邻单胞菌邻苯二酚1,2-双加氧酶基因(tfd C)的克隆及其在大肠杆菌中表达

根据已报道的邻苯二酚１,２－双加氧酶基因（ｔｆｄ Ｃ）序列,设计ＰＣＲ引物,从一株邻单胞菌（Ｐｌｅｓｉｏｍｉｎａｓ）的ｐＬ１质粒上扩增到ｔｆｄ Ｃ基因片段,连接到ｐＧＥＭ－Ｔ载体上,并转化大肠杆菌ｌＭ１０９菌株,筛选到阳性克隆。序列分析结果表明,ＰＣＲ产物全长８０１ｂｐ,有一阅读框,编码２５５个氨基酸,与增氧产碱菌（Ａｌｃａｌｉｇｅｎｅｓ ｅｕｔｒｏｐｌｕｓ）的ｔｆｄ Ｃ基因相比,在６９３位相差一     

萘对川西亚高山森林土壤呼吸、可溶性有机质和微生物生物量的影响

通过原位控制试验,研究了萘对川西亚高山森林土壤动物抑制效率、土壤呼吸、可溶性有机质和微生物生物量的影响.结果表明:萘施用显著抑制了大型和中小型土壤节肢动物的个体密度和类群数量,个体密度分别下降76.3%~78.5%和83.3%~84.8%,类群数量分别降低48.3%~56.1%和45.8%~58.3%.萘处理与对照的土壤呼吸速率季节动态呈单峰曲线,分别以2月和8月为最低值和最高值,而且未受萘施用的显著影响.与对照相比,萘处理显著降低了8月和10月土壤可溶性碳和可溶性氮含量,以及4月和8月微生物生物量碳,增加了4月的微生物生物量碳氮比.萘处理和采样时间的交互作用显著影响了微生物生物量碳和微生物生物量氮,但对土壤动物个体密度和类群数量以及可溶性碳含量影响不显著.总体上,萘作为抑制剂,在川西亚高山森林土壤能够有效地抑制土壤动物节肢动物,且并未显著影响土壤呼吸,但对土壤碳氮组分造成了不同程度的影响.     

嗜酸硫氧化细菌消解元素硫的研究进展

浸矿酸性环境下,金属硫化矿在Fe3+作用下,经过硫代硫酸盐途径或多聚硫化氢途径而分解的过程中导致大量元素硫的累积,进而可能在金属硫化矿表面形成疏水元素硫层,阻碍金属离子的进一步浸出。酸性环境下,惰性元素硫的消解必须借助嗜酸硫氧化细菌来实现。该消解过程包括嗜酸硫氧化细菌对元素硫的吸附、转运以及氧化转化等过程。本文对近年来嗜酸硫氧化细菌消解元素硫过程的相关研究进行了全面评述,认为有关嗜酸硫氧化细菌消解元素硫的分子机制的清晰阐述还有待人们通过对消解过程的各个环节的分子机制进行大量研究来实现。     

苯胺双加氧酶基因的克隆与序列分析

通过设计苯胺双加氧酶基因特异引物,以苯胺降解菌株ANA5基因组DNA为模板,PCR扩增出目的基因片断。然后利用粘粒pLAFR3作为载体,以E．coliEPI100作为受体,构建了菌株ANA5的基因组粘粒文库。以PCR扩增产物作为探针,通过菌落原位杂交筛选得到两个阳性克隆,经Southern杂交及亚克隆测序分析,初步确认克隆到苯胺双加氧酶基因。同时完成了苯胺双加氧酶基因atdA3A4A5序列的测定,并对其核苷酸及其推导的氨基酸序列进行分析,结果表明克隆到的苯胺双加氧酶基因与GenBank报道的基因有一定的差异,同时体现了该基因在进化上的保守性。     

番茄(Solanum lycopersicum)类胡萝卜素降解关键基因筛选

类胡萝卜素衍生挥发物对提升番茄风味至关重要。为筛选调控类胡萝卜素衍生挥发物合成的关键基因,以90个番茄自交系中香气寡淡的TI4001和香气浓郁的CI1005为材料,分析了番茄类胡萝卜素裂解双加氧酶(SlCCDs)基因在不同组织及不同发育期果实中的表达量,果实不同成熟期类胡萝卜素及其衍生挥发物的含量。发现在7个SlCCDs基因中,SlCCD1A和SlCCD1B基因在番茄果实中表达量最高,且随着果实发育成熟表达量显著升高。果实中类胡萝卜素及其衍生挥发物含量也显著升高。SlCCD1A和SlCCD1B基因表达量与类胡萝卜素及其衍生挥发物含量之间极显著正相关。推测SlCCD1A和SlCCD1B基因是裂解类胡萝卜素合成挥发物的关键基因。     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Degradation of diphenylether by Pseudomonas cepacia Et4: enzymatic release of phenol from 2,3-dihydroxydiphenylether

2,3-Dihydroxybiphenyl dioxygenase from Pseudomonas cepacia Et 4 was found to catalyze the ring fission of 2,3-dihydroxydiphenylether in the course of diphenylether degradation. The enzyme was purified and characterized. It had a molecular mass of 240 kDa and is dissociated by SDS into eight subunits of equal mass (31 kDa). The purified enzyme was found to be most active with 2,3-dihydroxybiphenyl as substrate and showed moderate activity with 2,3-dihydroxydiphenylether, catechol and some 3-substituted catechols. The K _m-value of 1 M for 2,3-dihydroxydiphenylether indicated a high affinity of the enzyme towards this substrate. The cleavage of 2,3-dihydroxydiphenylether by 2,3-dihydroxybiphenyl dioxygenase lead to the formation of phenol and 2-pyrone-6-carboxylate as products of ring fission and ether cleavage without participation of free intermediates. Isotope labeling experiments carried out with ¹⁸O₂ and H₂ ¹⁸O indicated the incorporation of ¹⁸O from the atmosphere into the carboxyl residue as well as into the carbonyl oxygen of the lactone moiety of 2-pyrone-6-carboxylate. Based on these experimental findings the reaction mechanism for the formation of phenol and 2-pyrone-6-carboxylate is proposed in accordance with the mechanism suggested by Kersten et al. (1982).Non-standard abbreviations DPE diphenylether - 2,3-dihydroxy-DPE 2,3-dihydroxydiphenylether - PCA 2-pyrone-6-carboxylic acid - 2,3-dihydroxy-BP dioxygenase 2,3-dihydroxybiphenyl dioxygenase - GC gas chromatography     

Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity

Crystals have been obtained for a 2,3-dihydroxybiphenyl dioxygenase (conventionally called BphC) from a polychlorinated biphenyl (PCB)-degrader, Pseudomonas sp. strain KKS1O2. The crystals were grown using both ammonium sulfate and MPD as the precipitating agents. The crystals belonged to a tetragonal space group (I422) and diffracted to 2.5 Å. © 1995 Wiley-Liss, Inc.     

Nascent α2-macroglobulin-trypsin complex: Incorporation of amines and water at the thiol esterified GLX-residues of α2-macroglobulin during activation with trypsin

During complex formation between α₂-macroglobulin and trypsin the internal thiol esters (one in each of the four M_r 180,000 subunits) are activated. In this activated state (nascent α₂-macroglobulin-trypsin complex) addition of low M_r amines lead to their covalent incorporation into α₂M. Evidence is presented showing that covalent binding of added amines occurs at the γ-carbonyl group of the Glx-residue in the thiol ester sequence:

     

Dynamic response of naphthalene biodegradation in a continuous flow soil slurry reactor

Periodic perturbations were used to evaluate the system stability and robustness of naphthalene biodegradation in a continuous flow stirred tank reactor (CSTR) containing a soil slurry. The experimental design involved perturbing the test system using a sinusoidal input either of naphthalene or non-naphthalene organic carbon at different frequencies during steady state operation of the reactors. The response of the test system was determined by using time series off-gas analysis for naphthalene liquid phase concentration and degradation, total viable cell counts, and gene probe analysis of naphthalene degradative genotype, and by batch mineralization assays.Naphthalene biodegradation rates were very high throughout the experimental run (95 to >99% removed) resulting in very low or undetectable levels of naphthalene in the off-gas and reactor effluent. Attempts to reduce the rate of naphthalene biotransformation by either reducing the reactor temperature from 20°C to 10°C or the dissolved oxygen level (>1 mg/L) were unsuccessful. Significant naphthalene biodegradation was observed at 4°C. While variable, the microbial community as measured by population densities was not significantly affected by temperature changes. In terms of naphthalene biotransformation, the system was able to adapt readily to all perturbations in the reactor.Department of Chemical EngineeringDepartment of Microbiology and The Graduate Program in EcologyDepartment of Civil Engineering, New Orleans University