Klebsiella oxytoca HP1 adhE基因插入失活法构建产氢重组菌

乙醇是产酸克雷伯氏菌(Klebsiella oxytoca)HP1厌氧发酵产H2的主要副产物,每生成1.0 mol的乙醇需要消耗2.0 mol NAD(P)H,从而降低了H2的产量.本研究以编码乙醇脱氢酶系(含乙醛脱氢酶和乙醇脱氢酶活性)的adhE基因为改造目标,利用同源重组技术获得了以提高产氢为目标的K.oxytoca重组菌.构建工作包括:根据adhE基因保守序列框克隆K.oxytoca HP1 adhE基因片段,以质粒pMHE6为模板进行链霉素抗性基因表达盒的扩增,表达链霉素抗性的aadA基因片段和adhE基因片段分别与载体pMD18-T相连构建重组质粒,同源整合质粒pTA-Str的构建,以链霉素作为筛选标记筛选重组菌.菌落PCR鉴定结果表明,aadA基因表达盒通过质粒pTA-Str的介导已定点插入K.oxytoca HP1基因组中,成功地构建了adhE基因部分片段缺失的重组菌.葡萄糖发酵实验结果表明,相同发酵条件下,重组菌比野生菌的产氢量提高了16.07%,乙醇产量下降了70.47%.利用基因工程技术提高产氢初步获得成功.国家自然科学基金(批准号:30470395);; 福建省重点科技项目(批准号:2005I106

A study on antioxidation of synechococcus sp. PCC 7942 withTrans-thymosin α1-gene in Mice

本试验室已在蓝藻聚球藻中高效表达了人源胸腺素α1(thymosinα1,Tα1)基因,为研究转Tα1基因聚球藻口服后的生物活性,本研究给小鼠灌服转Tα1基因聚球藻14d,研究其对小鼠谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(Cat)和超氧化物歧化酶(SOD)活力以及丙二醛(MDA)含量的影响,结果表明:转胸腺素α1基因聚球藻可显著提高小鼠心、肝与肾中GSH-Px活力(P<0.01);明显提高心脏Cat活性(P<0.01);显著降低肝脏中MDA的含量(P<0.01);但对SOD活力无明显作用。提示转胸腺素α1基因聚球藻较强的抗氧化作用。Human thymosin α1 gene was expressed effectively in Synechococcus sp. PCC 7942 and antioxidant effect of Synechococcus sp. PCC 7942 with trans-thymosin α1-gene in mice were investigated. Synechococcus sp. PCC 7942 with trans-thymosin al-gene were administrated orally 14d,Laters the results showed that the activities of glutathione peroxidase (GSH-Px) in heart、liver and kidney were increased significantly (P<0. 01);the activity of catalase (Cat) in heart was increased markedly;the content of malondialdehyde (MDA) in liver was decreased obviously (P<0.01). But no significant change in the activity of super-oxide dismutase (SOD) was observed. It indicated that Synechococcus sp. PCC 7942 with trans-thymosin α1-gene had obvious antioxidation in vivo.国家海洋863课题(项目编号:819-04-03);福建省自然科学基金项目(项目编号:c0010002

Hydrogen Production by Chromatium vinosum with Fermentation Waste Produced by Klebsiella oxytoca

研究了酒色着色菌(Chromatiumvinosum DSM185)利用产酸克雷伯氏菌(Klebsiellaoxytoca HP1)发酵产氢废液进行光发酵和暗发酵产氢的可行性,以达到对产氢底物的充分利用和对产氢废液的进一步处理。研究结果表明C.vinosum可以利用K.oxytoca的发酵废液进行光发酵产氢和暗发酵产氢。C.vinosum发酵产氢后废液中残余还原糖和主要有机酸(丁酸)的含量明显降低,发酵产氢的最佳pH为6.5,添加0.1%(W/W)NH4Cl能促进产氢。在光照条件下丁酸利用率可达54.38%,产氢量达36.97mL/mg;在黑暗条件下丁酸利用率可达36.01%,产氢量达37.50mL/mg。Photosynthetic bacteria(PSB) showed great promise in biohydrogen production. Chromatium vinosum was able to utilize the fermentation waste of Klebsiella oxytoca for both photo-fermentative and dark-fermentative hydrogen production. The content of residual sugars and main organic acids decreased obviously after hydrogen production by C.vinosum. The maximal hydrogen production of C.vinosum was obtained at pH 6.5 adding extra 0.1%(W/W) NH_4Cl. Under photo-fermentative conditions, the content of butyric acid decreased by 54.38%, and the maximal hydrogen yield was 36.97 mL/mg cell. Under dark-fermentative conditions, the content of butyric acid decreased by 36.1% and the maximal hydrogen production was achieved as 37.50 mL/mg cell.国家863计划项目(No.2006AA05Z111);; 福建省科技项目(No.2006H0091);; 厦门大学创新项目(No.2002xjkt033)

Screen of O_2-tolerate Phenotype of Klebsiella oxytoca HP1 Mutants with High H_2-evolving Activity by Selection with MNZ Combination O_2

氢酶是生物制氢的关键酶,大多数氢酶因对氧极敏感而易失活,因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1%甲基磺酸乙酯对Klebsiella oxytoca HP1进行了两轮诱变,经40mmol/L甲硝唑和21%氧联合处理1h(第一轮诱变)或2h(第二轮诱变)进行筛选。所得突变菌株经产氢测试,结果在15%氧浓度条件下,第一代突变菌株HP1-A15产氢活性为出发菌株Klebsiella oxytoca HP1的3.70倍,在21%氧浓度条件下第二代突变菌株HPA15-37产氢活性为HP1-A15菌株的2.75倍,是出发菌株的11倍。突变菌株HP1-A15和HPA15-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。Hydrogenases are key enzyme for bio-hydrogen production, most of them were rapidly inacti-vated by oxygen. It is important to bio-hydrogen production and hydrogen application that improve the O2-tolerance of hydrogenase. In this experiment, the hydrogen producing strain Klebsiella oxytoca HP1 was treated with 1% ethyl methanesulfonate(EMS) , the mutants with high O2-toleration ability were screened with 40mmol/L metronidazole (MNZ) and 21% oxygen. The H2-evolving activity of the first generation mu-tant HP1-A15 was increased 3.70 times than that of the wild-type (WT) under 15% oxygen. The H2-evolving activity of the second generation mutant HPA15-37 was enhanced 11 times than that of WT under the condi-tion of 21% oxygen. The mutants HP1-A15 and HPA15-37 had steady heredity. These results suggest that MNZ and in addition oxygen is a good way to screen of O2-tolerate phenotype of facultativeanaerobe withhigh H2-evolving activity.福建省青年科技人才创新项目(No.12005J003);; 厦门市科技项目(No.13502Z20041070

Study on Hydrogen Production Activity of Klebsielal oxytoca HP1 and Stability of Its Soluble Hydrogenase under Atmosphere with O_2

目的:考察产酸克雷伯氏菌(Klebsielal oxytoca HP1)耐氧产氢特性及其可溶性氢酶的氧耐受特性。方法:研究K.oxytoca HP1在不同气相氧浓度条件下利用葡萄糖(1%,m/v)、丙酮酸钠(0.5%,m/v)及甲酸(0.1%,v/v)等底物产氢活性的以及K.oxyto-ca HP1可溶性氢酶在空气及氧饱和溶液中催化产氢活性。结果:K.oxytoca HP1在葡萄糖(1%,m/v)底物中具有较高耐氧产氢活性,6h内在气相氧浓度为5%、10%和21%条件下的氢产量分别为厌氧条件下的20.9%、13.7%、8.3%;K.oxytoca HP1可溶性氢酶在空气中孵育12h后,其活性残余85.4%,在氧饱和溶液中活性损失一半约3h。结论:试验结果提示K.oxytoca HP1具有耐氧产氢特性,其可溶性氢酶具有较高氧耐受性,在氢能源的开发中具有潜在的应用前景。Objective: To study the hydrogen production activity of K. oxytoca HP1 under condition with oxygen stress and the oxygen tolerance ability of its soluble hydrogenase. Method: The hydrogen production activity of K. oxytoca HP1 incubated in glucose (1%,m/v), sodium pyruvate (0.5%,m/v)or formate (0.1%,v/v) under atmosphere with 0%,5%,10% and 21% O2 were studied on, and the remain activity of its soluble hydrogenase incubated in air or pure O2 during 0-12h were studied on. Result: The results show that K. oxytoca HP1 has high hydrogen production activity in glucose (1%,m/v) substrate under atmosphere with oxygen. Compared to in anaerobic condition, the hydrogen production of K. oxytoca HP1 under atmosphere with 5%, 10% and 21% oxygen was 20.9%,13.7% and 8.3% respectively in 6h. The soluble hydrogenase from K. oxytoca HP1 exhibits considerable oxygen toleration ability, the remain activity was 85.4% after the hydrogenase exposure to air 12h and its activity lost half after the hydrogenase exposed to oxygen 3h. Conclusion:These results suggest that K. oxytoca HP1 and its soluble hydrogenase have potential to be applied in hydrogen production and application.福建省青年科技人才创新项目资助(No.12005J003

Isolation and Characterization of a H2 -producing Strain Enterbacter sakazakii HP

通讯作者 Tel 0592-2185731, E-mail： longmn@ xmu.edu.cn[中文文摘]在自然环境中分离到一株具有高产氢活性的微生物菌株,经细菌鉴定仪及16S rRNA基因序列分析,鉴定该菌株为Enterbacter sakazakii HP。分析了起始pH值、反应温度、碳源、起始糖浓度、起始氧浓度及菌体密度等因素对菌株产氢活性的影响。研究表明,该菌株发酵产氢较适合的条件为:以葡萄糖为产氢底物,起始pH值8.0,菌体密度OD600=0.7,反应温度35℃,糖浓度为0.1mol/L,氧浓度为0%的条件下,此时产氢菌株的最高产氢活性为5.34μmolH2/h.mgdw,氢的得率为1.94molH2/mol葡萄糖。[英文文摘]A H2 -producing bacterial strain was newly isolated and identified as Enterbacte sakazakii HP by 16S rDNA sequence analysis and detection by BBLCRYSTAL AUTOREADER.Various factors,including substrates and its concentration,initial pH,temperature and oxygen,on the hydrogen production of E.sakazakii HP have been studied extensively.Among several sugars,glucose was the favorite substrate for hydrogen production.The optimum condition for hydrogen production by Enterbacte sakazakii HP was achieved as:initial pH8.0,cell density OD600 =0.7,temperature 35℃,glucose concentration 0.1 mol/L,oxygen concentration 0%.Under batch fermentative hydrogen production conditions,the maximal hydrogen production activity and hydrogen yield were obtained as 5.34μmol H2 /h·mg dw and 1.94mol H2 /mol glucose,respectively.The research results suggest that Enterbacter sakazakii HP is an ideal candidater for biological hydrogen production.国家自然科学基金项目(No30470395);厦门市科技项目(No3502Z20041070);厦门大学新世纪优秀人才计划项目;福建省青年科技人才创新项目(No2005J003

Isolation and Characterization of H_2-producing Strains Enterobacter sp. and Clostridium sp.

在高温水体中分离得到2株具有较高产氢活性的微生物菌株Z-16和C-32。根据两菌株的16SrDNA序列分析,初步鉴定菌株Z-16为Enterobactersp.,菌株C-32为Clostridiumsp.。研究了起始pH值、反应温度、碳源等对菌株放氢活性的影响。菌株Z-16的最适产氢条件为:反应系统起始pH7·0,反应温度35℃,以蔗糖为产氢底物。在最适条件下,菌株Z-16的氢转化率为2·68molH2/mol蔗糖。菌株C-32的最适产氢条件为:反应系统起始pH8·0,反应温度35℃,以麦芽糖为产氢底物。在最适条件下,菌株C-32的氢转化率为2·71molH2/mol麦芽糖。以葡萄糖为碳源时,菌株Z-16和菌株C-32的氢转化率分别为2·35和2·48molH2/mol葡萄糖。Two hydrogen-producing bacterial strains were newly isolated and identified as Enterobacter sp. Z-16 and Clostridium sp. C-32 by 16S rDNA sequence analysis. Various parameters for hydrogen production, including substrates, initial pH and temperature, have been studied. The optimum condition for hydrogen production of strain Z-16 were achieved as: initial pH7.0, temperature 35℃, sucrose as the favorite substrate. In comparison, The optimum condition for hydrogen production of strain C-32 were obtained as: initial pH8.0, temperature 35℃, maltose as the favorite substrate . Under batch fermentative hydrogen production conditions, the maximal hydrogen conversion rate for strain Z-16 and strain C-32 were 2.68 mol H_2/mol sucrose and 2.71mol H_2/mol maltose, respectively. Using glucose as substrate, the hydrogen conversion rate of strain Z-16 and strain C-32 were 2.35 and 2.48 mol H_2/mol glucose, respectively. This research suggest a good application potential of strain Z-16 and C-32 in the future biological hydrogen production.福建省重点科技项目(No.2005I016);; 厦门市科技项目(No.3502Z20041070)资助~

Purification and characterization of hydrogenase from Synechococcus sp. PCC 7942

报道了室温、空气环境下聚球藻Synechococcus sp.PCC 7942氢酶的分离纯化。经过超声破碎、超速离心、离子交换层析、疏水层析及凝胶层析等步骤,氢酶被纯化了218倍,得率为6.5%,比活为1.46 U·mg-1蛋白。纯化氢酶的SDS-PAGE图显示五条蛋白带,分子量约为83kDa,60kDa,47kDa,30kDa和27kDa。该氢酶为可溶性的双向氢酶,其催化放氢的最佳电子供体为还原态的甲基紫精,最适温度50℃,最适pH 8.0。Hydrogenase from Synechococcus sp.PCC 7942 was purified to close homogeneity aerobically at room temperature.The hydrogenase-containing crude extract was collected after ultrasonic disruption and removal of cell debris by ultracentrifugation.Subsequently,three steps of column chromatographies(anion exchange,hydrophobic interaction and gel filtration)were performed.Hydrogenase was purified about 218-fold with a yield of 6.5% finally.The purified enzyme has a specific activity for hydrogen evolution of 1.46 U.mg-1 protein.SDS-PAGE gel of the purified enzyme revealed five predominant protein bands with estimated molecular weights of 83,60,47,30 and 27 kDa,respectively.The enzyme is a soluble bidirectional hydrogenase and shows maximum activity while using reduced methyl viologen as an electron donor.The optimum temperature and pH value for hydrogen evolution catalyzed by the purified hydrogenase are 50 ℃ and pH 8.0.福建省自然科学基金(C0410002)资

苯硫酚对固氮酶催化活性的影响

研究了苯硫酚对棕色固氮菌固氮酶催化底物还原活性的影响。结果表明:当反应体系中固氮酶钼铁蛋白与苯硫酚的摩尔比为1:4时,固氮酶的乙炔还原活性比对照组下降了62.3％,下降的幅度随着苯硫酚浓度的升高而增大;但在乙炔存在的情况下,固氮酶的放H_2活性随着苯硫酚的浓度的升高而升高。在氩气氛下,苯硫酚浓度的升高对固氮酶放H_2活性的影响不明显。这一现象有可能是由于苯硫酚取代与铁钼辅基的Fe_1原子连接的半胱氨酸的巯基而引起的

钼酸盐柠檬酸盐络合物及有机酸对棕色固氮菌生长的影响

将经过0.9%NaCl溶液处理8h的棕色固氮菌(AzotobactervinelandiiOP)作为菌种分别接入Burk′s培养基和用不同有机酸替代柠檬酸三钠或用不同等摩尔的钼络合物替代钼酸钠的各种改良的Burk′s培养基中,分别测定菌体生长曲线和固氮活性.结果发现,与Burk′s培养基相比,以高柠檬酸、苹果酸、马来酸替代柠檬酸三钠或以K6[Mo2O5(cit)2]·5H2O、K4[Mo2O5(Hcit)2]·4H2O和Na2[MoO2(Hcit)]·3H2O替代柠檬酸三钠和钼酸钠的培养基能促进菌体的生长;以乙醇酸替代柠檬酸三钠的培养基则会抑制固氮菌的生长;各种培养条件下菌体细胞的C2H2还原活性表现了类似的规律.讨论了固氮酶活性中心FeMoco在装配过程中钼的可能运输方式和装配机理