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

Cloning and Characterization of Hydrogenase Gene hydSL from Chromatium vinosum

光合细菌酒色着色菌(Chromatium vinosum)至少含有一种膜结合态氢酶(623、2 ku)和一种可溶性氢酶(52、21.5ku).其膜结合态氢酶是一种催化吸氢的氢酶,而可溶性氢酶则是一种催化放氢的氢酶.本研究分离提纯了C.vinosum膜结合态氢酶,并对其大、小亚基N-末端氨基酸序列进行测定;根椐氢酶亚基N-末端氨基酸序列及氢酶保守序列设计引物,通过PCR扩增分别获得了1.1和3.5 kb的DNA片段.对该片段进行克隆和序列分析,结果表明该基因编码氢酶HydSL,其结构不同于典型的氢酶基因结构,在氢酶大、小亚基基因间插入了ISP基因.The photosynthetic bacteria Chromatium vinosum contained at least a membrane-bound hydrogenase and a soluble hydrogenase.A membrane-bound hydrogenase from C.vinosum was purified and identified to consist of two subunits(62,32 ku).According to the N-terminal sequences of the large subunit:N′-SEEIVVDPITRIEGH and the small subunit:N′-SEQARRPSVIWLSFQE,and the conserved motif of NiFe-hydrogenase,two pairs of primers were synthesized and applied for PCR to amplify the fragment encoding hydrogenase.The sequence analysis results showed that the hydSL contained the unusual gene organization which existed one ISP gene between the small and large subunit.It was suggested that there are more than one membrane-bound hydrogense existed in C.vinosum.国家863计划项目(2006AA05Z111);; 福建省自然科学基金(C0410002);; 福建省科技项目(2006H0091,2005I016);; 厦门大学新世纪优秀人才计划项目资

Bioaugmentation Study on UASB Biohydrogen Production System

采用上流式厌氧污泥床(UASB)反应器,以糖蜜废水为底物,利用厌氧活性污泥发酵产氢.向反应器中投加高产氢微生物产酸克雷伯氏菌HP1,探讨了生物强化作用对反应器产氢能力的影响.研究表明:在污泥接种量为30.0 gVSS/L、启动负荷为6.0 kgCOD/(m3.d)、水力停留时间(HRT)为9 h、投菌量为3%的条件下对生物制氢系统进行强化,可使反应系统产氢能力提高25%,并形成丁酸型发酵产氢,液相末端发酵产物中丁酸和乙酸的含量占挥发酸总含量的63%以上,气相中氢气含量在40%~52%之间,最大产氢量达4.52 L/d.Bioaugmentation with hydrogen producing strain Klebsiella oxytoca HP1 in up-flow anaerobic sludge bed(UASB) reactor was studied.The optimum start-up condition for reactor was obtained as: sludge inoculants of 30.0 gVSS/L,initial organic loading rate of 6.0 kgCOD/(m3·d),hydraulic retention time of 9 h,and bacteria dosing inoculants of 3%.Under the optimal conditions,the hydrogen production increased by 25%,the hydrogen content in the biogas was about 40%～52%,the maximal hydrogen rate was 4.52 L/d.福建省自然科学基金(C0410002);; 福建省科技项目(2005I016,2006H0091);; 厦门大学新世纪优秀人才计划项目资

1H NMR Study on the Products of the Catalytic Reduction of Ethyne by Nitrogenase in D_2O

用--1H nMr研究了固氮酶在重水中催化还原乙炔的反应产物氘代乙烯.利用群对称性对--1HnMr谱图进行了归属,计算了几种可能的C_2H_2d_2结构以及C_2H_3d结构的nMH谱线频率和强度,得出了理论谱.通过理论谱与实验谱的比较,表明固氮酶在重水中催化还原乙炔的产物主要以顺式结构C_2H_2d_2为主,并含有较多的单氘代乙烯.单氕代乙烯相对乙烯的化学位移往高场移动约4.0Hz,而双氘代乙烯向高场的位移大约是单氘代乙烯的2倍左右.The products of the catalytic reduction ethyne by nitrogenase in D2O were studied by 1H NMR.In order to assign the H NMR spectrum, the theoretical spectra of C2H2D2 were calculated using group symmetry.The final eigenfunctions, eigenvalues, allowed transitions and intensities of C2H2D2 and C2H3D were obtained.It can be concluded that the products comprised mainly of cis-C2H2D2 There was also some C2H3D.1 H chemical shift in C2H3D moved by ca.4.0Hz to the upfield compared with that in C2H4.The upfield shift in C2H2D2 was about twice as large as that in C2H3D.The theoretical results are in good agreement with experimental ones.国家攀登(29892166)预选项目;国家自然科学基金(19605004)项目;固体表面物理化学国家重点实验室基金项

Screening and Condition Optimization of a Xylanase-producing Strain

以甘蔗渣半纤维素为碳源,从垃圾场土壤中分离到6株分解半纤维素的菌株。通过固态发酵的木聚糖酶活力比较,筛选到1株木聚糖酶活力较高的菌株。该菌株18SrDNA序列与曲霉(Aspergillussp.)的同源性达97%,根据对菌株形态学分析和18SrDNA序列分析的结果,将该菌株鉴定为曲霉HQ3。HQ3的最佳产酶条件为:甘蔗渣:麸皮为7:3(W/W),固液比为1:4(W/W),尿素0.4%,pH7.0,温度30℃,发酵产酶时间4d。在最佳产酶条件下,其木聚糖酶活最高可达3421U/g干曲。Six hemicellulose-decomposing strains were isolated from the refuse soil samples using bagasse hemicellulose as the sole carbon.The strain with high xylanase activity was screened and identified as Aspergillus sp.HQ3 by analysis of 18S rDNA sequences (97% similarity to Aspergillus sp.)and phenotypic properties.The optimum solid-state fermentation conditions for xylanase production were obtained as: bagasse:bran=7∶3 (W/W),solid:liquid=1∶4 (W/W),urea 0.4%,pH 7.0,fermentation temperature 30 ℃,fermentation time 4d.The activity of xylanase reached 3421 U/g koji under the optimum conditions.国家“863”计划资助项目(2006AA05Z111);; 福建省重点科技平台资助项目(2006H0091

Advances of molecular imprinted fluorescence sensing technology and its applications

Molecular imprinting is a technique to synthesize molecularly imprinted polymers(MIPs) with specific and selective recognition ability for a given template molecule. As a molecular recognition component, MIPs combined with highly sensitive fluorescent detection to construct molecular imprinting-based fluorescence sensors(MI-FL sensors),which have attracted much attention in the field of trace detection of environmental organic pollutants. According to the expression of fluorescence emission signal, this review introduces the various strategies for MI-FL sensors. The applications of MI-FL sensors in pesticide residues, estrogen, and antibiotic in the environment are described briefly,and the challenges and development are prospected

H-1 NMR study on the products of the catalytic reduction of ethyne by nitrogenase in D2O

The products of the catalytic reduction ethyne by nitrogenase in D2O were studied by H-1 NMR. In order to assign the H-1 NMR spectrum, the theoretical spectra of C2H2D2 were calculated using group symmetry. The final eigenfunctions, eigenvalues, allowed transitions and intensities of C2H2D2 and C2H3D were obtained. It can be concluded that the products comprised mainly of cis-C2H2D2. There was also some C2H3D. 1H chemical shift in C2H3D moved by ca. 4.0Hz to the upfield compared with that in C2H4 The upfield shift in C2H2D2 was about twice as large as that in C2H3D The theoretical results are in good agreement with experimental ones