葡萄糖氧化酶产生菌Z—l—C的分批发酵与恒化培养

本文对葡萄糖氧化酶产生菌z—I—c的分批发酵与恒化培养进行了研究。实验发现,在以葡萄糖为生长限制性基质的恒化培养中,该产生菌的维持系数为O．04 g葡萄糖／g菌体．H,生长得率系数Y^max_G=O．714 g菌体／g葡萄糖,最大比生长速率μmax=O．385h^-1,饱和常数Ks=4．76g/L,理论最适稀释度Dm=0．260h^-1,最大酶比活(E／x)max为2．16×10³μ／mg,其值较分批发酵的最大酶比活(1．5l×10³μ／mg)提高43％。当向恒化培养的补料培养基中添加0．02％的α-甲基-D-葡萄糖时,由于该葡萄糖结构类似物的诱导作用,可使(E／x)max达3．11×10³μ／mg,较分批发酵之值提高106％。     

球形红杆菌L2的生物学特性及其应用

从淀粉废水中分离获得一株光合细菌,经形态特征,培养特征,生理生化特征及Ｇ＋Ｃｍｏｌ％含量等生物学特性分析,确定为球形红杆菌（Ｒｈｏｄｏｂａｃｔｅｒｓｐｈａｅｒｏｉｄｅｓ）Ｌ２。该菌应用于淀粉废水处理,ＣＯＤ去除率达９５．７％发酵产类胡萝卜素,产量达２９５ｍｇ／Ｌ;作为饲料添加剂进行肉鸡饲喂,增重１６．４０％。     

鸟苷补料分批发酵的研究

目的:以枯草芽孢杆菌TA208为出发菌株,研究了补料分批发酵方式下各种参数对鸟苷产量的影响。方法:采用补料分批发酵工艺,利用纸层析法测定发酵液中鸟苷的产量。结果:确定了葡萄糖、酵母粉和次黄嘌呤的最优补料方式,使鸟苷产量达到32.05g/L,较分批发酵方式提高了36.3%。结论:发酵工艺过程控制对发酵生产鸟苷具有重大影响。     

球形红杆菌积累聚β-羟基链烷酸(PHA)的研究

通过对球形红杆菌(Rhodobacter sphaeroides)生长和积累聚卢-羟基链烷酸(PHA)条件的研究,确定采用两段培养法提高PHA的产量。第一阶段提供适合菌体生长的条件:以葡萄糖作碳源,尿素为氮源,光照微好氧培养。第二阶段则提供使菌体积累PHA的条件:补加乙酸钠厌氧光照培养。经两段培养后菌体PHA含量可占细胞干重的45％,PHA产量每升发酵液可达1.7g。     

灭蚊球形芽孢杆菌Ts-1中大质粒pNT-1的分离和电子显微镜观察

由我国筛选的灭蚊球形芽孢杆菌Ts-1中分离到一种大分子质粒pNT-1,通过电子显微镜观察到环状DNA分子,根据轮廓周长推算其分子量为114.13×106道尔顿。本文还报道了球形芽孢杆菌中大质牡的分离纯化方法。     

球形芽孢杆菌TS-1发酵条件的研究

本文用正交试验设计方法选出的球形芽孢杆菌发酵培养基配方,菌体数量可达到100亿/ml,芽孢形成率达到70—80%,杀蚊幼的半致死剂量在0.125ppm以下。上述的三个指标明显优于以前的报道。通过摇瓶发酵验证和50L³小罐的放大试验,证明所有的实验结果都很稳定。使用现在的发酵配方,使每吨发酵液的成本降低了33%。     

球形红杆菌积累聚β-羟基链烷酸(PHA)的研究

通过对球形红杆菌(Rhodobacter sphaeroides)生长和积累聚卢-羟基链烷酸(PHA)条件的研究,确定采用两段培养法提高PHA的产量。第一阶段提供适合菌体生长的条件:以葡萄糖作碳源,尿素为氮源,光照微好氧培养。第二阶段则提供使菌体积累PHA的条件:补加乙酸钠厌氧光照培养。经两段培养后菌体PHA含量可占细胞干重的45％,PHA产量每升发酵液可达1.7g。     

球形芽孢杆菌TS—1原生质体电诱导质粒转化研究

This report gave the best conditions of Bacillus sphaericus Ts-1 protoplast-plasmid pHV33 electroporation. The highest transformation frequency and transformation efficiency induced by three pulse of 21 KV/cm and 10 microseconds duration applied at an interval of one sec., was 2.44 x 10(2) transformants/micrograms DNA and 3.16 x 10(-6) respectively. The saturated concentration of DNA absorbed by the protoplast was 5 micrograms DNA/10(9) cells/ml. By means of this method, pJB417, a recombinant mosquito larvicide clone, was introduced into B. subtilis 168M and B. sphaericus Ts-1. The transformants of B. subtilis 168M with biocide activity were obtained, but the toxicity of B. sphaericus Ts-1 was not increased.     

浑球红细菌谷氨酰胺合成酶基因(glnA)及PⅡ蛋白基因(glnB)的序列分析

测定了浑球红细菌的glnA和／glnB基因DNA序列,共2707nt。其中glnA基因编码区为1401nt,编码467个氨基酸;glnB基因编码区为336nt,编码112个氨基醚。DNA序列的G＋C百分含量为65％,它们的密码子第三位GC利用率高达89．1％。在氨基酸序列上,GS酶和PⅡ蛋白与其它不同属的细菌间有较好的同源性,尤其是固氮类细菌间同源性较高。     

含硒类球红细菌的研究

为了确定类球红细菌转硒培养的最佳条件 ,研究了无机硒的加入浓度、时间以及分批补料培养对菌体生长和转硒效率的影响。实验表明 ,无机硒的浓度低于 1× 10 -5mol/L时 ,对类球红细菌的生长基本没有影响 ,并能将6 3.9%的无机硒转化为有机硒。转硒的最佳时间是在接种后 12h左右 ,此时转硒效率最高。实验还表明 ,分批补料培养可以提高菌体浓度 ,可使转硒效率和绝对量增加。体内试验表明 ,用 5mL/kgbw和 10mL/kgbw剂量的含硒类球红细菌灌养小鼠 ,可以使其全血GSH Px酶活性提高 2 0 .9%和 2 5 .5 % ,使其血清丙二醛 (MDA)含量降低2 1.0 %和 2 3.2 %。     

光合细菌Rhodobacter sphaeroides glnBlacZ 融合子的构建与表达

亚克隆了Ｒｈｏｄｏｂａｃｔｅｒ ｓｐｈａｅｒｏｉｄｅｓ ｇｌｎＢ启动子,以ｐＭＰ２２０ 为载体构建成ｇｌｎＢｌａｃＺ融合子。将ｇｌｎＢｌａｃＺ、ｎｉｆＨｌａｃＺ、ｎｉｆＡｌａｃＺ分别导入Ｒ． ｓｐｈａｅｒｏｉｄｅｓ 谷氨酸合酶突变株ｇｌｔＢ－ 、ｇｌｔＤ－ 和野生型菌株中,分析了突变对固氮基因转录表达的影响。试验证明,在ｇｌｔＢＤ 突变株中ｎｉｆＨ 的表达受阻遏,ｎｉｆＡ 表达水平很低。这证明ｇｌｔ 基因的突变引起固氮酶结构基因和固氮正调节基因的转录被阻遏,而ｇｌｎＢ 基因的表达几乎不受影响。试验还测定了环境中结合态氮和有机酸等信号分子对ｇｌｎＢ 和ｎｉｆＨ 表达的影响,发现加入氨或谷氨酰胺后,ｎｉｆＨ 的表达受到明显的阻遏作用,ｇｌｎＢｌａｃＺ的β半乳糖苷酶活性虽下降３０ ％ 左右但不随结合态氮浓度升高而变化,仍维持在一个较高的水平。α酮戊二酸和丙酮酸对ｎｉｆＨ 的表达有部分去阻遏作用而对ｇｌｎＢ的表达无诱导作用     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.     

Growth and Antithraquinone Biosynthesis by Galium mollugo L. Cells in Batch and Chemostat Culture

The kinetics of growth, nutrient uptake, and anthraquinone biosynthesisby suspension cultures of Galium mollugo L. cells were examinedin batch and continuous (chemostat) culture. In batch culture,although the initial growth rate was constant (minimum doublingtime = 35 h) characteristic changes in cell composition wereobserved during the growth cycle particularly cell dry weight(between 3.9 and 9.2 g/10⁹ cells), cell anthraquinone (22–80mg/10⁹ cells), and cell protein (0.7–1.6 g/10⁹ cells).Using a chemostat steady state growth was established at twodifferent specific growth rates with mean doubling times of40 h and 25 h. Phosphate was established as the growth-limitingnutrient in chemostat culture at a concentration of 11 µgP ml^–1. In steady state growth at a doubling time of 40h the cell composition remained constant although this was differentfrom any cells grown in batch culture. The cell anthraquinonelevel in steady state growth was between 7 and 30 times lowerthan in batch culture. This result raises the question of therelative importance of growth rate and the growth-limiting nutrientin determining accumulation of secondary products by culturedplant cells.     

Engineered citrate synthase alters Acetate Accumulation in Escherichia coli

Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.     

Control of nitrogenase in chemostat cultures of Rhodobacter capsulatus grown on ammonium at different illuminations

Rhodobacter capsulatus strain 37b4 was grown phototrophically in chemostat cultures with 2 mM of ammonium chloride and 30 mM of malate at a constant dilution rate of 0.075 h^-1. When illumination was raised from 3000 to 30000 lx, steady state biomass levels as well as malate uptake increased linearly with increasing illumination. Yet, in no case external ammonium could be detected in the culture fluid. Specific nitrogenase activity increased by a factor of ten between 3000 and 15000 lx and approached constancy above 15 000 lx. When samples were anaerobically withdrawn from the chemostat and subsequently grown in batch cultures under saturating light conditions, biomass increased to a constant level, independently of the illumination used in the previous chemostat culture. In fact, the specific nitrogen contents of cells were 0.195 and 0.154 (g of N per g of protein) with chemostat cultures adapted to 3000 and 30000 lx, respectively. With the former cultures, specific nitrogen contents decreased to 0.142 g of nitrogen per g of cell protein upon incubation in a batch system. This suggests the existence of free nitrogen compounds in cells of chemostat cultures, the concentrations of which decrease while protein levels increase with increasing energy supply. Intracellular amino acid pools revealed slightly elevated levels of major amino acids in low-light cultures as compared to high-light cultures. On the basis of intracellular levels of ammonium, however, no significant differences could be detected. Since, in addition, malate consumption increased linearly with increasing illumination, it is proposed that light controls nitrogenase in Rhodobacter capsulatus via the C/N ratio, as represented by malate and ammonium consumption, rather than directly.     

Response of four rice varieties to Rhodobacter capsulatus at seedling stage

The phototrophic purple nonsulphur bacterium (PPNSB) Rhodobacter capsulatus was used to inoculate seedlings of four rice varieties Giza 159, Giza 171, Giza 176 and Giza 181, grown in hydroponic culture with or without nitrogen. After three weeks the seedling growth parameters were measured. Inoculation with R. capsulatus enhanced seedling growth of all rice varieties tested. The response to inoculation as compared to control plants (no nitrogen, no R. capsulatus) were 52% to 75% for shoot height, 47% to 100% for aerial part dry weight, 45% to 78% for aerial part N content, –37% to –9% for maximum root length, –4% to 8% for root system dry weight and 50% to 62% for root N content.     

球形红假单胞菌反应中心中蛋白的量子化学研究

采用重叠二体近似方法和建立在从算水平上的扩展负本征值因数计算方法（extended negative factor counter method）研究了球形红假单胞菌（Rhodobacter sphaeroides(Van Niel)Imhoff,Truper et Pfennin）中兴合反应中心中蛋白链L及M的电子结构。结果表明：（1）对组成蛋白链L（M）的前线轨道有重要贡献的氨基酸残基分布在L链的自由螺旋区（M链的α螺旋区）。由于自由螺旋是有柔曲性的,它易于在电子转移的过程中改变其构象并降低体系的能量,而α螺旋结构却相对稳定,这种差别有可能是光合反庆中心中电子转移只沿L支进行的原因之一。（2）与特殊对分子及辅助叶绿素分子形成轴向配位的组氨酸残基对于特殊对P和辅助叶绿素分子的ELUMO有重要影响,但此组氨酸的相应分子轨道的贡献并没有出现在蛋白链的前线轨道组成中。这意味着色素分子与蛋白链之间的相互作用对蛋白链前线轨道的贡献没有影响,但却能影响相应色素分子的ELUMO能级。     

Quantum Chemical Studies on Proteins in the Reaction Center of Rhodobacter sphaeroides

The electronic structure of protein chains L and M in photosynthetic reaction center (PRC) of Rhodobacter sphaeroides (Van Niel) Imhoff, Truper et Pfennig) was studied by using the Overlapping Dimer Approximation method and the Extended Negative Factor Counter method at ab initio level. The result indicated that: (1) Amino acid residues, the molecular orbitals of which composed the main components of frontier orbitals of protein chain L (M ), are located at the random coil areas of chain L (α helix areas of chain M ). Since the random coil is flexible and more easy to change its conformation in the electron transfer process and to reduce the energy of the system, and the structure of the α helix is reletively stable, this difference might be one of the causes for the electron transfer in photosynthetic reaction center (PRC) only takes place along the L branch. (2) The His residues which axially coordinated to the “special pair” P and accessory chlorophyll molecules （ABChls） are essentially important for the ELUMO levels of P and ABChl. But, the corresponding molecular orbitals of these His residues do not appear in the composition of frontier orbitals of protein chains. It means that the interaction between pigment molecules and protein chains do not influence the contribution to the frontier orbitals of protein chains explicitly, but influences the corresponding ELUMO levels significantly.