沼泽红假单胞菌Rhodopseudomonas palustris 2-8的亚硝酸盐还原酶基因克隆和序列分析

沼泽红假单胞菌2-8具有亚硝酸盐还原能力, 根据不同类型亚硝酸盐还原酶保守序列设计引物, 通过PCR扩增的方法对2-8菌株的亚硝酸盐还原酶类型进行鉴定, 发现该菌株的亚硝酸盐还原酶为Cu型亚硝酸盐还原酶。从2-8菌株基因组中克隆出编码该Cu型亚硝酸盐还原酶的基因(nirK), 该基因由1 154个碱基对组成, 在GenBank数据库的登录号为GU332847, 与沼泽红假单胞菌(Rhodopseudomonas palustris TIE和CGA009) 的nirK序列相似性为90%。互联网数据库及生物信     

Cu-NiR与cd1-NiR——两类反硝化亚硝酸还原酶研究进展

亚硝酸还原酶(Nitrite Reductase,NiR)是自然界氮循环过程中催化亚硝酸盐还原的一类关键酶.其中,nirS和nirK基因编码合成的亚硝酸还原酶Cu-NiR和cd1-NiR是反硝化作用中的限速酶,二者功能类似但结构和辅助因子组成截然不同.研究发现这两类酶对不同环境梯度具有不同的响应机制.因此,本文详述了C...     

硝化细菌中亚硝酸盐氧化还原酶的研究进展

亚硝酸盐氧化还原酶(nitrite oxidoreductase,NXR)是硝化细菌将亚硝酸盐氧化为硝酸盐的关键酶,广泛存在于亚硝酸盐氧化菌中.由于它是可溶性的膜内酶,其催化机理与膜内电子传递密切联系,给它的研究带来了一定的困难.本文综述了多年来国内外研究者从不同方面对NXR研究的成果,详细论述了NXR的组成结构、工作机理以及不同因子对其活性的影响,总结了近几年应用于研究NXR的新方法,并展望了对NXR研究的发展方向及其意义.     

过表达亚硝酸盐还原酶的重组大肠杆菌辅助污水短程硝化

【目的】为了体现并突出亚硝酸盐还原酶在污水脱氮以及短程硝化中的重要性,对过表达亚硝酸盐还原酶的大肠杆菌进行了污水脱氮的研究。【方法】通过转化带有亚硝酸盐还原酶基因的重组质粒,将亚硝酸盐还原酶在大肠杆菌中过表达,通过分析重组大肠杆菌的产物研究了该酶的表达及还原亚硝酸盐的情况,通过将该重组菌与已报道的硝化-反硝化细菌或生活污水进行混合培养,研究重组菌用于辅助氨氮去除的短程硝化能力。【结果】重组大肠杆菌能正确表达亚硝酸盐还原酶,OD600=2.0的菌悬液在2 h内还原约1 mmol/L的亚硝酸盐,并产生几乎等量的一氧化氮;重组大肠杆菌与Acinetobacter sp.YF14菌株等比例混合时,12 h能够提高氨氮脱氮效率约(36.0±7.4)%,且在4 h时,最大亚硝酸盐的积累量减少37%;重组大肠杆菌(OD600=1.0)12 h内能够提高污水厂活性污泥的脱氮效率约(31.0±5.7)%,且未检测到亚硝酸盐和硝酸盐的积累;溶氧水平对于亚硝酸盐还原酶重组菌辅助脱氮具有明显的影响,中等溶氧量[(6.4?0.7)mg/L]时脱氮效果最好。【结论】过表达亚硝酸盐还原酶的大肠杆菌可以提高污水脱氮的短程硝化能力。     

植物体内一氧化氮合成途径研究进展

一氧化氮(NO)作为一种气体信号分子,在植物生理过程中发挥重要作用,它参与调节植物的生长、发育及对外界环境的应激反应.植物体内主要通过酶催化途径和非酶催化途径合成NO.酶催化途径合成NO的主要酶包括一氧化氮合酶(nitric oxide synthase,NOS)和硝酸还原酶(nitrate reductase,NR),以及在某些植物的特定组织或器官或在特殊环境条件下存在的一氧化氮氧化还原酶(nitric oxide oxidoreductase,Ni-NOR)和黄嘌呤氧化还原酶(xanthine oxidoreductase,XOR).非酶催化合成途径主要是在酸性和还原剂存在条件下将亚硝酸盐还原成NO.该文主要结合研究方法,综述了植物体内NO合成途径的研究进展,为植物体内NO信号的作用机理的深入研究提供信息资料.     

亚硝酸盐影响Lactobacillus brevis 4903发酵的研究

通过研究可知,亚硝酸盐对Lactobacillusbrevis4903发酵有抑制作用,环境中亚硝酸盐一旦分解掉,这种抑制作用就会被解除。分析其原因:①亚硝酸盐抑制了乳酸菌生长,从而抑制了乳酸发酵;②在发酵初期可能因亚硝酸盐还原酶的作用,使亚硝酸盐酶解生成NH3,NH3中和了乳酸菌生成的酸(H ),从而使环境pH值的下降和酸的积累变得缓慢。     

Acinetobacter sp. Y1的氨氮去除性能及其关键酶活性

【目的】研究Acinetobacter sp.Y1的氨氮(NH_4~+-N)去除性能及其关键酶的提取与酶活性。【方法】以柠檬酸钠为碳源,硫酸铵为氮源,研究菌株Y1的NH_4~+-N去除性能;采用正交实验优化超声波破碎法提取粗酶的条件,SDS-PAGE分析比较渗透压休克法和超声波破碎法获得的粗酶;检测关键酶——羟胺氧化还原酶(HAO)、亚硝酸盐还原酶(NIR)、硝酸盐还原酶(NAR)的酶活性。【结果】24 h内菌株Y1的菌密度(OD600)可达1.280,对NH_4~+-N、总氮(TN)和COD的降解率分别达到98%、94%和92%,硝化过程中羟胺、亚硝酸盐氮、硝酸盐氮不积累,反硝化产生N2;超声波破碎法提取粗酶的最佳工作条件为:破碎功率50 W,工作与间歇时间分别为4 s和7 s,OD600为1.250,总工作时间20 min,关键酶HAO、NIR和NAR的比活力分别为0.011、0.002和0.018 U/mg;渗透压休克法得到的HAO比活力是0.067 U/mg。【结论】Acinetobacter sp.Y1能同时高效去除NH_4~+-N、TN和COD。优化超声波破碎法提取粗酶的条件,检测到HAO、NIR和NAR的酶活性,且渗透压休克法比超声波破碎法更适合用来提取HAO。     

羟胺氧化还原酶在生物脱氮中的研究进展

羟胺氧化还原酶(hydroxylamine oxidoreductase,HAO)属于多血红素蛋白酶家族,每个单体由7个电子转移血红素和1个催化血红素组成.HAO既可分别催化羟胺和肼的氧化反应,也可催化羟胺、一氧化氮及亚硝酸盐的还原反应.不同硝化细菌中,HAO的最适温度、pH、底物、产物特异性及酶抑制剂等存在差异.作为...     

Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶活性

【背景】异养硝化-好氧反硝化菌由于能够同时实现硝化反硝化作用而备受关注,但由于菌的种类不同,其脱氮途径不尽相同,研究菌株脱氮关键酶的种类及其活性可以推测菌株的脱氮途径,从而为菌株在生产上的应用提供技术支撑。【目的】研究Pseudomonas alcaliphila AD-28的脱氮性能及其关键酶的活性,为菌株脱氮分子机理研究奠定基础。【方法】以柠檬酸钠为碳源,以硫酸铵、亚硝酸钠、硝酸钾为氮源,研究菌株AD-28的脱氮性能并检测其关键酶氨单加氧酶(AMO)、羟胺氧化还原酶(HAO)、亚硝酸盐还原酶(NIR)、硝酸盐还原酶(NAR)的酶活性。【结果】菌株AD-28培养24h的菌密度(OD600)可达1.971,对初始浓度为18.85mg/L的氨氮、26.13mg/L的硝酸盐氮、19.47mg/L的亚硝酸盐氮、66.11 mg/L的总氮去除率均达到96%以上;关键酶AMO、HAO、NIR和NAR的比活力分别为0.028、0.003、0.011、0.027 U/mg。【结论】Pseudomonas alcaliphila AD-28能同时进行异养硝化-好养反硝化作用,该菌在AMO作用下将NH4+-N氧化为羟胺,然后由HAO氧化为NO2--N,NO2--N和NO3--N在NIR、NAR等酶的催化作用下脱氮。     

Sortase A介导的(S)-羰基还原酶Ⅱ寡聚体高效立体选择性转化(S)-苯基乙二醇

【目的】以金黄色葡萄球菌(Staphylococcus aureus)的sortase A为"分子订书机",用于(S)-羰基还原酶Ⅱ分子之间的连接,获得催化功能与稳定性增强的氧化还原酶寡聚体,高效催化2-羟基苯乙酮,合成(S)-苯基乙二醇。【方法】从S.aureus基因组中克隆sortase A基因,在大肠杆菌中表达,通过镍柱和凝胶层析纯化重组酶,获得纯酶sortase A。通过基因工程手段在(S)-羰基还原酶Ⅱ的C末端添加GGGGSLPETGG序列,蛋白纯化获得(S)-羰基还原酶Ⅱ-GGGGSLPETGG,摸索了sortase A催化(S)-羰基还原酶Ⅱ-GGGGSLPETGG的分子连接,形成(S)-羰基还原酶Ⅱ寡聚体的最佳条件,并研究了寡聚体酶学性质及生物转化(S)-苯基乙二醇的效率。【结果】(S)-羰基还原酶Ⅱ寡聚体比酶活力为38.5 U/mg,比原始型(S)-羰基还原酶Ⅱ提高了6倍,最适反应温度为50°C,最适pH为6.0,在50°C放置1 h后酶活仍旧保持90%以上;蛋白质变性实验结果显示,(S)-羰基还原酶Ⅱ寡聚体的变性温度为60.1°C,比原始酶提高了10°C;生物转化结果显示(S)-羰基还原酶Ⅱ寡聚体在3 h内完全转化5 g/L 2-羟基苯乙酮,产生光学纯度为100%的(S)-苯基乙二醇,相比于重组大肠杆菌(S)-羰基还原酶Ⅱ全细胞催化时间缩短了16倍。【结论】本研究首次将sortase A应用于氧化还原酶的分子连接,显著提高了酶的催化效率和热稳定性,表明sortase在手性催化中有很大的潜在应用价值。     

A Comparison of Nitrite Reductase Enzymes from Green Leaves, Scutella, and Roots of Corn (Zea mays L.)

Nitrite reductase from green leaves of corn (Zea mays L.) is eluted from a diethylaminoethyl-cellulose column in one peak of activity by a chloride gradient, while nitrite reductase from scutellum tissue is resolved into two peaks of activity, apparently representing two forms of the enzyme NiR1 and NiR2. One of these (NiR2) elutes at the same concentration of chloride as the leaf nitrite reductase. Roots and etiolated shoots also exhibited both forms of the enzyme, however, lesser amounts of NiR1 is extractable from these tissues than from scutellum. Comparison of green leaf nitrite reductase with NiR2 from scutellum tissue shows similar or identical properties with respect to molecular weight, isoelectric point, electron donor requirements, inhibition properties, pH optima, thermal stability, and pH tolerance. The significance of these similarities in relation to probable differences in the biochemical mechanism of nitrite reduction between leaf and scutellum tissues is discussed. Although ferredoxin is considered, with some reservations, to be the electron donor for nitrite reductase in green tissue, the reductant for nongreen tissue is not known. The possibility that nitrite reductases from green and non-green tissues uses the same electron donor, in vivo, is considered.     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

Diversity of culturable denitrifying bacteria

Sequence divergence in the ribosomal genes of known strains and isolates of aquatic denitrifying bacteria was investigated using restriction fragment length polymorphism (RFLP) analysis. The same cultures were characterized for their homology with antibody and gene probes for nitrite reductase (NiR), a key enzyme in the denitrification pathway, and for amplification with a set of polymerase chain reaction primers designed to amplify a portion of the NiR gene. The NiR probes were developed from Pseudomonas stutzeri (ATCC 14405) and several P. stutzeri strains were included in the analyses. The RFLP analysis clustered most of the P. stutzeri strains together, but detected considerable diversity within this group. Isolates from three aquatic environments exhibited within —and among — habitat diversity by RFLP. Hybridization with the NiR probes and amplification with the NiR primers were not correlated with the clustering of strains by rDNA RFLP analysis. The relationships among strains deduced from ribosomal DNA RFLP reflect heterogeneity within the P. stutzeri group and among other pseudomonads, and the patterns differ from those inferred from specificity of the NiR probes.Abbreviations NiR Nitrite reductase - PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

木本植物阳生和阴生叶片叶绿体O_2和NO_2~-光还原作用

在有ＰＣＲ和ＰＣＯ环活性抑制剂甘油醛和光合磷酸化解偶联剂ＮＨ４ＣＬ存在下,比较了生长于 ３种 光环境的乔木黧蒴和灌木九节幼苗阳生和阴生叶片叶绿体的Ｏ２和ＮＯ２光还原速率。全自然光下两种 植物阳生叶片的叶绿体Ｏ２的光还原速率最高,占总光合电子传递活性的６６％－６８％,ＮＯ２光还原速率 也有类似趋势占总电子传递的１１％－１５％左右。３６％和１６％自然光下阴生叶片Ｏ２和ＮＯ２光还原 速率及Ｏ２光还原电子传递的比例显著降低,但ＮＯ２光还原电子传递的比例不受影响。与ＮＯ２光还原 相关的叶片ＮｉＲ和ＮＲ活性及ＮｉＲ／ＮＲ活性比也因叶片接受光强度大小而异,随光强减弱,黧蒴的 ＮｉＲ活性降低,九节的ＮＲ活性增高,但黧蒴的ＮＲ活性和九节的ＮｉＲ活性变化未达差异显著性。     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Nitrite reduction in barley-root plastids: Dependence on NADPH coupled with glucose-6-phosphate and 6-phosphogluconate dehydrogenases,and possible involvement of an electron carrier and a diaphorase

Plastids from roots of barley (Hordeum vulgare L.) seedlings were isolated by discontinuous Percoll-gradient centrifugation. Coinciding with the peak of nitrite reductase (NiR; EC 1.7.7.1, a marker enzyme for plastids) in the gradients was a peak of a glucose-6-phosphate (Glc6P) and NADP⁺-linked nitrite-reductase system. High activities of phosphohexose isomerase (EC 5.3.1.9) and phosphoglucomutase (EC 2.7.5.1) as well as glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44) were also present in the isolated plastids. Thus, the plastids contained an overall electron-transport system from NADPH coupled with Glc6PDH and 6PGDH to nitrite, from which ammonium is formed stoichiometrically. However, NADPH alone did not serve as an electron donor for nitrite reduction, although NADPH with Glc6P added was effective. Benzyl and methyl viologens were enzymatically reduced by plastid extract in the presence of Glc6P+ NADP⁺. When the plastids were incubated with dithionite, nitrite reduction took place, and ammonium was formed stoichiometrically. The results indicate that both an electron carrier and a diaphorase having ferredoxin-NADP⁺ reductase activity are involved in the electron-transport system of root plastids from NADPH, coupled with Glc6PDH and 6PGDH, to nitrite.Abbreviations Cyt cytochrome - Glc6P glucose-6-phosphate - Glc6PDH glucose-6-phosphate dehydrogenase - MVH reduced methyl viologen - NiR nitrite reductase - 6PG 6-phosphogluconate - 6PGDH 6-phosphogluconate dehydrogenase     

Effects of freezing on purified nitrite reductase from a denitrifier, Alcaligenes sp. NCIB 11015

The effects of freezing on Alcaligenes sp. nitrite reductase [nitric-oxide: ferricytochrome c oxidoreductase, EC 1.7.2.1] dissolved in sodium phosphate (pH 7.2) were investigated. The nitrite reductase was gradually activated with time in the frozen state, resulting in an increase in its activity of 2.5-4.5 times. The final freezing temperature influenced the enzyme activation, maximal activation being observed at around -20 degrees C. All the enzymatic activities that the nitrite reductase is known to catalyze were enhanced by freeze-thawing. The activation was followed by neither association-dissociation nor any gross conformational change of the enzyme molecule, but was accompanied by an increase in the fluorescence intensity of 2-p-toluidinonaphthalene-6-sulfonate used as a hydrophobic probe. The results are consistent with the hypothesis that the activation of the NiR is due to a limited conformational change of the enzyme molecule, particularly in the hydrophobic region. The mechanism of the activation of NiR by freeze-thawing is discussed, in comparison with the mechanisms of inactivation by freeze-thawing of many enzymes reported by previous workers.     

Inorganic nitrogen assimilation in the non-N2-fixing cyanobacterium Phormidium laminosum. II. Effect of the nitrogen source on the nitrite reductase levels

The effect of different inorganic nitrogen sources on the cellular levels of nitrite reductase (NiR. EC 1.7.7.1) activity has been studied in the filamentous non-N₂-fixing cyanobacterium Phormidium laminosum (strain OH-1-p.Cl,). Nitrate-grown cells gave the highest NiR in cell-free extracts [ca 165 nmol of nitrite reduced (mg protein)-1 min-], whereas no activity could be detected in extracts from ammonium-grown cells. The in vivo effect of ammonium on NiR was similar to that exerted by chloramphenicol, suggesting that de novo synthesis of protein was probably repressed by this ion. When ammonium was removed from the culture medium, a rapid increase of de novo synthetized NiR occurred, and the appearance of the enzyme was slightly stimulated by the presence in the medium of either nitrate or nitrite. However, remarkably high levels of NiR [around 1.2 μmol of nitrite reduced (mg protein) ⁻¹min⁻¹] could be routinely measured in nitrogen-deficient cells, indicating that the enzyme was ammonium-repressible rather than nitrate- or nitrite-inducible.     

Expression of a fully functional cd1 nitrite reductase from Pseudomonas aeruginosa in Pseudomonas stutzeri

Nitrite reductases are redox enzymes catalysing the one electron reduction of nitrite to nitrogen monoxide (NO) within the bacterial denitrification process. We have cloned the gene for cd(1) nitrite reductase (Pa-nirS) from Pseudomonas aeruginosa into the NiRS(-) strain MK202 of Pseudomonas stutzeri and expressed the enzyme under denitrifying conditions. In the MK202 strain, denitrification is abolished by the disruption of the endogenous nitrite reductase gene; thus, cells can be grown only in the presence of oxygen. After complementation with Pa-nirS gene, cells supplemented with nitrate can be grown in the absence of oxygen. The presence of nitrite reductase was proven in vivo by the demonstration of NO production, showing that the enzyme was expressed in the active form, containing both heme c and d(1). A purification procedure for the recombinant PaNir has been developed, based on the P. aeruginosa purification protocol; spectroscopic analysis of the purified protein fully confirms the presence of the d(1) heme cofactor. Moreover, the functional characterisation of the recombinant NiR has been carried out by monitoring the production of NO by the purified NiR enzyme in the presence of nitrite by an NO electrode. The full recovery of the denitrification properties in the P. stutzeri MK202 strain by genetic complementation with Pa-NiR underlines the high homology between enzymes of nitrogen oxianion respiration. Our work provides an expression system for cd(1) nitrite reductase and its site-directed mutants in a non-pathogenic strain and is a starting point for the in vivo study of recombinant enzyme variants.