嗜盐古菌Haloferax sp. D1227中超氧化物歧化酶功能鉴定及其增强细菌耐盐性的研究

【背景】细菌、酵母或植物来源的超氧化物歧化酶(Superoxide dismutase,SOD)编码基因在异源宿主中表达并提高宿主耐盐性的研究已有一些报道,其异源宿主也多为植物,而古菌来源的超氧化物歧化酶编码基因在细菌中成功表达并提高其耐盐性的研究尚无报道。【目的】寻找嗜盐古菌Haloferax sp.D1227中的超氧化物歧化酶编码基因并鉴定其功能,将其在4-硝基苯酚降解细菌Burkholderia sp.SJ98中表达,研究该古菌的超氧化物歧化酶对菌株SJ98耐盐性和降解4-硝基苯酚功能的影响。【方法】通过生物信息学方法寻找嗜盐古菌D1227中潜在的超氧化物歧化酶编码基因,利用表达载体p ET-28a和广泛宿主载体p BBR1MCS-2将其分别在E.coli BL21(DE3)和4-硝基苯酚的降解菌株SJ98中异源表达,检测细胞抽提液和纯化蛋白的超氧化物歧化酶比活力。分别以葡萄糖和4-硝基苯酚为碳源,在M9培养基和添加500 mmol/L Na Cl(Na Cl含量约3%)的M9培养基中分别培养细菌SJ98的重组菌株和空载体重组菌株,利用全自动生长曲线分析仪和高效液相色谱等方法检测重组菌株的生长能力和对4-硝基苯酚的降解能力。【结果】通过生物信息学分析,在嗜盐古菌D1227基因组中发现了潜在的超氧化物歧化酶编码基因sod A,其在E.coli BL21(DE3)和菌株SJ98中分别异源表达均具有超氧化物歧化酶活力[细胞抽提液的比活力分别为21.07±0.02 U/mg和84.56±0.16 U/mg,从BL21(DE3)菌株纯化的蛋白Sod AD1227比活力为179.46±3.43 U/mg]。在添加500 mmol/L Na Cl的M9培养基中培养时,以葡萄糖为碳源,重组菌株SJ98[p BBR-sod A]仍可正常生长,而空载体对照菌株SJ98[p BBR1MCS-2]几乎丧失了生长能力;以4-硝基苯酚为碳源,菌株SJ98[p BBR-sod A]保持了利用底物生长和降解底物的能力,而菌株SJ98[p BBR1MCS-2]的生长和降解能力几乎丧失。用软件Phyre2模拟分析Sod AD1227的单体结构,该蛋白拥有Fe/Mn-SOD家族的典型结构特征,推测其属于Fe/Mn-SOD家族。【结论】本研究为利用古菌SOD对细菌进行改造以适应高盐环境中降解有机污染物的应用提供了潜在的可行性。     

邻苯二酚-1,2-双加氧酶的催化性质

邻苯二酚-1,2-双加氧酶能催化邻苯二酚的两个羟基之间裂解,生成顺,顺-己二烯二酸,加水很容易转化为尼龙-6,6的原料己二酸。此外,它有极易起化学反应的共轭双键和羧基,可成为新功能树脂的原料。1955年发现邻苯二酚-1,2-双加氧酶,此后进行了大量研究工作,但是直到近几年由于合成顺,顺-己二烯二酸的需要,才引起人们的高度重视。我们对胞外酶产生菌的发酵条件及其所产的邻苯二酚-1,2-双加氧酶性质进行了研究,为工业规模的生产应用作了准备。     

嗜盐古菌启动子的研究及应用

嗜盐古菌是古菌域的一个重要代表类群,在遗传与代谢、进化与适应、前沿生物技术及合成生物学领域都显示了其重要的研究价值。嗜盐古菌启动子的认识和利用,可以为嗜盐古菌的基础和应用研究提供必要的条件。本文从古菌启动子的结构与功能出发,就启动子的研究方法、嗜盐古菌启动子的特征及嗜盐古菌启动子的应用3个方面综述了嗜盐古菌启动子的研究现状,并对嗜盐古菌启动子未来研究的重点和方向进行了展望。     

极端嗜盐古菌中CRISPR结构的生物信息学分析

摘要：【目的】利用生物信息学方法了解目前拥有全基因组序列的极端嗜盐古菌中CRISPR结构的特征。【方法】通过比对,保守性分析,GC含量分析,RNA结构预测等方法对已有全基因组序列的嗜盐古菌基因组进行研究。【结果】在5株嗜盐古菌基因组中发现CRISPR结构,在leader序列内得到具有回文性质的保守motif。发现在大CRISPR结构内repeat序列具有很强的保守性。同时根据第四位碱基的不同,repeat序列可形成两类不同的RNA二级结构。【结论】leader序列中回文结构的发现对其可能为蛋白结合位点的假     

邻苯二酚1,2-双加氧酶的结构、功能及同工酶现象研究进展

邻苯二酚是芳香族化合物多条生物降解途径中共有的一种重要的中间产物,根据开环方式的不同,可分为邻位降解途径和间位降解途径,其中邻位降解途径中的关键酶是邻苯二酚1,2-双加氧酶。本文主要综述了邻苯二酚1,2-双加氧酶的结构、酶学性质,以及它在芳香烃降解菌中存在的同工酶现象及其功能研究进展。     

产胞外邻苯二酚1，2-双加氧酶的菌种筛选和发酵条件的研究

从112株细菌中筛选出两株产胞外邻苯二酚1,2一双加氧酶的假单胞菌(Pseubomonassp.)。 进行了该菌产酶的发酵条件试验。产酶的最适温度为30℃,最适起始pH为6．8—7-0。葡萄糖、麦芽糖和甘油对产酶有明显的抑制作用,苯甲酸钠对产酶有促进作用。氨态氮对菌体生长和产酶是必需的。琥珀酸钠是酶形成的有教诱导物。采用0.15％苯甲酸钠培养基(pH6.8—7.0),于30℃振荡培养72h,每毫升发酵液酶活力可达10单位。     

联苯利用节杆菌YC-RL1中2,3-二羟基联苯-1,2-双加氧酶BphC功能验证

【目的】通过节杆菌(Arthrobacter sp.)YC-RL1对多氯联苯降解过程中关键基因bph C的克隆与原核表达,鉴定其编码的2,3-二羟基联苯-1,2-双加氧酶Bph C的酶活特性与功能。【方法】以菌株YC-RL1全基因组为模板进行PCR扩增获得bph C基因,将该基因转入Escherichia coli BL21(DE3)感受态细胞后进行原核表达;利用镍柱亲和层析法对Bph C酶进行纯化并分别测定该酶在不同条件下对底物2,3-DHBP的催化特性,确定其最适反应pH、温度及不同金属离子对酶活特性的影响;进一步根据米氏方程对该酶的动力学参数进行测定与分析。【结果】通过PCR扩增获得了bphC基因,其大小为930 bp;对该基因进行原核表达,所得重组蛋白BphC携带有6个组氨酸标签,经纯化后体外仍具有活性,该酶作用于2,3-DHBP时的最适pH与温度分别为pH 7.4和30°C,且在最适条件下,Fe~(2+)、Cu~(2+)及Cd~(2+)等金属离子可明显促进其酶活作用,但多数金属离子对该酶有不同程度的抑制作用;该酶在与底物2,3-DHBP作用过程中,酶促动力学常数分别为K_m:8.67 mmol/L,V_(max):27.32μmol/s,k_(cat):15.55 s~(–1),k_(cat)/K_m:1.79 L/(mmol·s),其催化效率同有关报道中同类酶的动力学特性比较均有所提高。【结论】菌株YC-RL中的bphC基因对于多氯联苯的生物降解具有至关重要的作用,其编码的BphC是重要的芳香环裂解酶,该酶对其底物具有较高的亲和性,可在体外环境中发挥高效的酶促作用,具有良好的应用价值。     

嗜盐古菌R1中与真核同源基因rad25启动子的序列分析和启动活性研究

在盐生盐杆菌(Halobacterium halobium)R1中分析了真核同源基因rad25的转录,分析了rad25同源基因启动子片段的序列特征,用β_半乳糖苷酶基因(bgaH)为报告基因,利用启动子探针检测技术验证了rad25同源基因启动子片段在嗜盐古生菌WFD11中的启动功能;缺失分析进一步确认了rad25同源基因启动子具有嗜盐古菌启动子典型特征。从启动子和转录水平上证明盐生盐杆菌R1中真核同源基因rad25存在生物学功能,推测其可能在核苷酸切除修复(NER)起作用。     

倭蜂猴粪便微生物苯酚羟化酶和邻苯二酚1,2-双加氧酶基因多样性研究

【目的】分析倭蜂猴粪便微生物中苯酚羟化酶(Phenol hydroxylase,PH)和邻苯二酚1,2-双加氧酶(Catechol 1,2-dioxygenase,C12O)的基因多样性。【方法】利用简并引物,以倭蜂猴粪便微生物宏基因组DNA为模板,通过PCR扩增,分别构建PH和C12O基因克隆文库,并对克隆进行测序分析。【结果】倭蜂猴粪便微生物来源的PH和C12O基因序列经BLAST比对分析,与GenBank中相应酶的序列一致性分别介于92%?100%和87%?100%。系统进化树分析表明PH基因序列与Neisseria、Burkholderia、Alcaligenes、Acinetobacter 4个属来源的PH序列相关;C12O基因序列全部与Acinetobacter来源的C12O序列相关。序列比对结果表明PH序列具有LmPH (Largest subunit of multicomponent PH)中高保守的两个DEXRH结构域;C12O序列具有能被Ag+和Hg2+抑制的位点(半胱氨酸)。【结论】倭蜂猴粪便微生物来源的PH为多组分PH,其降解苯酚的中间产物邻苯二酚可以被C12O通过邻位开环途径裂解。     

洋葱伯克霍尔德氏菌产邻苯二酚2,3-双加氧酶的研究

对洋葱伯克霍尔德氏菌L 68生长及产邻苯二酚2,3 双加氧酶(C23D)的条件进行了研究,其最适产酶pH7.2;最适生长温度30～35℃;最适培养时间48h;苯酚浓度0.09%最有利于菌体产酶.对菌株L 68产生的C23D酶进行了纯化,超声波破碎后的细胞提取液经硫酸铵分级沉淀、DEAESepharoseFastFlow层析、Hydroxyapatite层析、SephadexG 150层析后,收率为20%,酶比活力提高了230倍.SDS PAGE检测得到了分子量为(34±1)kDa的蛋白.     

Gentisate 1,2-dioxygenase from Haloferax sp. D1227

Gentisate 1,2-dioxygenase from the extreme halophile Haloferax sp. D1227 (Hf. D1227) was purified using a three-step procedure. The enzyme was found to be a homotetramer of 42 000 ± 1000 Da subunits, with a native molecular weight of 174 000 ± 6000 Da. The optimal salt concentration, temperature, and pH for enzyme activity were 2 M KCl or NaCl, 45°C, and pH 7.2, respectively. The gene encoding Hf. D1227 gentisate 1,2-dioxygenase was cloned, sequenced, and expressed in Haloferax volcanii. The deduced amino acid sequence exhibited a 9.2% excess acidic over basic amino acids typical of halophilic enzymes. Four novel histidine clusters and a possible extradiol dioxygenase fingerprint region were identified. Received: November 19, 1997 / Accepted: May 12, 1998     

Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.     

NMR-derived folate-bound structure of dihydrofolate reductase 1 from the halophile Haloferax volcanii

Folate binds to dihydrofolate reductase (DHFR) to form a binary complex whose structure maintains the overall configuration of the enzyme; however, some significant changes are evident when a comparison is made to the enzyme. The structure of DHFR1 from the halophilic Halopherax volcanii was solved in its folate-bound form using nuclear magnetic resonance spectroscopy. NOE data obtained from the (15)N-NOESY-HSQC and (13)C-NOESY-HSQC experiments of the triply labeled ((1)H, (13)C, and (15)N) binary complex were used as input for the structure calculation with the Crystallography and Nuclear Magnetic Resonance System program. The resulting family of structures was compared with the enzyme solved by both nuclear magnetic resonance and X-ray crystallography and also to the mesophilic folate-bound enzyme from Escherichia coli. The binary complex exhibited less convergence of structure in the alpha2-helix and differences in the hinge residues D39 and A94. In comparison to the previously reported mesophilic binary complex solved by X-ray crystallography, the halophilic binary complex reported here does not agree with the convergence of the M20 loop to a single structure. The corresponding L21 loop of the halophilic binary complex family of structures solved by nuclear magnetic resonance indicates variability in this region.     

Identification of residues essential for the catalytic activity of Sec11b, one of the two type I signal peptidases of Haloferax volcanii

Sec11b is one of two signal peptidases (SPases) in the haloarchaeon Haloferax volcanii. Site-directed mutagenesis revealed Ser-72, His-137 and Asp-187 as essential for signal peptide cleavage. Thus, like the SPase of the methanoarchaeon Methanococcus voltae, H. volcanii Sec11b uses a catalytic mechanism reminiscent of its eukaryal rather than its bacterial counterpart. The availability of an additional model system to study the archaeal SPase, now in the form of the purified protein, promises additional insight into the behavior of this enzyme.     

Ionic strength‐dependent conformations of a ubiquitin‐like small archaeal modifier protein (SAMP1) from Haloferax volcanii

Eukaryotic ubiquitin and ubiquitin‐like systems play crucial roles in various cellular biological processes. In this work, we determined the solution structure of SAMP1 from Haloferax volcanii by NMR spectroscopy. Under low ionic conditions, SAMP1 presented two distinct conformations, one folded β‐grasp and the other disordered. Interestingly, SAMP1 underwent a conformational conversion from disorder to order with ion concentration increasing, indicating that the ordered conformation is the functional form of SAMP1 under the physiological condition of H. volcanii. Furthermore, SAMP1 could interact with proteasome‐activating nucleotidase B, supposing a potential role of SAMP1 in the protein degradation pathway mediated by proteasome.     

Identification of a novel gentisate 1,2-dioxygenase from <Emphasis Type="Italic">Silicibacter pomeroyi</Emphasis>

A 1,125-bp long ORF encoding a novel gentisate 1,2-dioxygenase with two-domain bicupins was cloned from Silicibacter pomeroyi DSS-3 and expressed in Escherichia coli. The resulting product was purified to homogeneity and partially characterized. Non-reductive SDS-PAGE and gel filtration showed that the active recombinant gentisate 1,2-dioxygenase had an estimated molecular mass of 132 kDa, and reductive SDS-PAGE indicated an approximate size of 45 kDa. The enzyme thus appears to be a homotrimeric protein. This is in contrast to the homotetrameric or dimeric protein of the gentisate 1,2-dioxygenases that have been characterized thus far. The K (m) and K (cat)/K (m) for gentisate were 12 muM and 653 x 10(4) M(-1 )s(-1); the pI was 4.6-4.8. It was optimally active at 40 degrees C and pH 8.0.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Crystal structure and mutagenic analysis of GDOsp, a gentisate 1,2-dioxygenase from Silicibacter pomeroyi

Dioxygenases catalyze dioxygen incorporation into various organic compounds and play a key role in the complex degradation pathway of mono- and polycyclic aromatic and hetero-aromatic compounds. Here we report the crystal structure of gentisate 1,2-dioxygenase from Silicibacter pomeroyi (GDOsp) at a 2.8 Å resolution. The enzyme possessed a conserved three-dimensional structure of the bicupin family, forming a homotetramerization. However, each subunit of GDOsp unusually contained two ferrous centers that were located in its two homologous cupin domains, respectively. Further mutagenic analysis indicated that the enzyme activity of GDOsp depends on the microenvironment in both metal-binding sites. Moreover, homologous structural comparison and functional study on GDOsp variants unveiled a group of functionally essential residues and suggested that the active site of the enzyme is located in the amino-terminal domain, but could be influenced by changes in the carboxyl domain. Therefore, GDOsp may provide a working model for studying long-distance communication within a protein (or its complex).     

Site-directed mutagenesis of gentisate 1,2-dioxygenases from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2

Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is the first enzyme in gentisate pathway that catalyses the ring fission of gentisate to form maleylpyruvate. Phylogenetic tree of amino acid sequences from 11 GDOs demonstrates that the GDOs from different genus share identities between 12.1% and 64.8%. According to the alignment result, four highly conserved histidine residues in GDO from Klebsiella pneumoniae M5a1 and Ralstonia sp. strain U2 were chosen to be substituted with aspartate residues. Enzyme analysis indicated that substitution of any of these four histidine residues had resulted in the complete loss of its catalytic activity.