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1.
供试菌株Alcaligenes faecalis A15,Enterobacter cloacae EnSs.E·cl-oacae E26,Klebsiella planticola DWUL2.K·oxytoca NG13和Pseudomonassacharophila均能还原2,3,5-氯化三苯基四氮唑(TTC),说明它们含有吸氢酶。以Rhizobium japonicum的hup基因为探针进行杂交的结果表明。除E·oloacae E26和K·oxytoca NG13外,其它菌株的DNA与hup探针具有同源性。A·faecalis A15 hup基因位于染色体上‘E·cloacae EnSs则位于较大的一个质粒上。A·faecalis A15和E·Cloacae EnSs的nif基因与hup基因位于同一复制子上。hup基因不具必然的保守性,因此异源hup基因探针不一定都适宜于探测hup基因。 相似文献
2.
Summary Hydrogen oxidation in soil was measured at low (1 ppmv) and high (300 ppmv) H2 concentrations to distinguish between the activities of abiontic soil hydrogenases and Knallgas bacteria, respectively. The two activities also showed distinctly different pH optima, temperature optima, and apparent activation energies. The pH optima for the soil hydrogenase activities were similar to the soil pH in situ, i.e., pH 8 in an slightly alkaline garden soil (pH 7.3) and pH 5 in an acidic cambisol (pH 4.6–5.4). Most probable number determinations in the alkaline acidic soils showed that Knallgas bacterial populations grew preferentially in neutral or acidic media, respectively. However, H2 oxidation activity by Knallgas bacteria in the acidic soil showed two distinct pH optima, one at pH 4 and a second at pH 6.4–7.0. The soil hydrogenase activities exhibited temperature optima at 35–40°C, whereas the Knallgas bacteria had optima at 50–60°C. The apparent activation energies of the soil hydrogenases were lower (11–23kJ mol-1) than those of the Knallgas bacteria (51–145 kJ mol-1). Most of the soil hydrogenase activity was located in the upper 10 cm of the acidic cambisol and changed with season. The seasonal activity changes were correlated with changes in soil moisture and soil pH. 相似文献
3.
H2-oxidizing activities were assayed in slurries of four soils by measuring the consumption of H2 and the exchange of 3H2 with H2O at increasing mixing ratios of H2 or 3H2. Both H2 consumption and 3H2 exchange were abolished by autoclaving or the addition of formaldehyde. The rates of H2 consumption and 3H2 exchange were proportional to the quantity of soil used. Both activities increased with increasing concentrations of H2 or 3H2 and displayed biphasic kinetics, demonstrating the existence of two different H2-oxidizing activities, one with a relatively low K
m and V
max, and a second with a relatively high K
m und V
max. The first type of activity was characteristic of abiontic soil hydrogenases, and the second of aerobic H2-oxidizing bacteria. In contrast to H2 consumption, which required the presence of either O2 or ferricyanide, 3H2 exchange operated equally well without an external electron acceptor. The 3H2 exchange assay may thus be particularly useful for enrichment of soil hydrogenases which have not yet been isolated and for which no natural electron acceptor is known. 相似文献
4.
根据物种间同源基因相对保守的特点,利用生物信息学的方法,以拟南芥牻牛儿基牻牛儿基氯化酶（GGH）基因cDNA序列为信息探针,搜索GenRank中的HTGs数据库,找到一个与之高度匹配的日本百脉根基因组DNA序列LJT46M09,用GENSCAN软件分析该序列得到一个包含2个外显子和1个内含子的基因。以此基因序列BLAST检索GenBank中的est-others数据库获得了同源的EST片段。进行EST拼接后得到该基因的cDNA序列,GenBank登录号为DQ013361,由1389bp核苷酸组成,具有完整的开放阅读框架（ORF）,推测编码蛋白为462个氨基酸。推导的氨基酸序列与大豆、截形苜蓿、烟草、拟南芥、小麦、水稻的牻牛儿基牻牛儿基氢化酶基因氨基酸序列的一致率分别为91％、89％、84％、81％、73％、74％。访基因与叶绿素等的生物合成有关。 相似文献
5.
为了克隆氢化酶3大亚基（HyA3L）和甲酸氢酶抑制因子（hycA）,研究其在阴沟肠杆菌产氢代谢中的作用和机制,笔者利用CODEHOP设计阴沟肠杆菌NRRL B-414 HyA3L和hycA简并引物,选用引物HyA3J和HycAJ扩增基因组DNA,分别得到约1500、150 bp的PCR产物,该产物连接pMD18-T载体,并克隆转化至E.coli DH5α感受态细胞中,经筛选后测序。推导的HyA3J扩增产物氨基酸序列与Enterobacter sp. 638的HyA3L蛋白一致性最高达到99%,仅有3个氨基酸的差异,表明其为阴沟肠杆菌的HyA3L;推导的HycAJ扩增产物氨基酸序列与Enterobacter cloacae subsp. cloacae ATCC 13047的HycA蛋白一致性最高达到92%,仅有4个氨基酸的差异,表明其为阴沟肠杆菌的hycA。通过以上分析可得出,采用CODEHOP程序设计的简并引物可信性强,阳性率高。阴沟肠杆菌NRRL B-414的氢化酶3大亚基和甲酸氢酶抑制因子部分基因的成功克隆,为通过代谢工程手段敲除氢化酶3大亚基和甲酸氢酶抑制因子的基因全长克隆奠定基础,不但增加了这2个基因的资源,也可为其基因敲除等代谢工程研究提供科学依据和工作基础。 相似文献
6.
在吸氢培养基(HuM)和氢气培养下,镍明显地促进了花生根瘤菌(Rhizobiumarachis)L8-3菌株的氢酶活性.5μM NiCl_2能提高吸氢活性两倍以上。螯合剂EDTA和邻菲啰啉抑制氢酶活性,在抑制浓度的螯合剂存在下.加镍可使吸氢活性恢复。除镍外其他二价金属离子如钼、铜、锌、钴、铁、镁、锰、钙等未发现对吸氢活性有促进作用。氢酶活性表达的时间进程受镍的调节,氯霉素阻遏氢酶活性。镍和EDTA对无细胞提取液的氢酶活性不产生影响。研究结果初步认为镍是花生根瘤菌L8-3菌株氢酶活性表达所必需的,可能参与氢酶蛋白的合成。 相似文献
7.
Two complementary experimental approaches were utilized to examine the extent to which free soil hydrogenases and H2-oxidizing bacteria contribute to the soil uptake of atmospheric H2. First, high affinity hydrogenase activity and H2-oxidizing bacteria were fractionated in non-axenic soil and axenic soil colonized with the high affinity H2-oxidizing bacterium Streptomyces sp. PCB7. Non-axenic soil was fractionated by buoyant density centrifugation. High affinity H2 oxidation activity measured in individual fractions was proportional to the copy number of hhyL gene, specifying the large subunit of putative high affinity [NiFe]-hydrogenases. 2.5% of the hydrogenase activity was recovered in bacteria-free soil extract. Similarly, sequential centrifugation and wet filtrations of strain PCB7-colonized soil dispersed in solubilization buffer caused a loss of the activity, at a ratio proportional to the number of living cells removed. No abiontic hydrogenase activity was detected in bacteria-free fractions. The second experimental approach was designed to verify whether or not the [NiFe]-hydrogenase of strain PCB7 retains high affinity H2 oxidation activity in soil, under the abiontic state. H2 oxidation rates of crude enzyme extract of strain PCB7 measured under aerobic and anaerobic conditions were indistinguishable, indicating that the high affinity hydrogenase of strain PCB7 is oxygen-tolerant. The hydrogenase activity of sterile soil spiked with as much as 0.14 mg(protein) g(soil-dw)−1 was equivalent to the H2-oxidation activity of only 106-107 CFU of strain PCB7 g(soil-dw)−1. Taken together, our results indicate that high affinity hydrogenase activity is proportional to the abundance of H2-oxidizing bacteria in soil and, that abiontic hydrogenases contribute only a few percent of the total high affinity H2 oxidation activity detected in soil. 相似文献
8.
氢气是目前最常用的清洁能源,具有能量含量高和清洁燃烧的特点。制氢的方式有多种,生物制氢与传统物理和化学工艺制氢相比,是最清洁的一种方法。然而,大规模生物制氢的产氢量与产氢率往往受到各种环境等因素的限制。近年来的许多研究突破了环境因素的限制,从微生物代谢、能源来源及微生物产氢关键酶等方面有效提高了微生物产氢效率。本文总结了生物制氢的几种主要方法,详细讨论了光合细菌产氢的影响因素,并对其有效促进途径的研究进展进行了综述,以期为生物制氢领域的深入研究提供参考,为工业大规模制氢、减轻环境污染做出贡献。 相似文献
9.
为更好地了解小麦维生素E的生物合成并将其应用于营养品质改良,以电子和实验克隆相结合的方法克隆分离了小麦牻牛儿牻牛儿基脱氢酶（GGH）基因的cDNA序列,序列号为DQ139268。序列分析结果表明,该cDNA序列含有一个完整的开放读码框,编码462个氨基酸,含有保守的ChlP结构域,氮端有信号肽序列,定位于叶绿体中。氨基酸序列比对和系统发育分析结果显示,不同物种之间GGH基因编码的氨基酸序列同源性都较高。用生物信息学工具将该基因定位于小麦第六部分同源群的长臂上。RTPCR和电子组织表达分析结果显示该基因在光合器官中表达量丰富,这可能与其亚细胞定位有关。 相似文献
10.
极端嗜热菌Thermotogahypogea(T.h.)是一株来自中非油田井下的,能够生长在90℃高温下的短杆状厌氧嗜热菌,经研究发现,在T.h.菌体细胞抽提液中存在一种特殊的NADP依赖性醇脱氢酶(ADH)。该酶在T.h.菌的醇醛代谢途径中起着关键性的催化作用,可催化多种醇醛底物的脱氢反应。同时,当以二氯联卞吡啶为作用底物时,T.h.菌的细胞抽提物显示很高的氢化活性(Km=0.29mmol·L-1,Vmax=41.69)。 相似文献