Transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by permanganate

The chemical oxidant permanganate (MnO(4)(-)) has been shown to effectively transform hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at both the laboratory and field scales. We treated RDX with MnO(4)(-) with the objective of quantifying the effects of pH and temperature on destruction kinetics and determining reaction rates. A nitrogen mass balance and the distribution of reaction products were used to provide insight into reaction mechanisms. Kinetic experiments (at pH ～ 7, 25 °C) verified that RDX-MnO(4)(-) reaction was first-order with respect to MnO(4)(-) and initial RDX concentration (second-order rate: 4.2 × 10(-5) M(-1) s(-1)). Batch experiments showed that choice of quenching agents (MnSO(4), MnCO(3), and H(2)O(2)) influenced sample pH and product distribution. When MnCO(3) was used as a quenching agent, the pH of the RDX-MnO(4)(-) solution was relatively unchanged and N(2)O and NO(3)(-) constituted 94% of the N-containing products after 80% of the RDX was transformed. On the basis of the preponderance of N(2)O produced under neutral pH (molar ratio N(2)O/NO(3) ～ 5:1), no strong pH effect on RDX-MnO(4)(-) reaction rates, a lower activation energy than the hydrolysis pathway, and previous literature on MnO(4)(-) oxidation of amines, we propose that RDX-MnO(4)(-) reaction involves direct oxidation of the methylene group (hydride abstraction), followed by hydrolysis of the resulting imides, and decarboxylation of the resulting carboxylic acids to form N(2)O, CO(2), and H(2)O.     

Abiotic transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by green rusts

The rate and extent of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transformation was measured in the presence of carbonate and sulfate green rust suspended in solutions containing common groundwater anions. Formaldehyde (HCHO), nitrous oxide gas (N2O(g)), and ammonium (NH4+) were the major end products, accounting for about 70% of the carbon mass balance and about half of the nitrogen mass balance. Results from experiments with both 14C-RDX and LC-MS analysis indicate that the remaining carbon products are soluble and most likely small (< 50 Da). The transient appearance of 1,3-dinitro-5-nitroso-1,3,5-triazacyclohexane (MNX), 1,3-dinitroso-5-nitro-1,3,5-triazacyclohexane (DNX), and 1,3,5-trinitroso-1,3,5-triazacyclohexane (TNX) indicate that some nitro-group reduction occurred. The kinetics of RDX transformation was rapid with a half-life of less than an hour in a pH 7.0 KBr solution. Little difference in rates of RDX transformation or product distribution was observed between carbonate and sulfate green rust, and an apparent reaction order of 1.0 was measured with respect to Fe(II) in both green rusts. Phosphate anions completely inhibited RDX reduction, and carbonate and sulfate anions resulted in slower kinetics, and in some cases, an initial lag period, compared to bromide and chloride. Our results suggest that green rusts may contribute to abiotic natural attenuation of RDX in Fe-rich subsurface environments, but that it will be important to consider groundwater composition when assessing rates of attenuation.     

Abiotic transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine by Fe(II) bound to magnetite

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), a nitramine explosive, is often found as a subsurface contaminant at military installations. Though biological transformations of RDX are often reported, abiotic studies in a defined medium are uncommon. The work reported here was initiated to investigate the transformation of RDX by ferrous iron (Fe(II)) associated with a mineral surface. RDX is transformed by Fe(II) in aqueous suspensions of magnetite (Fe3O4). Negligible transformation of RDX occurred when it was exposed to Fe(II) or magnetite alone. The sequential nitroso reduction products (MNX, DNX, and TNX) were observed as intermediates. NH4+, N2O, and HCHO were stable products of the transformation. Experiments with radiolabeled RDX indicate that 90% of the carbon end products remained in solution and that negligible mineralization occurred. Rates of RDX transformation measured for a range of initial Fe(II) concentrations and solution pH values indicate that greater amounts of adsorbed Fe(II) result in faster transformation rates. As pH increases, more Fe(II) adsorbs and k(obs) increases. The degradation of RDX by Fe(II)-magnetite suspensions indicates a possible remedial option that could be employed in natural and engineered environments where iron oxides are abundant and ferrous iron is present.     

Degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) using zerovalent iron nanoparticles

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a common contaminant of soil and water at military facilities. The present study describes degradation of RDX with zerovalent iron nanoparticles (ZVINs) in water in the presence or absence of a stabilizer additive such as carboxymethyl cellulose (CMC) or poly(acrylic acid) (PAA). The rates of RDX degradation in solution followed this order CMC-ZVINs > PAA-ZVINs > ZVINs with k1 values of 0.816 +/- 0.067, 0.082 +/- 0.002, and 0.019 +/- 0.002 min(-1), respectively. The disappearance of RDX was accompanied by the formation of formaldehyde, nitrogen, nitrite, ammonium, nitrous oxide, and hydrazine by the intermediary formation of methylenedinitramine (MEDINA), MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine). When either of the reduced RDX products (MNX or TNX) was treated with ZVINs we observed nitrite (from MNX only), NO (from TNX only), N2O, NH4+, NH2NH2 and HCHO. In the case of TNX we observed a new key product that we tentatively identified as 1,3-dinitroso-5-hydro-1,3,5-triazacyclo-hexane. However, we were unable to detect the equivalent denitrohydrogenated product of RDX and MNX degradation. Finally, during MNX degradation we detected a new intermediate identified as N-nitroso-methylenenitramine (ONNHCH2NHNO2), the equivalentof methylenedinitramine formed upon denitration of RDX. Experimental evidence gathered thus far suggested that ZVINs degraded RDX and MNX via initial denitration and sequential reduction to the corresponding nitroso derivatives prior to completed decomposition but degraded TNX exclusively via initial cleavage of the N-NO bond(s).     

Phytophotolysis of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in leaves of reed canary grass

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was degraded in reed canary grass leaves exposed to simulated sunlight to primary products nitrous oxide and 4-nitro-2,4-diazabutanal. This is the first time that 4-nitro-2,4-diazabutanal, a potentially toxic degradate, has been measured in plant tissues following phytotransformation of RDX. These compounds, along with nitrite and formaldehyde, were also detected in aqueous RDX systems exposed to the same simulated sunlight. Results showed that the initial products of RDX photodegradation in translucent plant tissues were similar to products formed from aqueous photolysis of RDX. Combustion analysis of leaves following 14C-RDX uptake and subsequent light exposure revealed the presence of tissue-bound material that could not be extracted with acetonitrile. No detectable formaldehyde was emitted from the leaves. The detection of similar RDX degradation products in both aqueous and plant-based systems suggests that RDX may be initially transformed by similar mechanisms in both systems. Direct photolysis of RDX via ultraviolet irradiation passing into the leaves is hypothesized to be responsible for the observed transformations. In addition, membrane-bound "trap chlorophyll" in the chloroplasts may shuttle electrons to RDX as an indirect photolysis transformation mechanism. Results from this study indicate that reed canary grass facilitates photochemical degradation of RDX, and this mechanism should be considered along with more established phytoremediation processes when assessing the fate of contaminants in plant tissues. Plant-mediated phototransformation of xenobiotic compounds is a process that may be termed "phytophotolysis".     

Metabolism and mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine inside poplar tissues (Populus deltoides x nigra DN-34)

Poplar tissue cultures and leaf crude extracts (Populus deltoides x nigra DN-34) were exposed to [U-14C]hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and incubated under light and in the dark. Poplar tissue cultures were able to partially reduce RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), regardless of the presence or absence of light. However, further transformation of RDX, MNX, and DNX required exposure to light and resulted in the formation of formaldehyde (CH2O), methanol (CH3OH), and carbon dioxide (CO2). Similarly, transformation of RDX by poplar leaf crude extracts required exposure to light. Neither reduction of RDX to MNX and DNX nor mineralization into CO2 were recorded in crude extracts, even when exposed to light, suggesting that both processes were light-independent and required intact plant cells. Control experiments without plant material showed that RDX was partially transformed abiotically, by the sole action of light, but to a lesser extent than in the presence of plant crude extracts, suggesting the intervention of plant subcellular structures through a light-mediated mechanism. Poplar tissue cultures were also shown to mineralize 14CH2O and 14CH3OH, regardless of the presence or absence of light. These results suggest that transformation of [U-14C]RDX by plant tissue cultures may occur through a three-step process, involving (i) a light-independent reduction of RDX to MNX and DNX by intact plant cells; (ii) a plant/light-mediated breakdown of the heterocyclic ring of RDX, MNX, or DNX into C1-labeled metabolites (CH2O and CH3OH); and (iii) a further light-independent mineralization of C1-labeled metabolites by intact plant cells. This is the first time that a significant mineralization of RDX into CO2 by light-exposed plant tissue cultures is reported.     

Biotransformation of quinazoline and phthalazine by Aspergillus niger

Cultures of Aspergillus niger NRRL-599 in fluid Sabouraud medium were grown with quinazoline and phthalazine for 7 days. Metabolites were purified by high-performance liquid chromatography and identified by mass spectrometry and proton nuclear magnetic resonance spectroscopy. Quinazoline was oxidized to 4-quinazolinone and 2,4-quinazolinedione, and phthalazine was oxidized to 1-phthalazinone.     

黑曲霉B0201产单宁酶生物转化没食子酸工艺研究

该文对微生物产单宁酶发酵五倍子合成没食子酸进行了研究.并对五倍子诱导黑曲霉产生单宁酶进行了生物转化实验研究,开展了产酶条件优化实验,对酶反应转化的最适pH值、温度、反应时间、底物浓度等进行研究,得出了最佳培养条件和微生物酶法制备没食子酸的工艺,为开发五倍子的生物化工加工技术提供参考.     

Aspergillus niger CGMCC No.6640全细胞生物转化制备蔗果低聚糖

蔗果低聚糖（FOS）作为一种益生元物质,由于其具有极好的生物和功能性质,受到人们极大的关注。具有独立知识产权的菌株Aspergillus niger CGMCC No. 6640被发现能利用蔗糖制备FOS。为利用A. niger 6640的全细胞制备FOS,以蔗糖为底物,利用高效液相法对该菌株的全细胞生物转化参数进行了研究。利用色谱柱Rezex RCM-Monosaccharife Cat的高效液相法能同时检测催化产物中蔗果四糖（或蔗果五糖）、蔗果三糖、蔗糖、葡萄糖和果糖的浓度,其保留时间分别为8.403 min,8.853 min,9.705 min,11.473 min和14.683 min。全细胞生物催化剂浓度和底物浓度对FOS产量有正面的影响。而CaCl2浓度对FOS含量有负面影响。当全细胞生物催化剂浓度,初始反应pH、反应温度、反应时间、蔗糖浓度分别为60 g/L、7.0、33 ℃、40 h和600 g/L的最佳条件下,FOS含量为314.60 g/L。总之,Aspergillus niger CGMCC No.6640全细胞生物催化剂能有效地制备FOS,为其工业化提供依据。     

黑曲霉H9-30全细胞催化合成低聚异麦芽糖

以黑曲霉H9-30全细胞为催化剂转化麦芽糖生产低聚异麦芽糖,通过单因素实验确定其摇瓶最佳转化条件为:温度48℃,初始p H值4. 2,麦芽糖质量浓度为600 g/L,黑曲霉细胞添加量为15 g/L。此条件下,有效三糖(异麦芽糖、潘糖和异麦芽三糖)占总糖质量分数的50. 5%,总低聚异麦芽糖含量为63. 3%,达到低聚异麦芽糖工业生产标准。研究结果表明,利用黑曲霉全细胞催化生产低聚异麦芽糖具有较好的操作稳定性,生产IMO-50的半衰期达到20批次(10 d),有望应用于工业化生产低聚异麦芽糖。     

Identification of the gene encoding a NAD(P)H-flavin oxidoreductase coupling with dibenzothiophene (DBT)-desulfurizing enzymes from the DBT-nondesulfurizing bacterium Paenibacillus polymyxa A-1

The gene encoding NAD(P)H-flavin oxidoreductase (flavin reductase), which couples efficiently with dibenzothiophene (DBT)-desulfurizing monooxygenases of Rhodococci, was cloned from a DBT-non-desulfurizing bacterium Paenibacillus polymyxa A-1 in Escherichia coli, and designated as flv. Cell-free extracts from the recombinant exhibited a flavin reductase activity about forty times higher than that of the E. coli carrying the vector DNA only. Nucleotide sequence analysis reveals that the gene product consists of 208 amino acids and showed about 27%, 32% and 21% identity in amino acid sequence with FRase I, the major flavin reductase of Vibrio fischeri, the NADH dehydrogenase of Thermus thermophilus and several members of the nitroreductase family, respectively. The coexpression of flv with two kinds of desulfurizing genes, dszABC and tdsABC, in E. coli enhanced the rate of DBT degradation by about 10 and 5 times as high as in the case without flv, respectively.     

黑曲霉Aspergillus niger H菌株所产酸性蛋白酶的质谱法鉴定及酶学特性

采用质谱指纹(MS mapping)分析方法,直接对黑曲霉Aspergillus niger H菌株发酵液中所产蛋白进行鉴定,通过与质谱数据库比对分析,确定此蛋白为曲霉酸性蛋白酶Aspergillopepsin I(EC.3.4.23.18).对其酶学性质的研究表明,此蛋白酶分子量约为47kDa,最适温度为50℃,温度稳定性范围为30～50℃,最适pH为3.0,pH稳定性为2～5,其表征与其它已报道的曲霉酸性蛋白酶基本一致,证实此质谱指纹分析方法可快速有效地鉴定混合发酵液中的未知蛋白,并省去中间繁琐的分离纯化步骤.     

单增李斯特菌nox基因的克隆、表达以及功能

以Gen Bank中报道的单增李斯特菌(Listeria monocytogenes,Lm)野生型菌株EGDe的nox基因(Gen Bank ID:986631)为研究对象,探讨在高等动植物中普遍存在的烟酰胺腺嘌呤二核苷酸磷酸氧化酶在Lm中是否也可以介导产生活性氧(reactive oxygen species,ROS)。首先通过诱导nox基因在BL21中表达产生Nox蛋白,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western blot鉴定该蛋白质分子质量;然后构建过表达菌株EGDe-nox,测定ROS产生情况,并使用实时荧光定量-聚合酶链式反应检测nox基因的过表达对Lm毒力基因表达的影响。结果表明,经鉴定Nox蛋白分子质量约为33 k D,过表达菌株EGDe-nox与对照组EGDe的ROS产生量相比并无多大变化,nox基因的过表达会导致与侵袭相关的基因act A、inlA和inlB以及毒力基因prfA的表达上调。由此推测,该Nox蛋白不能独立主导ROS的产量,但其过表达却可以增强毒力基因表达上调。本研究为继续探讨细菌中Nox的作用提供一定的参考依据。     

The aliphatic isothiocyanates erucin and sulforaphane do not effectively up‐regulate NAD(P)H:quinone oxidoreductase (NQO1) in human liver compared with rat

Isothiocyanate up‐regulation of hepatic NAD(P)H:quinone oxidoreductase (NQO1) and glutathione S‐transferases (GSTs) is an integral mechanism of their chemoprevention. In this paper, for the first time, the potential of the isothiocyanates erucin and sulforaphane to modulate these enzymes was investigated in two human livers and compared to rat liver. Precision‐cut liver slices were incubated with erucin or sulforaphane (1–50 μM). Both isothiocyanates elevated NQO1 activity in rat slices that was paralleled by a fourfold rise in protein levels. No change in activity was noted in human slices, and only a weak rise in protein levels, < 10% of that in rat, was observed in only one of the human livers, whereas the other was refractive. GST activity, assessed with three substrates, was elevated in rat slices treated with either isothiocyanate, and was accompanied by a rise in GSTα and GSTμ, but not GSTπ, protein levels. A rise in activity and in GSTα and GSTμ protein levels was also noted in one of the human livers. It appears that erucin and sulforaphane elevate GST expression in isoform‐specific manner in both rat and human liver, whereas NQO1 is inducible by these compounds only in rat liver and very poorly in human liver.     

离子液体中米曲霉果糖基转移酶催化合成蔗果三糖的研究

对来自于米曲霉的果糖基转移酶在离子液体中催化蔗糖反应生成蔗果三糖的过程进行了研究。考察了盐平衡、底物蔗糖和酶量的比率、温度、时间、pH、离子液体和酶量的比率对催化得率的影响,其中离子液体的水活度采用盐的高水合物和低水合物组成的水合盐进行控制。结果表明,最优的催化反应条件为:反应温度40℃,pH6.2,底物浓度60%,反应时间24h。关于离子液体与酶的重复利用方面,离子液体性质和酶的催化活力有待于进一步研究。     

Hexahydro-1,3,5-trinitro-1,3,5-triazine transformation by biologically reduced ferrihydrite: evolution of Fe mineralogy, surface area, and reaction rates

Microbial respiration of Fe(III) oxides has been shown to produce reduced Fe phases that are capable of transforming a variety of oxidized contaminants. Little data, however, are available on how these Fe phases evolve over time and how this evolution may affect their ability to reduce contaminants. Here,the evolution and reactivity of biologically reduced ferrihydrite were monitored over a period of 14 months. Solids were collected from a culture of Geobacter metallireducens (GS-15) thatwas incubated with ferrihydrite (as the electron acceptor) for 0, 7, 10, 20, 75, and 400 days. Mineralogical composition and surface area of the biologically reduced solids were characterized using M?ssbauer spectroscopy, X-ray diffraction, and BET with N2 adsorption. By day 10, ferrihydrite began to transform, and a nanoparticle magnetite/maghemite phase, as well as two ferrous phases, was observed. One of the ferrous phases was identified as siderite, whereas the other could not be positively identified. Likely candidates, however, include Fe(OH)2(s) or an adsorbed Fe(II) species. Over the next few months, ferrihydrite was completely reduced and evolved into a mixture containing about 70% magnetite/maghemite, 19% siderite, and 11% of the second Fe(II) phase. The effect of incubation time on the reactivity of the biologically reduced solids was evaluated by measuring the kinetics of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) transformation. The only products observed were the three reduced nitroso products. Rate coefficients (k) for RDX transformation were dramatically influenced by incubation time with half-lives of about 1 month observed in the presence of solids incubated for 10 and 20 days, 3 months with solids incubated for 75 days, and negligible removal with solids incubated for 400 days. The loss of reactivity was not directly correlated to any one mineralogical variable but may be due to particle size or surface chemistry changes in the reactive Fe phase or to cell die-off and the accumulation of cell lysis products after consumption of the electron acceptor. The dramatic effect of incubation time on the rate of RDX removal highlights a potential limitation of studying complex systems, as we have here, in batch reactors and suggests that incubation time is an important variable to consider when measuring and comparing rates of contaminant reduction.     

Production optimization,purification, and characterization of a novel acid protease from a fusant by Aspergillus oryzae and Aspergillus niger

The production of a novel acid protease was enhanced by 44 % through statistical optimization. The cultural parameters, such as inoculum size, temperature, moisture content, and incubation time, were 8.59 × 10⁵ g^?1 dry koji, 31 °C, 57 %, and 86 h, respectively. This novel acid protease was purified by 17 folds with a recovery yield of 33.56 % and a specific activity of 4,105.49 U mg^?1. Far-UV circular dichroic spectra revealed that this purified protease contained 7.1 % α-helix, 64.1 % β-sheet, and 32 % aperiodic coil. This novel acid protease was active over the temperature range of 35–55 °C with optimum temperature of 40 °C and was stable in the pH range of 2.5–6.5 with optimum pH of 3.5. Mn²⁺ enhanced its activity while Co²⁺ showed inhibitory effect. With casein as substrate, the kinetic parameters of K _m, V _max, energy of activation (E _a), and attenuation index of inactivation velocity by heat inducing (λ) were 0.96 mg mL^?1, 135.14 μmol min^?1 mg^?1, 64.11 kJ mol^?1, and 0.59, respectively.     

微波杀灭玉米黑曲霉工艺优化

应用响应面法对微波杀灭玉米黑曲霉工艺进行优化研究,考查微波功率、微波时间和装载量对玉米黑曲霉孢子减少对数周期和玉米裂纹率的影响,并建立相应的回归模型。结果表明最佳工艺条件为:微波功率119W,微波时间6min,装载量50g,在此条件下,玉米黑曲霉孢子减少对数周期为1.3769,玉米裂纹率为0,与理论预测值基本符合,为微波法杀灭玉米霉菌的工业应用提供理论参考。      

黑曲霉木聚糖酶（xynZF-2）基因克隆、表达及酶学性质分析

利用RT-PCR技术从黑曲霉（Aspergillusniger）XZ-3S中成功克隆出木聚糖酶基因（xynZF-2）的cDNA序列（Genbank登录号为：JQ700382）。该基因核苷酸序列阅读框全长678bp,编码225个氨基酸,其中成熟肽为107个氨基酸。以重组质粒pUCm-T—xynZF-2为模板,扩增得到编码xynZF－2成熟肽的cDNA片段（624bp）。将其与表达载体pET－28a连接,构建重组表达载体pET－28a—xynZF－2,并转化大肠杆菌（Escherichiacoh）BL21（DE3）,获得重组工程菌BL21／xynZF-2。经IPTG诱导表达,木聚糖酶的的比酶活最高可达42．33U／mg。重组表达的木聚糖酶最适反应温度为40℃,最适反应pH为5．0,在碱性条件下具有良好的稳定性。Fe^3＋对酶的激活作用最为明显。