大豆种子萌发过程中线粒体的发生和发育

从萌发一天的大豆种子中用蔗糖密度梯度离心,不能分离出线粒体,种子萌发两天后,线粒体才开始出现,然后迅速增长。电镜和线粒体标记酶(细胞色素C氧化酶)分析,也证明萌发一天的种子细胞中既不能见到线粒体的结构,也检测不出线粒体酶的活性。种子萌发第2天到第6天线粒体量成百倍地增加,但很少见到线粒体的分裂相,似乎说明线粒体主要不是依靠分裂增殖的。线粒体本身也有一个发育过程,萌发第2天的种子中线粒体结构简单,只有网状膜系统和较大的基质区,萌发第3～4天就开始出现嵴膜,基质区缩小,至第5～6天嵴膜密集,基质区更小,线粒体的出现似乎和氧供应有关,因为线粒体是在种皮破裂的同时出现的。高等植物线粒体在个体发育中似乎并非连续存在,主要也不是以自我分裂方式繁殖,因此推测原质体作为未分化的细胞器既是叶绿体的前体,也是线粒体的前体。     

氢化酶重组表达研究进展

氢化酶催化最简单的氧化还原反应,但蛋白结构却非常复杂,对其蛋白结构和催化功能的研究牵动着生物制氢、光电产氢催化剂及氢能源电池等相关绿色能源产业的发展。氢化酶通常可逆地催化质子还原产氢的反应,对氧化还原电位非常敏感,催化活性中心易于被氧化失活,活性蛋白的分离提纯十分不易,使得对其催化机制的认识推进缓慢。为了获取更多的氢化酶活性蛋白,许多研究团队先后对氢化酶开展了大量的同源或异源重组表达研究,就这类研究工作进行了扼要的总结和分析。     

低温胁迫对两种圆柏属植物亚细胞抗氧化酶活性的影响

以祁连圆柏和圆柏幼苗为材料,研究不同处理时间下低温胁迫对圆柏属植物叶片亚细胞抗氧化酶活性的影响,探讨其在圆柏属植物叶片中的亚细胞定位。结果表明：低温胁迫下,丙二醛(MDA)含量和抗氧化酶活性随时间变化均呈先升后降趋势,祁连圆柏中抗氧化酶的种类比圆柏的多且活性强,而 MDA 含量低于圆柏,表明祁连圆柏在低温胁迫下具有更广泛的适应性。此外,两种圆柏植物叶片超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)定位为叶绿体＞细胞溶质＞线粒体,过氧化氢酶(CAT)定位为线粒体＞叶绿体＞细胞溶质,谷胱甘肽还原酶(GR)定位为线粒体＞细胞溶质＞叶绿体,祁连圆柏过氧化物酶(POD)定位为细胞溶质＞叶绿体＞线粒体,圆柏POD定位为细胞溶质＞线粒体＞叶绿体,且抗氧化酶SOD、APX和 GR在亚细胞中分布差异达到极显著,这说明抗氧化酶在其中一种亚细胞中发挥主要作用,为克隆亚细胞组分中的抗氧化酶基因提供了理论依据。     

氢气生物学作用的生物酶基础

氢气具有广泛的生物学功能,近年来逐渐引起广泛关注。但是氢气发挥生物学作用的机理一直都有争论,制约了氢生物学的进一步发展。现在被广泛接受的是氢气选择性与毒性自由基反应的理论,但是生理条件下氢气与自由基直接反应的证据并不充分,多数属于间接证据,无法区分氢气是与自由基直接反应还是影响了自由基的产生。氢气具有抗氧化作用,本团队研究表明,氢气不是在自由基产生之后去清除,而是减少自由基的产生,类似于在自由基产生之初就关上“开关”;氢气可以提高包括线粒体复合物Ⅰ、乙酰胆碱酯酶、HRP在内的生物酶的活性,可以影响线粒体膜电位和调节神经细胞膜电位,细胞膜的氧化还原酶类及离子通道等都受到氢气的调节,这表明氢气的作用可能是多靶点的主要基于酶学反应的过程,高等生物具有产生和利用氢气的氢化酶活性。主要探讨了氢气和自由基的关系以及氢气作用的生物酶学基础,以期为揭示氢气发挥生物学作用的机理提供参考。     

微生物产氢机理的研究进展

微生物可以利用工业废弃物产生氢气,其产氢机理可以分成两种:光合产氢和发酵产氢。前者利用光能,后者利用代谢过程中产生的电子,分解有机物产氢。氢酶是产氢过程中的关键酶,催化氢的氧化或质子的还原。氢酶主要有[NiFe]氢酶和[Fe]氢酶两种,具有不同的结构,但催化机理是相似的。本文主要综述产氢微生物的种类、微生物产氢代谢途径和关键酶催化机理,并展望微生物产氢研究的发展方向。     

氢酶结构及催化机理研究进展

氢酶是一类催化氢的氧化或质子还原的酶,它在微生物产氢过程中扮演着重要角色。根据氢酶所含的金属元素,可分为NiFe_氢酶、Fe-氢酶和不含金属元素的metal_free氢酶。大多数氢酶含有金属原子,它们参与氢酶活性中心和[Fe_S]簇的形成。氢酶的活性中心直接催化氢的氧化与质子的还原,[Fe_S]簇则参与氢酶催化过程中电子的传输。目前已有数种NiFe_氢酶和Fe_氢酶的X射线衍射晶体结构被阐明。根据metal_free氢酶的序列特征,推断其结构与NiFe_氢酶和Fe_氢酶之间存在较大差异。对氢酶活性中心和[Fe_S]簇的深入研究,揭示了氢酶催化反应的机理。     

氢气对辣根过氧化物酶活性的影响及其作用机制的研究

氢气具有广泛的生物学功能,但氢气发挥生物学效应的靶点仍存在争论。我们前期发现,氢气可以与乙酰胆碱酯酶(AChE)直接作用并提高其活性,提示酶分子可能是氢气的潜在作用靶点。除AChE外,氢气是否还可与其它酶分子相互作用目前尚未见报道。本研究目的是探索氢气对辣根过氧化物酶(HRP)活性的影响。采用荧光光谱和紫外光谱方法检测氢气对HRP活性的影响,并对氢分子作用机制进行了深入探索。研究结果表明,当氢气浓度在饱和状态(800μmol/L)和半饱和状态(400μmol/L)下,HRP酶活性分别提高了18.42%和5.44%;荧光光谱检测表明,氢气使HRP内源性氨基酸荧光强度增强了4.15%,色氨酸到血红素中心的距离由2.85 m缩短至2.69 nm,荧光量子产率由72.8%增加至73.4%;紫外光谱检测表明,氢气可使HRP转化为高价铁的HRP I状态速率降低,同时在间歇性紫外照射后,波长在红移至HRP I状态后存在蓝移现象。以上结果提示,氢气可能改变了HRP中的组氨酸、色氨酸及天冬氨酸的微环境,导致氢键网络重构,从而使反应过程中向血红素中心传递能量的效率增加,His42-Asn70之间氢键增强,酶活增强;同时,氢气可使"H_2O-H_2O_2"为桥梁的氢键网络式催化发生变化,降低HRP I的形成速率。本研究不仅进一步证实了酶分子可能是氢分子发挥生物学作用的潜在靶点,同时对氢分子调节HRP酶活性机制的探索,为氢分子作用机制研究提供了更多的线索。     

在玉米线粒体基因组内的叶绿体DNA序列

目前,研究家们发现除了在核基因中具有叶绿体DNA片段外,在许多种高等植物的线粒体基因组中也具有叶绿体DNA序列。采用限制性内切酶进行研究发现,在玉米线粒体基因组内至少存在三个重要的叶绿体DNA序列:(i)1,5-二磷酸核酮糖羧化酶的大亚     

高等植物叶绿体和线粒体免疫亲近性的研究

以火箭免疫电泳分析表明：大豆叶绿体抗体与大豆线粒体有免疫交叉反应,同时大豆线粒体抗体与大豆叶绿体也有免疫交叉反应,但是大豆线粒体的抗体与鼠肝线粒体之间无免疫交叉反应。这说明高等植物线粒体对叶绿体比之对动物线粒体在免疫特性上有更大的亲近性;亦即高等植物线粒体和高等植物的叶绿体有更大的同源性。经火箭免疫电泳,交叉免疫电泳和线状免疫电泳进一步分析表明：菠菜偶联因子抗体和大豆线粒体,大豆叶绿体间,大豆线粒     

大麦脂肪酶的研究进展

脂肪酶以其独特的优势在各个领域都有其潜在的应用前景。在高等植物中,种子萌发过程中脂肪酶活性较高,其活性与食品的加工方法有关。应用不同的方法测定大麦脂肪酶酶活力,得到的结果也不同。详细介绍了大麦脂肪酶的部分酶学性质。     

High Yields of Hydrogen Production Induced by Meta-Substituted Dichlorophenols Biodegradation from the Green Alga Scenedesmus obliquus

Hydrogen is a highly promising energy source with important social and economic implications. The ability of green algae to produce photosynthetic hydrogen under anaerobic conditions has been known for years. However, until today the yield of production has been very low, limiting an industrial scale use. In the present paper, 73 years after the first report on H₂-production from green algae, we present a combinational biological system where the biodegradation procedure of one meta-substituted dichlorophenol (m-dcp) is the key element for maintaining continuous and high rate H₂-production (>100 times higher than previously reported) in chloroplasts and mitochondria of the green alga Scenedesmus obliquus. In particular, we report that reduced m-dcps (biodegradation intermediates) mimic endogenous electron and proton carriers in chloroplasts and mitochondria, inhibit Photosystem II (PSII) activity (and therefore O₂ production) and enhance Photosystem I (PSI) and hydrogenase activity. In addition, we show that there are some indications for hydrogen production from sources other than chloroplasts in Scenedesmus obliquus. The regulation of these multistage and highly evolved redox pathways leads to high yields of hydrogen production and paves the way for an efficient application to industrial scale use, utilizing simple energy sources and one meta-substituted dichlorophenol as regulating elements.     

Cyanobacterial H<Subscript>2</Subscript> production — a comparative analysis

Several unicellular and filamentous, nitrogen-fixing and non-nitrogen-fixing cyanobacterial strains have been investigated on the molecular and the physiological level in order to find the most efficient organisms for photobiological hydrogen production. These strains were screened for the presence or absence of hup and hox genes, and it was shown that they have different sets of genes involved in H₂ evolution. The uptake hydrogenase was identified in all N₂-fixing cyanobacteria, and some of these strains also contained the bidirectional hydrogenase, whereas the non-nitrogen fixing strains only possessed the bidirectional enzyme. In N₂-fixing strains, hydrogen was mainly produced by the nitrogenase as a by-product during the reduction of atmospheric nitrogen to ammonia. Therefore, hydrogen production was investigated both under non-nitrogen-fixing conditions and under nitrogen limitation. It was shown that the hydrogen uptake activity is linked to the nitrogenase activity, whereas the hydrogen evolution activity of the bidirectional hydrogenase is not dependent or even related to diazotrophic growth conditions. With regard to large-scale hydrogen evolution by N₂-fixing cyanobacteria, hydrogen uptake-deficient mutants have to be used because of their inability to re-oxidize the hydrogen produced by the nitrogenase. On the other hand, fermentative H₂ production by the bidirectional hydrogenase should also be taken into account in further investigations of biological hydrogen production.Abbreviations Chl chlorophyll - MV methyl viologen     

Mitochondria and hydrogenosomes are two forms of the same fundamental organelle

Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.     

Reinvestigation of the steady-state kinetics and physiological function of the soluble NiFe-hydrogenase I of Pyrococcus furiosus

Pyrococcus furiosus has two types of NiFe-hydrogenases: a heterotetrameric soluble hydrogenase and a multimeric transmembrane hydrogenase. Originally, the soluble hydrogenase was proposed to be a new type of H₂ evolution hydrogenase, because, in contrast to all of the then known NiFe-hydrogenases, the hydrogen production activity at 80°C was found to be higher than the hydrogen consumption activity and CO inhibition appeared to be absent. NADPH was proposed to be the electron donor. Later, it was found that the membrane-bound hydrogenase exhibits very high hydrogen production activity sufficient to explain cellular H₂ production levels, and this seems to eliminate the need for a soluble hydrogen production activity and therefore leave the soluble hydrogenase without a physiological function. Therefore, the steady-state kinetics of the soluble hydrogenase were reinvestigated. In contrast to previous reports, a low K_m for H₂ (~20 μM) was found, which suggests a relatively high affinity for hydrogen. Also, the hydrogen consumption activity was 1 order of magnitude higher than the hydrogen production activity, and CO inhibition was significant (50% inhibition with 20 μM dissolved CO). Since the K_m for NADP⁺ is ~37 μM, we concluded that the soluble hydrogenase from P. furiosus is likely to function in the regeneration of NADPH and thus reuses the hydrogen produced by the membrane-bound hydrogenase in proton respiration.     

Inhibition of respiration and nitrate assimilation enhances photohydrogen evolution under low oxygen concentrations in Synechocystis sp. PCC 6803

In cyanobacterial membranes photosynthetic light reaction and respiration are intertwined. It was shown that the single hydrogenase of Synechocystis sp. PCC 6803 is connected to the light reaction. We conducted measurements of hydrogenase activity, fermentative hydrogen evolution and photohydrogen production of deletion mutants of respiratory electron transport complexes. All single, double and triple mutants of the three terminal respiratory oxidases and the ndhB-mutant without a functional complex I were studied. After activating the hydrogenase by applying anaerobic conditions in the dark hydrogen production was measured at the onset of light. Under these conditions respiratory capacity and amount of photohydrogen produced were found to be inversely correlated. Especially the absence of the quinol oxidase induced an increased hydrogenase activity and an increased production of hydrogen in the light compared to wild type cells. Our results support that the hydrogenase as well as the quinol oxidase function as electron valves under low oxygen concentrations. When the activities of photosystem II and I (PSII and PSI) are not in equilibrium or in case that the light reaction is working at a higher pace than the dark reaction, the hydrogenase is necessary to prevent an acceptor side limitation of PSI, and the quinol oxidase to prevent an overreduction of the plastoquinone pool (acceptor side of PSII). Besides oxygen, nitrate assimilation was found to be an important electron sink. Inhibition of nitrate reductase resulted in an increased fermentative hydrogen production as well as higher amounts of photohydrogen.     

Enhanced Biological Hydrogen Production from Escherichia coli with Surface Precipitated Cadmium Sulfide Nanoparticles

The precipitation of cadmium sulfide nanoparticles is induced on the surface of Escherichia coli , and the biological hydrogen production efficiency under visible light (VL) irradiation is investigated. When endogenous [Ni–Fe]‐hydrogenase is anaerobically induced, an additional 400 µmol of hydrogen gas is generated within 3 h from the hybrid system suspension (50 mL) under VL irradiation (2000 W m^?2), corresponding to an increase in hydrogen production of ≈30%. The apparent quantum efficiencies of the hybrid system under 470 and 620 nm VL irradiation are 7.93% and 9.59%, respectively, which are higher than those of many photoheterotrophic bacteria. Furthermore, the mechanism of the enhanced hydrogen evolution is investigated. The interaction between photogenerated electrons and cells of E. coli is confirmed by heat‐treatment, electron‐scavenger, and separation studies. The acceleration of pyruvate generation, inhibition of lactate fermentation, increase of formate concentration, stimulation of hydrogenase activity, and elevation of nicotinamide adenine dinucleotide (NAD)H/NAD ratio in the hybrid system are responsible for the enhanced hydrogen production. A feasibility study is also conducted using wastewater and natural sunlight for the hydrogen production by the hybrid system. An additional 120 µmol of hydrogen is generated from the hybrid system within 3 h under these conditions using natural resources.     

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe⿿Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production.     

Nickel is essential for active hydrogenase in free-living Frankia isolated from Casuarina

Five free-living Frankia strains isolated from Casuarina were investigated for occurrence of hydrogenase activity. Nitrogenase activity (acetylene reduction) and hydrogen evolution were also evaluated. Acetylene reduction was recorded in all Frankia strains. None of the Frankia strains had any hydrogenase activity when grown on nickel-depleted medium and they released hydrogen in atmospheric air. After addition of nickel to the medium, the Frankia strains were shown to possess an active hydrogenase, which resulted in hydrogen uptake but no hydrogen evolution. The hydrogenase activity in Frankia strain KB5 increased from zero to 3.86 μ mol H₂ (mg protein)⁻¹ h⁻¹ after addition of up to 1.0 μ M Ni. It is likely that the hydrogenase activity could be enhanced even more as a response on further addition of Ni. It is indicated in this study that absence of hydrogenase activity in free-living Frankia isolated from Casuarina spp. is due to nickel deficiency. Frankia living in symbiosis with Casuarina spp. show hydrogenase activity. Therefore, the results also indicate that the hydrogenase to some extent is regulated by the host plant and/or that the host plant supplies the symbiotic microorganism with nickel. Moreover, the result shows that this Frankia is somewhat different from Frankia isolated from Alnus incana and Comptonia peregrina ., i.e., Frankia isolated from A. incana and C. peregrina showed a small hydrogen uptake activity even without addition of nickel.     

Hydrogen Oxidation by the Host-Controlled Uptake Hydrogenase Phenotype of Bradyrhizobium japonicum in Symbiosis with Soybean Host Plants

Symbioses between uptake hydrogenase host-regulated (Hup-hr) phenotypes of Bradyrhizobium japonicum and exotic, agronomically unadapted soybean germ plasm were examined for expression of uptake hydrogenase activity. Determinations for hydrogen evolution and uptake hydrogenase activity identified five plant introduction (PI) lines which formed hydrogen-oxidizing symbioses with strains USDA 61 and PA3 6c. Hup-hr strains belonging to serogroup 94 expressed uptake hydrogenase activity in symbioses with PI 181696 and PI 219655 at rates sufficient to prevent hydrogen from escaping the nodules. The identification of soybean germ plasm forming hydrogen-oxidizing symbioses with Hup-hr bradyrhizobia potentially has implications for enhancing nitrogen fixation efficiency in soybean production.