亚心形四爿藻氢酶的稳定性及初步纯化

亚心形四爿藻Tetraselmis subcordiformis是一种具有高产氢能力的海洋绿藻,在厌氧环境中经暗诱导调控,可进行一定时间的连续产氢。氢酶是亚心形四爿藻进行光合产氢的关键酶,本文研究了在厌氧环境中连二亚硫酸钠、β-巯基乙醇和丙三醇等试剂对氢酶的稳定性影响,考察了硫酸铵分级沉淀对氢酶的纯化效果及回收情况。结果表明,连二亚硫酸钠和丙三醇均能对氢酶起到较好的保护作用。60%～70%饱和度硫酸铵沉淀出的蛋白样品比酶活较高,且所含其他蛋白较少,可用于氢酶的进一步纯化。     

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。     

蓝藻Anabaena 7120的吸氢及其和固氮活力的关系

分子氢通过氢酶活动支持蓝藻固氮。氢酶活性高低变化规律与蓝藻固氮活性大小在趋势上是一致的。氢对固氮产生的效用与蓝藻本身所处的内外界生理条件也有一定的联系。但是,正如前文曾经指出过的,蓝藻的吸氢特性和包括固氮在内的其它生理生化功能的关系,都还没有得到完全的阐明。本文以具有不同固氮活力的蓝藻Ana-     

分子氢支持蓝藻Anabaena7120固氮的生理基础研究

1.在一般条件下供给分子氢,蓝藻的乙炔还原活性相对增长百分率不如加入光合及代谢抑制剂那样显著。弱光和暗处理条件下,分子氢对蓝藻的增益作用较明显。2.分子氢对年轻蓝藻固氮作用的促进比对年老的大。3.在氨和尿素等结合氮影响下,蓝藻固氮活性受到抑制,同时加入分子氢,则其活性便有所提高。4.用氮气、二氧化碳和二氧化碳+氮气处理蓝藻,其固氮活性受到削弱,同时供给分子氢,则蓝藻的乙炔还原活性便得到明显的促进。5.经以乙炔预先处理过的蓝藻,无论添加光合和代谢抑制剂与否,其固氮活性均下降。加分子氢与不加分子氢的乙炔还原活性相近,分子氢对固氮的增益作用显著被削弱以至消失,加和不加抑制剂的氢耗也都下降,不加抑制剂的氢耗相对下降百分率更大。这表明分子氢支持蓝藻固氮和氢酶活动有联系。     

缺钼培养的蓝藻吸氢

培养液中缺钼时,蓝藻(Anabaena 7120)吸氢量显著下降,补加钼时吸氢量恢复到有钼培养的蓝藻水平。缺钼培养的蓝藻吸氢对氧和CO都敏感,暗中经乙炔或光下以分子氢预处理后,吸氢量减少。光下缺钼培养的蓝藻吸氢比暗中高,光合抑制剂和结合态氮明显地抑制此种蓝藻吸氢。     

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。     

光合细菌光合产氢的研究进展

光合细菌 (Photosyntheticbacteria ,PSB)光合产氢的研究是国内外普遍关注的热点问题。就PSB光合产氢的机理、条件及光合细菌生态应用等方面进行综述 ,并着重论述了光合细菌产氢过程中两种主要的酶—固氮酶和氢酶以及影响酶活性的因素。     

蓝藻(Anabaena 7120)的光合放氢和参与放氢的酶

蓝藻Anabaena 7120放氢是一个依赖于光的过程,暗中几乎测不出放氢活性。静置方式培养的蓝藻预先进行强化培养是测得高放氢活性的重要条件。年轻的蓝藻放氢活性比年老的高。氯化铵和一系列光合作用抑制剂对蓝藻放氢有抑制作用,弱光加剧氯化铵对放氢的抑制。在弱光加光合抑制剂的条件下,受氯化铵抑制的放氢活性恢复速度比强光下慢。CO_2、N_2、NaN_3和KNO_3与放氢竞争电子而抑制蓝藻的放氢。C_2H_2促进蓝藻放氢,CO则抑制放氢,C_2H_2和CO一起加入时,放氢受到的促进显著比单加C_2H_2的大。经分子氢预处理过的蓝藻,其放氢活性在光下可以得到明显的促进。     

分子氢在鱼腥蓝藻Anabaena 7120固氮活性受氧损伤中的保护作用

分子氢在一定程度上可以消除氧对蓝藻固氮活性的损伤而起一种保护作用。氧对蓝藻固氮活性的损伤主要在固氮反应初期,且是不可逆的。分子氢的保护作用也在反应初期,分子氢过迟加入,其保护作用就会受到削弱或消失,其最大保护作用是在与氧同时加入之时。分子氢的保护作用和生理条件有一定联系。年老的蓝藻中,分子氢支持的固氮活性对氧敏感小些。温度、CO_2、氮气和一系列抑制剂都只影响固氮活性的高低,并不改变分子氢的有益行为。蓝藻经乙炔预处理导致氢酶受抑时,分子氢对固氮活性受氧损伤时的保护作用即受到削弱或消失。     

应用免疫和质谱法对亚心形扁藻氢酶蛋白进行亚细胞定位和分类

亚心形扁藻(Platymonas subcordiformis)是新发现的一株产氢海洋单细胞绿藻,经过胁迫调控可实现一定时间的持续产氢。氢酶是亚心形扁藻在胁迫条件下进行光合产氢的一个关键酶。但到目前为止,亚心形扁藻氢酶相关信息仍不清楚。利用蛋白合成抑制剂氯霉素和放线菌酮对亚心形扁藻氢酶活性进行考察,同时利用免疫印迹技术和免疫胶体金电镜对亚心形扁藻氢酶蛋白进行亚细胞定位分析。结果表明:亚心形扁藻氢酶蛋白可能由胞浆内合成,在叶绿体行使功能。采用免疫共沉淀技术富集亚心形扁藻细胞氢酶蛋白,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)对免疫共沉淀复合物进行分离,从胶中切取目的蛋白条带,胶内酶解后进行基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析,得到相应的肽指纹图谱,通过搜索数据库检索初步断定亚心形扁藻氢酶蛋白为铁氢酶。     

Nitrogen Fixation and Hydrogen Metabolism in Cyanobacteria

Summary: This review summarizes recent aspects of (di)nitrogen fixation and (di)hydrogen metabolism, with emphasis on cyanobacteria. These organisms possess several types of the enzyme complexes catalyzing N₂ fixation and/or H₂ formation or oxidation, namely, two Mo nitrogenases, a V nitrogenase, and two hydrogenases. The two cyanobacterial Ni hydrogenases are differentiated as either uptake or bidirectional hydrogenases. The different forms of both the nitrogenases and hydrogenases are encoded by different sets of genes, and their organization on the chromosome can vary from one cyanobacterium to another. Factors regulating the expression of these genes are emerging from recent studies. New ideas on the potential physiological and ecological roles of nitrogenases and hydrogenases are presented. There is a renewed interest in exploiting cyanobacteria in solar energy conversion programs to generate H₂ as a source of combustible energy. To enhance the rates of H₂ production, the emphasis perhaps needs not to be on more efficient hydrogenases and nitrogenases or on the transfer of foreign enzymes into cyanobacteria. A likely better strategy is to exploit the use of radiant solar energy by the photosynthetic electron transport system to enhance the rates of H₂ formation and so improve the chances of utilizing cyanobacteria as a source for the generation of clean energy.     

The AbrB2 Autorepressor, Expressed from an Atypical Promoter, Represses the Hydrogenase Operon To Regulate Hydrogen Production in Synechocystis Strain PCC6803

We have thoroughly investigated the abrB2 gene (sll0822) encoding an AbrB-like regulator in the wild-type strain of the model cyanobacterium Synechocystis strain PCC6803. We report that abrB2 is expressed from an active but atypical promoter that possesses an extended -10 element (TGTAATAT) that compensates for the absence of a -35 box. Strengthening the biological significance of these data, we found that the occurrence of an extended -10 promoter box and the absence of a -35 element are two well-conserved features in abrB2 genes from other cyanobacteria. We also show that AbrB2 is an autorepressor that is dispensable to cell growth under standard laboratory conditions. Furthermore, we demonstrate that AbrB2 also represses the hox operon, which encodes the Ni-Fe hydrogenase of biotechnological interest, and that the hox operon is weakly expressed even though it possesses the two sequences resembling canonical -10 and -35 promoter boxes. In both the AbrB2-repressed promoters of the abrB2 gene and the hox operon, we found a repeated DNA motif [TT-(N(5))-AAC], which could be involved in AbrB2 repression. Supporting this hypothesis, we found that a TT-to-GG mutation of one of these elements increased the activity of the abrB2 promoter. We think that our abrB2-deleted mutant with increased expression of the hox operon and hydrogenase activity, together with the reporter plasmids we constructed to analyze the abrB2 gene and the hox operon, will serve as useful tools to decipher the function and the regulation of hydrogen production in Synechocystis.     

Towards optimization of cyanobacteria as biotechnologically relevant producers of molecular hydrogen, a clean and renewable energy source

In discussions about alternatives to our current fossil energy sources, basic and applied research leading to biological production of molecular hydrogen utilizing cyanobacteria deserves serious attention. In these oxygenic phototrophic bacteria, hydrogen can be produced by the activity of either nitrogenases or reversible/bidirectional hydrogenases. Knowledge of the physiological and molecular basis of some of the processes involved in hydrogen metabolism in these peculiar microorganisms has increased during the last decade. However, further efforts are required in basic as well as applied research in order to obtain a clear impression of these processes and their regulation. This information might then constitute the basis for optimizing the efficiency of hydrogen evolution by cyanobacteria. Progress might be achieved by screening more cyanobacterial strains for their ability to produce and evolve hydrogen, by genetically manipulating specific strains as well as by improving the conditions for cultivation in bioreactors. Received: 17 February 1998 / Received revision: 24 April 1998 / Accepted: 27 April 1998     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocystous cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.     

Genetic control of hydrogen metabolism in cyanobacteria

The development of methods for the use of phototrophic cyanobacteria as producers of molecular hydrogen via bioconversion of solar energy is a promising filed of hydrogen energetics. Artificial optimization of hydrogen formation and release is based on studying the genetic control of hydrogen metabolism and the use of genetic approaches for obtaining efficient producer strains. Data on genes coding for the hydrogenases that are responsible for hydrogen uptake and production in cyanobacteria are summarized. Bioinformatic methods have been used to construct the scheme of the hydrogen metabolism gene network of nitrogen-fixing heterocyst cyanobacteria. The possible approaches to constructing the cyanobacterium strains producing molecular hydrogen that would be promising for photobiotechnology by mutagenesis and genetic engineering methods are discussed in terms of this model and analysis of the data on hydrogen-producing mutants.