?4.3 nm. X-ray photoelectron spectroscopy was used to determine surface chemistry of the functionalized and purified AuNPs. The ligand-exchange process was monitored within the time range from 30 min to 61 days. By the fourth day of exchange all the C2 NH(2)-thiol molecules had been replaced by C11 NH(2)-thiol molecules. C11 NH(2)-thiol molecules continued to be incorporated into the C11 NH(2) self-assembled monolayer between days 4 and 14 of ligand-exchange. As the length of the exchange time increased, the functionalized AuNPs became more stable against aggregation. The samples were purified by a centrifugation and resuspension method. The C2 NH(2) covered AuNPs aggregated immediately when purification was attempted. The C11 NH(2) covered AuNPs could be purified with minimal or no aggregation. Small amounts of unbound thiol (～15%) and oxidized sulfur (～20%) species were detected on the ligand-exchanged AuNPs. Some of the unbound thiol and all of the oxidized sulfur could be removed by treating the functionalized AuNPs with HCl.     

不同浓度配比的PEG、二甲基亚砜和水杨酸引发对玉米种子抗寒性的影响

以玉米杂交种创奇518新种子为材料,以不同浓度配比的聚乙二醇（PEG）、二甲基亚砜和水杨酸组合作为引发剂,采用L₉（3⁴）正交试验设计,通过低温种植处理,研究提高玉米种子抗寒性的3种引发剂的最佳浓度配比。结果表明,不同配比引发处理的玉米种子发芽势、发芽率、苗鲜重、活力指数、过氧化物酶（POD）和过氧化氢酶（CAT）活性均表现出明显差异。以浓度为20%的PEG、浓度为0.5%的二甲基亚砜和浓度为0.007%的水杨酸组合处理种子的发芽率、活力指数最高,幼苗长势整齐一致,POD和CAT较对照有明显提高。