热稳定性丙酮酸:铁氧还蛋白氧化还原酶异源表达及其在乙酰辅酶A合成中的应用

乙酰辅酶A被广泛应用到生物医学研究中,使用TPP代替昂贵的ATP为辅因子合成乙酰辅酶A受到广泛关注。新阿波罗栖热袍菌(Thermotoga neapolitana)来源的丙酮酸:铁氧还蛋白氧化还原酶(Tn PFOR)在大肠杆菌中进行了重组表达,分析了其酶学特性,并探讨了利用嗜热酶(TnPFOR)酶法合成乙酰辅酶A。采用pET-20b(+)载体,将新阿波罗栖热袍菌来源的四亚基组成的嗜热酶(Tn PFOR)在大肠杆菌中进行异源表达;通过热处理和阴离子交换层析法纯化嗜热酶(TnPFOR);重组表达的嗜热酶(TnPFOR)的最适反应温度和pH分别为90℃和6. 5,TnPFOR在90℃下孵育1h时保留了50%活性。利用嗜热酶(Tn PFOR),以TPP为辅酶合成了乙酰辅酶A,并探讨了不同温度、丙酮酸钠底物浓度和反应时间对乙酰辅酶A合成的影响。得到的优化条件为:最适反应温度为90℃,丙酮酸钠浓度为1. 5mmol/L,反应时间为2min。     

嗜热栖热菌α-葡萄糖苷酶基因的表达及酶学性质研究

用PCR方法从嗜热栖热菌(Thermus thermophilus)HB27中扩增出编码α-葡萄糖苷酶基因hbg,将其克隆到大肠杆菌(Escherichia coli)表达载体pET28a( )上,电击转化E.coliBL21(DE3),获得高效表达hbg基因的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测表达蛋白相对分子质量约为59kD,与预期分子量相符。经镍柱和阴离子交换柱纯化的重组表达的α-葡萄糖苷酶HBG最适温度为95℃,最适pH值为5.0。     

嗜热厌氧乙醇杆菌α-葡萄糖苷酶基因的表达及酶学性质的研究

用PCR方法从嗜热厌氧乙醇杆菌(Thermoanaerobacter ethanolicus)JW200中扩增出编码a-葡萄糖苷酶的基因,将其克隆到大肠杆菌(Escherichia coli)表达载体pTrc99A上并获得表达a-葡萄糖苷酶的大肠杆菌重组菌。重组菌经IPTG诱导表达,SDS-PAGE检测出蛋白相对分子量约89kDa,经阴离子交换层析和凝胶层析纯化后的a-葡萄糖苷酶最适反应温度为70℃,最适反应pH为5～5.5,且在pH 5.5～6.5之间有较高的稳定性。重组a-葡萄糖苷酶在70℃下105 min后酶活仍达到80%。     

嗜热糖化酶基因在重组黑曲霉工程菌中的表达及酶学性质初步研究

为了提高糖化酶的耐热性能,降低淀粉糖化发酵工艺的生产成本,构建了同源整合载体pEasy-glaAdir以及pEasyssg,将黑曲霉(Aspergillus niger)的糖化酶基因(glaA)灭活,并将硫磺矿硫化叶菌(Sulfolobus solfataricus)的嗜热糖化酶基因(ssg)插入到黑曲霉基因组中,筛选得到表达嗜热糖化酶的重组黑曲霉工程菌(A.nigerWW1)。重组菌的发酵结果显示,嗜热糖化酶在黑曲霉中得到了分泌表达,发酵液酶活达到3 030 U/mL。重组嗜热糖化酶的最适反应温度为90℃,最适pH为6.0,该酶具有较高的热稳定性,在80℃时的半衰期在60 min以上,具有良好的工业应用前景。     

嗜热真菌纤维素酶的CBD与海栖热袍菌的纤维素酶融合表达

将嗜热真菌毛壳菌纤维素酶Cel7A的纤维素结合结构域编码区与极端嗜热厌氧菌海栖热袍菌的纤维素酶CelB基因进行融合, 构建重组质粒pHsh-CBD-CelB, 并在大肠杆菌中表达。对融合蛋白进行纯化, 通过热处理和离子交换层析, 纯化到的融合蛋白SDS-PAGE 电泳图谱显示为单一条带。对融合蛋白的特性研究, 结果表明融合蛋白降解CMC的最适反应温度为90°C, 结晶纤维素吸附实验表明该融合蛋白具有结合结晶纤维素的能力, 并且融合蛋白降解CMC与结晶纤维素的能力得到提高。     

Aeropyrum pernix K1磷脂酶A2的克隆、表达及其性质

从超嗜热需氧古细菌AeropyrumpernixK1中抽提出染色体基因组,经PCR扩增得到磷脂酶A2基因,用带有His-tag标记的pET15b作为表达载体,在大肠杆菌BLP中成功地诱导表达。表达产物经过Ni-螯合柱一步得到纯化。SDS-PAGE检测只有一条带,其准确分子量为17,871kD。对纯化后的磷脂酶A2测定其酶活性和生物活性,得出其最适反应温度为90℃,最适pH范围为7·8~8·2。至此首次成功地在大肠杆菌中表达了古细菌嗜热磷脂酶A2,这将为以后对该酶的结构和功能以及耐热机制研究打下很好的基础,同时有利于古细菌研究领域的扩展。     

乙酰辅酶A合成酶家族中保守的色氨酸残基的生化功能（英文）

甲烷菌与甲烷八叠球菌是仅有的两种已知利用乙酸盐进行甲烷生成的菌属。稻田以及厌氧的废物分解物是甲烷生物生成的主要来源。甲烷菌在自然界广泛分布,相比甲烷八叠球菌,在低乙酸盐的环境中对乙酸盐仍有高亲和力。在甲烷生成第一步即将乙酸盐转化为乙酰辅酶A的过程中,与甲烷八叠球菌利用乙酸激酶与磷酸转乙酰酶激活途径不同,甲烷菌通过腺嘌呤形成乙酰辅酶A合成酶进行催化。在甲烷菌一属(Methanosaeta concilii)中,共发现5个乙酰辅酶A合成酶的编码基因,其中3种乙酰辅酶A合成酶的生化及酶活特性已被确定。该3种乙酰辅酶A合成酶均以乙酸盐为其最优底物。尽管在短链乙酰辅酶A合成酶家族中,发现酰基底物结合位点高度保守,但乙酰辅酶A合成酶家族的酰基底物范围极为广泛。本研究对甲烷菌中不同种乙酰辅酶A合成酶的酰基底物结合位点的关键氨基酸进行识别与比较,从而对乙酰辅酶A合成酶家族的酶活特性有更全面深入的了解。首先,我们对甲烷菌一属中乙酰辅酶A合成酶4进行生化性质测定。结果表明,该酶无催化一系列酰基底物为酰基辅酶A或其中间产物酰基腺苷酸的活性。通过序列对比发现,嗜热自养甲烷杆菌的乙酰辅酶A合成酶1中高度保守的416位色氨酸残基在甲烷菌一属的乙酰辅酶A合成酶4中被替换成528位苯丙氨酸残基。将甲烷菌一属的乙酰辅酶A合成酶4中的528位苯丙氨酸残基点突变为色氨酸残基后,进行酶学性质测定,未检测到该突变体具有乙酰辅酶A/乙酰腺苷酸合成活性。我们进一步对嗜热自养甲烷杆菌的乙酰辅酶A合成酶1中的416位色氨酸残基点突变为苯丙氨酸残基,酶活性质结果显示,突变酶对于乙酸盐以及丙酸盐作为底物时的活性未有明显差异。然而,以丙酸盐为底物时,释放丙酰腺苷酸中间产物。该结果表明,热自养甲烷杆菌的乙酰辅酶A合成酶1对于底物乙酸盐或丙酸盐的催化作用不甚相同,苯丙氨酸中的苯甲酰环降低该酶保留中间产物丙酰腺苷酸,从而转化为丙酰辅酶A的能力。     

乙酰辅酶A合成酶家族中保守的色氨酸残基的生化功能（英文）北大核心CSCD

甲烷菌与甲烷八叠球菌是仅有的两种已知利用乙酸盐进行甲烷生成的菌属。稻田以及厌氧的废物分解物是甲烷生物生成的主要来源。甲烷菌在自然界广泛分布,相比甲烷八叠球菌,在低乙酸盐的环境中对乙酸盐仍有高亲和力。在甲烷生成第一步即将乙酸盐转化为乙酰辅酶A的过程中,与甲烷八叠球菌利用乙酸激酶与磷酸转乙酰酶激活途径不同,甲烷菌通过腺嘌呤形成乙酰辅酶A合成酶进行催化。在甲烷菌一属(Methanosaeta concilii)中,共发现5个乙酰辅酶A合成酶的编码基因,其中3种乙酰辅酶A合成酶的生化及酶活特性已被确定。该3种乙酰辅酶A合成酶均以乙酸盐为其最优底物。尽管在短链乙酰辅酶A合成酶家族中,发现酰基底物结合位点高度保守,但乙酰辅酶A合成酶家族的酰基底物范围极为广泛。本研究对甲烷菌中不同种乙酰辅酶A合成酶的酰基底物结合位点的关键氨基酸进行识别与比较,从而对乙酰辅酶A合成酶家族的酶活特性有更全面深入的了解。首先,我们对甲烷菌一属中乙酰辅酶A合成酶4进行生化性质测定。结果表明,该酶无催化一系列酰基底物为酰基辅酶A或其中间产物酰基腺苷酸的活性。通过序列对比发现,嗜热自养甲烷杆菌的乙酰辅酶A合成酶1中高度保守的416位色氨酸残基在甲烷菌一属的乙酰辅酶A合成酶4中被替换成528位苯丙氨酸残基。将甲烷菌一属的乙酰辅酶A合成酶4中的528位苯丙氨酸残基点突变为色氨酸残基后,进行酶学性质测定,未检测到该突变体具有乙酰辅酶A/乙酰腺苷酸合成活性。我们进一步对嗜热自养甲烷杆菌的乙酰辅酶A合成酶1中的416位色氨酸残基点突变为苯丙氨酸残基,酶活性质结果显示,突变酶对于乙酸盐以及丙酸盐作为底物时的活性未有明显差异。然而,以丙酸盐为底物时,释放丙酰腺苷酸中间产物。该结果表明,热自养甲烷杆菌的乙酰辅酶A合成酶1对于底物乙酸盐或丙酸盐的催化作用不甚相同,苯丙氨酸中的苯甲酰环降低该酶保留中间产物丙酰腺苷酸,从而转化为丙酰辅酶A的能力。     

腾冲嗜热杆菌热稳定海藻糖磷酸化酶的基因表达与酶的分子生物学性质研究

分离克隆了腾冲嗜热杆菌(Thermoanaerobacter tengcongensis)海藻糖磷酸化酶(TreP)的编码基因(treP), 该酶可催化以葡萄糖和α-1-磷酸葡萄糖为底物的海藻糖合成反应及其逆向的分解反应. 反向mRNA点杂交实验表明, 腾冲嗜热杆菌中treP基因在高盐胁迫条件下表达量增加, 而在海藻糖诱导条件下表达量降低. 将该基因导入不含TreP的大肠杆菌中进行诱导表达, SDS-PAGE表明, 异源表达的TreP分子量约为90 kD, 与预期值相同. 通过葡萄糖氧化酶法测定分解产物葡萄糖的产率表明: TreP催化海藻糖分解反应的最适温度是70℃, 最适pH值为7.0; 通过HPLC检测合成产物海藻糖的产率表明: TreP催化合成反应的最适温度为70℃, 最适pH值为6.0. 在最适反应条件下, 50 μg的TreP粗酶可催化25 mmol/L α-1-磷酸葡萄糖与葡萄糖在30 min合成11.6 mmol/L海藻糖; 而同量的酶在同样时间内仅能将250 mmol/L海藻糖分解生成1.5 mmol/L葡萄糖. 以上体内胁迫和诱导表达分析及体外酶学性质分析均证明该酶的主要功能是催化海藻糖的合成反应. 热稳定性实验表明, 该酶性质比较稳定, 在50℃下温育7 h还能保持77%以上的活性, 是一个有潜在工业用途的新的热稳定海藻糖合成酶.     

油菜叶绿体乙酰辅酶A羧化酶单交换表达载体的构建

根据已知序列设计引物,通过PCR扩增获得质体定位的乙酰辅酶A羧化酶的4个亚基的基因序列。先将该酶4个亚基的基因进行拼接,然后将这4个拼接好的片段,克隆到pMD18-T载体上,得到质粒pH BM714。再以质粒pHBM714 DNA为模板,用分别带有CpoI和Asc I酶切位点的引物进行PCR扩增,PCR产物在dTTP的保护下经T4 DNA聚合酶处理,与将质粒pHBM720DNA纯化后经CpoI和AscI双酶切后得到的大片段连接,连接产物转化大肠杆菌Xl_(10)-gold,得到正确的重组子命名为pHBM726。此质粒pH BM726,即为带有壮观霉素抗性基因(aadA)筛选标记的质体定位的乙酰辅酶A羧化酶基因油菜叶绿体单交换表达载体;在此载体中壮观霉素抗性基因(aadA)、乙酰辅酶A羧化酶的4个亚基的基因(ACC)和绿色荧光蛋白基因(gfp)共6个基因串联在一起,共用一个启动子序列,一起来进行表达;通过酶切检测、PCR验证和测序验证,均表明该表达载体构建成功。最后此载体在大肠杆菌中表达时,发现重组菌能够在含壮观霉素的培养基上生长,且在可见光下,能看到绿色荧光,表明壮观霉素抗性基因和绿色荧光蛋白基因均在大肠杆菌中成功表达;表达产物通过Western印迹验证表明组成乙酰辅酶A羧化酶的4个亚基的基因在大肠杆菌中成功表达。以上结果表明,该表达载体中串联排列的这6个基因均在大肠杆菌中成功表达。该研究结果可为质体定位的乙酰辅酶A羧化酶转叶绿体的研究奠定基础,为油菜油脂代谢研究提供参考。     

白蚁与动物及微生物的共生类型及其机制

综述了白蚁螱客的主要种类、共生关系及相关机制的研究进展。白蚁螱客中,已报道的动物种类达170种。在与动物的共生关系中存在偏利共生（宾主共栖和异种共栖）、互利共生和无关共生三种;在与微生物的共生关系中,存在与内生菌（原生动物、细菌、真菌和放线菌）和外生菌（蚁巢伞菌等）间的互利关系。指出了白蚁与螱客研究中存在的问题,给出了解决方案,并提出了今后可能的研究热点或方向,为白蚁的综合利用（如纤维素酶）及今后研究物种间的协同进化提供了基础资料。     

New amino-dithiaphospholanes and phosphoramidodithioites and their rhodium and iridium complexes

New sulfur derivatives of phosphoramidite ligands were synthesized and the impact of the sulfur unit on the spectroscopic properties of their rhodium and iridium complexes was investigated. The new ligands Bn₂NPSCH₂CH₂S^a(P-S^a) (Bn = benzyl, 4), Bn₂NPSCHCHS^a(CH₂)₃C^aH₂(P-S^a)(C^a-S^a) (6) and Bn₂NP(4-XC₆H₄OMe)₂ (X = S, 7a; X = O, 7b) were converted to the rhodium and iridium complexes trans-[Rh(CO)Cl(L)₂] (L = 4, 6, 7), [RhCl(COD)(L)] (L = 4, 6, 7), [IrCl(COD)(7a)] and [IrCl₂Cp∗(6)]. For comparison, some phosphoramidite complexes of these formulations also were synthesized. The new metal complexes were spectroscopically analyzed. For the carbonyl complexes, the ν_CO IR stretching frequencies were lower than for the corresponding phosphite and phosphoramidite ligands. The ¹J_PRh coupling constants for the rhodium complexes with the new ligands were also smaller than for the respective phosphoramidite and phosphite complexes. Finally, the ¹J_PSe coupling constants of the selenides of the new ligands were lower than those of the phosphoramidite ligands but higher than for PPh₃. The spectroscopic data reveal that the new thio ligands 4, 6 and 7a are more electron donating than phosphites and phosphoramidites but less electron donating than PPh₃.     

Astrocytes and Synaptosomes Transport and Metabolize Lactate and Acetate Differently

Astrocytes transport the monocarboxylate acetate, but synaptosomes do not. The reason for this is unknown, because both preparations express monocarboxylate transporters (MCT). The transport and metabolism of lactate, another monocarboxylate, was examined in these two preparations, and the results were compared to those for acetate. Lactate transport is more rapid in astrocytes than in synaptosomes, but of lower affinity (Kms of 17 and 4 mM, respectively). Lactate (0.2 mM) is metabolized to CO2 more rapidly in synaptosomes than in astrocytes (rates of 0.37 and 0.07 nmol x mg protein(-1) x min(-1), respectively). The reason for this is unclear, but cellular differences in lactate dehydrogenase isotype expression may be involved. Acetate is metabolized to CO2 more rapidly in astrocytes than in synaptosomes (rates of 0.43 and 0.02 nmol x mg protein(-1) x min(-1), respectively). This is likely due to cellular differences in the expression of monocarboxylate transporter subtypes.     

Rapporteur report: Basics and technology and metrology and standards

The first and second sessions of the Workshop focussed on the basics of ultrasound and infrasound, their applications in both industry and medicine, and metrology and protection standards for ultrasound applications.     

Relative and combined effects of ethanol and protein deficiency on strontium and barium bone content and fecal and urinary excretion

To elucidate accumulation of minerals in human iliac arteries with aging, the content of minerals was analyzed by inductively coupled plasma atomic emission spectrometry. Bilateral common, internal, and external iliac arteries of 16 men and 8 women, ranging ages from 65 to 93 yr, were examined. It was found that an extremely high accumulation of calcium and phosphorus occurred in the common iliac artery at old age, being higher than that of the internal and external iliac arteries. It should be noted that the accumulation of calcium and phosphorus is the highest in the common iliac artery among the human arteries examined to date. Regarding sexual differences, the content of calcium and phosphorus in the common and internal iliac arteries was higher in women than in men, whereas their content in the external iliac artery was lower in women than in men.     

Prostaglandin and thromboxane production by human and guinea-pig macrophages and leucocytes

The ability of partially purified human and guinea-pig haematogenous cell populations, when cultured in vitro, to metabolise arachidonic acid (AA) has been studied. Supernatants from 24 hour cell culture have been subjected to analysis for products of AA metabolism by gas chromatography with electron-capture detection.The cell types studied were human peripheral blood monocytes (both glass adherent and non-adherent), neutrophils, eosinophils and leukemic leucocytes; thoracic duct lymphocytes and lung alveolar macrophages. From the guinea-pig, induced and non-induced macrophage or neutrophil enriched peritoneal exudate populations, lymph node cells, peritoneal eosinophils and peripheral blood platelets were examined. Supernatants were assayed for the presence of PGE₂, PGD₂, PGF_2α, TXB₂ and 6-keto-PGF_1α. In all types studied PGE₂ and TXB₂ were the major products formed. The identification of PGE₂ and TXB₂ was confirmed by GC/MS with multiple ion monitoring.The results have been compared with other reports and their possible significance discussed in relation to the proposed role of prostaglandins as mediators and modulators in immunopathology.     

Chemokines and cytokines: axis and allies in asthma and allergy

Allergic asthma can be precipitated by many factors. For the atopic person, fungus, pollen, dust mites, cockroach antigens, and diesel exhaust are all agents that may trigger an allergic attack. Cytokines and chemokines are integral mediators of fungal asthma. From the earliest time points, they recruit and activate the cells required for the clearance of fungus as well as being critical factors involved in the immunopathology of this disease. In the final analysis, it is clear that these mediators can act to the benefit or the detriment of the host.     

Oxidation and nitration of ribonuclease and lysozyme by peroxynitrite and myeloperoxidase

In spite of the many studies on protein modifications by reactive species, knowledge about the products resulting from the oxidation of protein-aromatic residues, including protein-derived radicals and their stable products, remains limited. Here, we compared the oxidative modifications promoted by peroxynitrite and myeloperoxidase/hydrogen peroxide/nitrite in two model proteins, ribonuclease (6Tyr) and lysozyme (3Tyr/6Trp). The formation of protein-derived radicals and products was higher at pH 5.4 and 7.4 for myeloperoxidase and peroxynitrite, respectively. The main product was 3-nitro-Tyr for both proteins and oxidants. Lysozyme rendered similar yields of nitro-Trp, particularly when oxidized by peroxynitrite. Hydroxylated and dimerized products of Trp and Tyr were also produced, but in lower yields. Localization of the main modified residues indicates that peroxynitrite decomposes to radicals within the proteins behaving less specifically than myeloperoxidase. Nitrogen dioxide is emphasized as an important protein modifier.