Evaluación agronómica con enfoque agroecológico en un sistema diversificado de guayaba (Psidium guajava L.), nopal (Opuntia ficus L.), piña (Ananas comosus L.) y papaya (carica papaya L.) utilizando vermicompost, Managua, Nicaragua, 2009-2011

Con el propósito de responder a preguntas de orden nutricional y biológico del suelo con un enfoque agroecológico se desarrolló el presente trabajo, en un terreno calcáreo, no salino, que permaneció en barbecho cinco años antes de iniciado el estudio. Se evaluó la implementación de una diversificación de cultivos (guayaba, nopal, piña y papaya) y tres dosis de vermicompost (a 1 :10, a 2 :15 y a 3 :20 t (ha año) - 1 . La investigación se inició en mayo del 2009 hasta diciembre del 2011 y el diseño usado fue cuasiexperimental en franjas pareadas. Las características físicas - químicas de suelo analizadas fueron: pH en H 2 O, materia orgánica , N, arena, limo y arcilla en %; P disponible, Fe, Cu, Mn y Zn en ppm; K disponible, Ca y Mg en meq, conductividad eléctrica en μS/cm y la relación C/N. Desde el punto de vista biológico del suelo se determinaron presencia de hongos, bacterias y actinomicetos. Antes de iniciar el estudio en 2009, y durante el ensayo, se determinaron las variables físicas, químicas y biológicas del suelo. Para la interpretación agronómica de la nutrición para cada cultivo se hizo un análisis combinado entre los resultados de las propiedades físico - químicas y los rangos definidos para cada cultivo por la metodología propuesta por Quintana (1983) . La diversificación de cultivos, influyó sobre las propiedades físicas, químicas y biológicas del suelo. En el cultivo de guayaba si se aplica 20 t (ha año) - 1 hay que realizar aplicaciones suplementarias de Cu. Aplicaciones de a 1 y a 2 en nopal y papaya respectivamente, además de aplicaciones complementarias de Cu, también necesitan suplemento de P. Sin efecto antropogénico los microorganismos que predominaron fueron bacterias del género Bacillus y esporádicamente Pseudomona y Sarcina . Con la diversificación de cultivos y aplicaciones de vermicompost se incrementó la diversidad de géneros de microorganismos. El total de géneros de microorganismos, al concluir el estudio fueron 13; de éstos 10 fueron diferentes (Cinco hongos: Curvularia, A spergillus, Pestalotia, Fusarium, Penicillium ; cuatro bacterias: Serratia, Erwinia, Sporolactobacillus y Caryophanon y un actinomiceto: Streptomyces). Variables del crecimiento en el cultivo de guayaba demostraron que con un mayor número de ramas terciarias existe un 70 % de probabilidad de tener más frutos comerciales. En nopal, el tratamiento con los mejores resultados para variables del crecimiento es 15 t (ha año) - 1 cosechado cada 90 días. Para piña, se encontró resultados superiores con 10 t (ha año) - 1 . Papaya obtuvo los mejores resultados con 20 t (ha año) - 1 . Con el cultivo de guayaba el mejor rendimiento se alcanzó utilizando 20 t (ha año) - 1 . En nopal un mayor número de cladodios se alcanza aplicando 15 t (ha año) - 1 cosechado cada 30 días. Un mayor peso en el cladodio se obtiene al aplicar 20 t (ha año) - 1 , cosechado cada 90 días. La piña obtiene mayor número de frutos aplicando 15 t (ha año) - 1 . En el cultivo de papaya un mayor número de frutos cosechados fue alcanzado con 15 - 20 t (ha año) - 1.

Caracterização molecular de bastonetes Gram positivos irregulares e actinomicetos aeróbios obtidos de espécimes clínicos, de ensaios de esterilidade e de áreas limpas

Os bastonetes Gram positivos irregulares (BGPIs) compõem um grupo de espécies bacterianas com ampla diversidade fenotípica e que podem estar presente no meio ambiente, na microbiota humana e de animais. A identificação acurada de BGPIs em nível de gênero e espécie empregando métodos bioquímicos convencionais é bastante limitada, sendo recomendado, portanto, o uso de técnicas moleculares. No presente estudo, foram identificadas amostras de BGPIs oriundas de espécimes clínicos de humanos, de produtos farmacêuticos e de áreas limpas através da análise de sequencias do gene 16S rRNA e de outros genes conservados (housekeeping genes). Os resultados obtidos pelo sequenciamento dos genes 16S rRNA e rpoB demonstraram C. striatum multi-resistente (MDR) como responsável por surto epidêmico em ambiente hospitalar da cidade do Rio de Janeiro. Quinze cepas de C. striatum foram isoladas em cultura pura a partir de secreção traqueal de pacientes adultos submetidos a procedimentos de entubação endotraqueal. A análise por eletroforese em gel de campo pulsado (PFGE) indicou a presença de quatro perfis moleculares, incluindo dois clones relacionados com cepas MDR (PFGE I e II). Os dados demonstram a predominância de PFGE I entre cepas MDR isoladas de unidades de terapia intensiva e enfermarias cirúrgicas. Uma potencial ligação causal entre a morte e a infecção por C. striatum MDR (PFGE tipos I e II) foi observada em cinco casos. Adicionalmente, acreditamos que este seja o primeiro estudo de identificação de espécies de Nocardia relacionadas com infecções humanas pela análise da sequencia multilocus (MLSA) no Brasil. Diferente dos dados observados na literatura (1970 a 2013) e obtidos pelos testes fenotípicos convencionais, a caracterização molecular de quatro lócus (gyrB-16S-secA1-hsp65) permitiu a identificação das espécies N. nova, N. cyriacigeorgica, N. asiatica e N. exalbida/gamkensis relacionadas com quadros de nocardiose em humanos. Cepas de N. nova isoladas de diferentes materiais clínicos de um único paciente apresentaram padrões de susceptibilidade antimicrobianos idênticos e dois perfis PFGE, indicando a possibilidade de quadros de co-infecção por N. nova em humanos. Em outra etapa da investigação, amostras de BGPIs obtidos de ambientes de salas limpas que não puderam ser identificadas por critérios convencionais foram submetidas a análise da sequência do gene 16S rRNA e caracterizadas 95,83% em nível de gênero e 35,42% em espécies. Para gêneros mais encontrados no estudo, foram analisados os genes rpoB e recA de dezessete cepas de Microbacterium e utilizado o MLSA para a identificação de sete cepas identificadas como Streptomyces. Os ensaios permitiram a identificação de três cepas de Microbacterium e de uma única amostra de Streptomyces ao nível de espécie. A análise da sequencia do gene rpoB também se mostrou eficaz na identificação de espécies de cepas de Corynebacterium. Finalmente, para as cepas ambientais pertencentes à classe Actinobacteria os dados morfológicos, bioquímicos e genotípicos permitiram documentar a cepa 3117BRRJ como representante de uma nova espécie do gênero Nocardioides, para o qual o nome Nocardioides brasiliensis sp. nov. e as cepas 3712BRRJ e 3371BRRJ como representante de um novo gênero e espécie para o qual o nome Guaraldella brasiliensis nov. foi proposto.

Isolatin [sic] the bacteria produced biactive [sic] metabolits [sic] cytotoxic compound in order to prduction [sic] anticancer substance in the coral regin [sic] the Persian Gulf (Larak Island)

In the present investigation the marine bacteria isolated from corals, sponges sea water and sediments of coral regions in the larak Island located in the Persian Gulf and were examined for ability to produce cytotoxic metabolits in order to use as an anticancer compounds. Cytotoxic effect were isolated bacteria from different samples and were examined by Artemia Cytotoxic Bioassay test, in which 4.5 percent of sea waters, 12 percent of sediments and 28 percent of marine invertebrat showed cytotoxic activity, using Brine Shrimp Bioassay test. Streptomyces S-2004 isolated from soft coral specified as Sinularia erecta had LC50=0.5mg/m1 in Brine Shrimp Bioaassay test. The streptomyces S-2004 produced cytotoxic metabolits in low nutrient condition and sea water medium after 7 days on 250 rpm shaken in vitro condition. The extract partially were semipurified. Then ethyl acetate extraction from aceton extracted of bacterial plate had cytotoxic effect (LC50=4.19ktg/m1) in Human epidermoid carcinoma of mouth cells (KB) by using neutral red assay. Morphological effects of this extract on KB cells showed turgescence, cellular blebs and apoptosis which was a proof for anticancer compounds of the extract. It is seems that streptomyces S-2004 is a new strain and could be introduced as a talented bacteria, which produced cytotoxic metabolits.

Identification of triosephosphate isomerase (TIM) genes from Microcystis (Cyanobacteria : Chroococcales) and theoretical analyses of the potential of cyanobacterial TIM as a target for designing specific inhibitors

The genes encoding triosephosphate isomerase (TIM) in three species of Microcystis (M. aeruginosa, M. viridis and M. wesenbergii) were investigated. Reverse transcriptase-polymerase chain reaction indicated that they were transcribed in the cells. Analyses showed that their DNA and deduced amino acid sequences were highly conserved between all the three species, only a single nonsynonymous substitution was seen at position 31, from an Asp in M. aeruginosa and M. viridis to Glu in M. wesenbergii. Sequence alignment of these with 12 other known cyanobacterial TIM sequences showed that all the cyanobacterial TIMs had a very high level of amino acid identity (over 50% between each two). Comparison of the cyanobacterial TIMs with other reported TIMs (from diverse lineages of the three Domains) showed that they possessed common active-site residues and sequence motifs. All cyanobacterial TIMs have two common cysteine residues (Cys127 and Cys176), and the Cys176 is almost cyanobacteria-specific with only one exception in Streptomyces coelicolor. Both secondary structure alignment and comparative modelling of Synechocystis sp. TIM showed that Cys176 was located at the hinge region of the flexible loop-6 and might therefore be critical to the movement of TIM's loop-6, which is important to the function of the enzyme. Thus, the cyanobacterial TIM-specific Cys176 may be a potential site for the discovery of suitable drugs against cyanobacteria, and such drugs may have utility in controlling water blooms due to cyanobacteria.

Actinomycetes and earthy-musty odorous compounds in brackish fishponds in Tianjin, China

The presence of the odorous compounds, 2-methylisoborneol (MIB) and geosmin, as well as causative microorganisms in brackish intensive cultivation fishponds in Tianjin, China that had a severe earthy-musty odor were evaluated. The results revealed that MIB was the primary odorous compound present in the Tianjin fishponds, with a concentration ranging from 0.53-5302.7 ng.L-1. Furthermore, the concentration of MIB was found to be closely correlated with the gross biomass of actinomycetes in the water, which ranged from 10.67-1528.24 x 10(6) cfu.ml(-1). Therefore, the sequences of the 16 SrRNA and morphological characteristics of the actinomycetes in the brackish fishponds were investigated. The results revealed that the actinomycetes in the brackish fishponds included 9 species of common and dominant actinomycetes belonging to 4 genera. Of these genera, Streptomyces were the dominant species, and Streptomyces, Nocardioides and Micromonospora were the most common species in the fishponds evaluated. Next, the ability of each of the isolated Streptomyces to produce MIB was measured under laboratory culture conditions. Streptomyces Sp2 was found to have a strong ability to produce MIB, which indicates that this strain may be the primary source of the earthy-musty odor reported in brackish intensive cultivation fishponds in Tianjin, China.

四株有潜在生物活性的放线菌分类地位和产物研究

放线菌是一种重要的微生物资源，广泛分布于自然界中。已经发现的微生物来源的生物活性物质中，由放线菌产生的约占三分之二。从我国云南省不同地方的土壤样品中分离到多株有潜在生物活性的放线菌，本文对从西双版纳原绐森林土壤样品中分得的JX-47菌株着重进行了研究，用形态观察、化学分类及分子分类等多项分类的方法确定了该菌株的分类地位，为链霉菌一新种，命名为Streptomyces autolyticus sp nov.。从该菌株的发酵液中分离得到三个化合物，用各种光谱实验鉴定了它们的结构式。这三个化合物均属于Ansamycin类抗生素；其中一个为新化合物，命名为autolytimycin，并申请了国家发明专利（申请号为：00102982. 7)。初步的实验结果证明这些化合物具有细胞毒性和一定的抗病毒活性。同时，对分离到的JX-42、JX-45、JX-46菌性的分类地位和次生代谢产物进行了初步研究。根据形态特征和细胞壁的化学组成，JX-42和JX-46菌株被确定属于链霉菌属；JX-45菌株被确定属于小单孢菌属。从它们的发酵液中，共分离出10个化合物，并鉴定了结构。

极端环境放线菌的分离及多相分类研究

本文综述了放线菌分类学研究的目的和作用，分析了放线菌分类学的历史和现状，介绍了当前放线菌多相分类研究中所采用的技术方法及适用范围。同时还重点介绍了极端高温、低温、高盐放线菌分离及分类研究的进展。从云南采集高温温泉水样、火山口土样，从云南、新疆等地采集雪山土样，从新疆、青海等地采集盐碱土样进行放线菌分离，对不同极端环境下的放线菌分离方法进行探讨，并对分离到的部分典型放线菌菌株采用形态特征、培养特征，生理生化测定，细胞化学组份分析，DNA G+C mol%和DNA同源性测定，以及16SrDNA全序列分析等相结合的多相分类技术进行系统的分类研究。从表型、基因型及系统发育三个不同层次对其分类地位进行了最终确定。其中，分离自云南洱源温泉的菌株YIM60013和腾冲火山口的菌株YIM60032分别确定为高温放线菌属的两个新种：白色高温放线菌（Thermoactznomyces albus sp. nov.）和云南高温放线菌（Termoactomyces yunnanensis sp. nov.）；分离自新疆北疆地区的一株低温放线菌菌株，结合其形态特征、细胞化学组份及16S rDNA序列分析将其鉴定为链霉菌的一个新种，北疆链霉菌（Streptomyces beijiangensis sp. nov.）；来自新疆盐碱土样的6株嗜盐放线菌菌株YIM90001-90006中，菌株YIM90001被命名为嗜盐普氏菌新种(Prauserella halophila sp. nov.），菌株YIM90005被 命名为脱卤普氏菌新种（Prauserella dehalogenans sp. nov.），菌株YIM90002和YIM90003鉴定为拟诺卡氏菌科中的链单抱菌新属Streptomonospora gen. nov.）和它的两个新种：菌株YIM90002定为盐生链单抱菌新种(Streptomonospora saline sp. nov.），菌株YIM90003定为白色链单抱菌新种(Streptomonospora alba sp. nov.）；菌株YIM90004和YIM90006分别被确定为拟诺卡氏菌属的一个新种和一个亚种：新疆拟诺卡氏菌新种（Nocardopsi sxiniangensis sp. nov.）和嗜阿拉伯糖新疆拟诺卡氏菌亚种( Noocardiopsi sxiniangensis subsp．arabicus subsp．nov，）。

调节LDLR及VCAM-1基因表达的微生物代谢产物的研究

为了筛选对靶基因LDLR和VCAM-1的表达具有调节作用的生物活性物质，建立了两个基于重组人细胞系的高通量的筛选模型，使用荧光素酶在96-孔版上来筛选对上述靶基因的表达具有调节作用的微生物代谢产物。模型之一是来自于人肝HepG2细胞系的重组L39细胞，用于筛选增加LDLR报告基因表达的生物活性物质，以期发现新的具有降胆固醇作用的药物。筛选之二为来源于细胞系ECV304的重组细胞株Nl-14,用于筛选抑制VCAM-1基因表达的活性物质，以期发现治疗风湿性关节炎等免疫性疾病治疗的药物。上述筛选系统均是稳定转染的细胞系，分别含有与荧光素酶报告基因相融合的LDLR或VCAM-1基因的转录调节元件。通过对6300株微生物的总计12600个样品的筛选，共发现和分离了17个活性化合物并进行了结构解析。其中两个被命名为Cladospolede D和Zelkovamycin的化合物被确定为新的化合物。由真菌 FO-6605的发酵液提取得到的一个化合物对LDLR报告基因的表达具有很强的上调作用，其SC200为1 Onmol/L a使用荧光标记的LDL检测到该化合物对于HepG2细胞膜上LDLR具有剂量依赖的增强作用。由真菌FO-5897的发酵液中分离到了一个已知的化合物Ascofuranone，该化合物曾经被报道具有降血脂抗肿瘤的活性。值得注意的是我们首次发现了该化合物同时具有抑制 VCAM-1报告基因表达和增强LDLR报告基因表达的作用，该发现有可能会对其降血脂作用的深入研究提供帮助。由海洋真菌FT-0012产生的化合物Cladospolede D为一个12-员环的大环内酷类的化合物，该化合物对两个测活系统均显示出无选择性的抑制作用形态学研究显示该真菌属于Cladosporiun属。另外一个由土壤放线菌K96-670产生的新化合物为一个环八肤类的化合物，经~1H~1-H COSY,~(13)C-H COSY,~(13)C-~1H HMQC, ~(15)N-~1H HMQC,~(15)-~1N HHMBC等波谱学研究得知该化合物的分子结构中含有六个非普通的氨基酸和两个普通氨基酸。该化合物对VCAM-I报告基因的表达显示出非常好的选择性的抑制活性，其IC50值为9.5ug/ml.形态学的研究表明该菌株属于链霉菌属。 在筛选过程中从来源于云南省西双版纳的土壤中分离到了一株编号为YIM1272的放线菌，经包括形态学、生理一生化和16S rDNA在内的分类学研究，确定该菌株为链霉菌属的一个新种，被命名为佩版纳链霉菌，(Streptomyces.bannaensis．sp.nov）。

繁茂膜海绵中放线菌的鉴定及S19抗菌素主要特性的研究

近年的研究表明，海绵微生物是某些海绵天然产物的真正产生者。因此，人们将海绵微生物作为开发海绵天然产物的重要来源之一。采用琼脂块法和液体扩散法，从分离自中国黄渤海大连海域的海绵优势种—繁茂膜海绵的28株放线菌中筛选到4株具有抗菌活性的放线菌，并对它们进行生物学鉴定。采用经典和现代分类鉴定方法，对4株具有抗菌活性的繁茂膜海绵放线菌的形态特征、培养特征、生理生化特征、细胞壁化学组分和16SrRNA序列进行了研究，得出种水平的鉴定结果：Hmp-S14为西唐氏链霉菌Streptomycessetonii；Hmp-S19为灰色链霉菌Streptomyces griseus；Hmp-S24为桔橙小单抱菌Micromonospora aurantiaca；Hmp-S26为生二素链霉菌Streptomyces ambifaciens。在四株具有抗菌活性的繁茂膜海绵放线菌中，菌株Hmp-S19的抗菌活性优于其它三株，并且与已报道的20多种灰色链霉菌菌株有不同的生理生化特性，故进一步优化其发酵条件并初步研究了S19抗菌素的理化性质。通过单因子和均匀设计实验，优化菌株Hmp-S19摇瓶发酵条件。确定最佳发酵培养基：玉米粉0.6％，葡萄糖0.1％，豆饼粉0.5％，NaCl 0.3％，KH2PO40.08％，CaCO30.08％，MgSO40.02％；最佳发酵条件：接种龄30h，接种量5％，初始pH7．0，发酵时间96h，装液量100ml／50ml，培养温度28 ℃。应用二剂量法测定519一抗菌素的相对效价，为5154μ/ml，较原始发酵培养基和发酵条件（3364μ/ml）提高了53％。通过pH纸层析和捷克八溶剂系统纸层析试验，初步判定519抗菌素为两性、非水溶性I型抗菌素。Hmp-S19发酵液经预处理、萃取、硅胶柱层析、制备薄层层析等步骤，对S19抗菌素进行分离纯化得粗制品，并进行了液 相色谱一质谱检测。

嗜盐和嗜碱放线菌的分离及多相分类研究

本文综述了放线菌分类学研究的意义和进展，分析了放线菌分类学研究特点及发展趋势；同时综述了嗜盐、嗜碱放线菌的研究进展，分析了嗜盐和嗜碱放线菌研究的重要性和意义。通过对采自渤海沿岸盐场、青海盐湖、张北盐湖、曲周、保定等地盐碱和高盐的样品进行嗜盐和嗜碱放线菌的分离，建立了适于嗜盐和嗜碱放线菌的分离培养条件；找到了广适性的嗜盐放线菌培养用培养基；发现了嗜盐放线菌多数耐碱的规律。采用形态培养特征、生理生化测定、细胞化学组分分析、DNA G＋C mol％测定以及DNA同源性分析和165 rDNA序列为基础的系统发育分析等相结合的多相分类技术对分离得到的部分典型菌株系统地进行了分类研究，从表型、基因型和系统发育三个层次对其分类地位进行了确定。本研究发现嗜盐放线菌新属新种1个，为波状盐场放线菌新属新种（Actinoosalterria undulata gen.nov．sp．nov），嗜盐放线菌新种4个，分别是：沧州拟诺卡氏菌(Nocardiopsl's cangzhouensis sp．nov．）、塘沽拟诺卡氏菌(Nocardiopsis tangguensis sp.nov）、茶卡拟诺卡氏菌(NocardioPs沽chakaensis)和盐场链霉菌(Streptomyces salinarum sP.nov.）；发现嗜碱放线菌新种2个，分别是；布尔津拟诺卡氏菌（NocardioPsis buerjinensis sp.nov．）和城墙拟诺卡氏菌（Nocardiopsis rampartis sp.nov．）；另有2株嗜碱放线菌鉴定为达松维尔拟诺卡氏菌，2株嗜碱放线菌鉴定为产气味拟诺卡氏。论文研究中还初步建立了放线菌磷酸类脂高效液相色谱测定的方法与测定条件。

吸水链霉菌、放线菌Sp.253和金色毛壳菌的次生代谢产物研究

链霉菌是十分重要的一类放线菌，绝大多数的抗生素都由该类细菌产生。毛壳属真菌是一类重要的丝状真菌，从中也发现有很多结构新颖、活性独特的活性物质。因此本论文对两株链霉菌的活性成分及一株金毛壳菌的次生代谢产物进行了研究。 1．从吸水链霉菌（Streptomyces hygroscopicus 1.358）液态发酵产物（乙酸乙酯提取物）中分离得到3个化合物，通过波谱方法鉴定为RK955A (1)、Nigericin（2）、Elaiophylin（3）。以青霉素耐药-金黄色葡萄球菌作为指示菌的抗菌活性测定表明，三者均具有较强抗菌活性。 2．通过抗肿瘤体外活性筛选模型筛选得到得到一株链霉属土壤放线菌，从中分离得到六个化合物：苯乙酰胺（4）、苯丙酰胺（5）、肉桂酰胺（6）、3-（N-（甲酰胺基）乙酰基）吲哚（7）、鸟苷磷酸（8）、鸟苷（9）。 3．从金色毛壳菌（Chaetomium aureus）的固态培养物中分离得到13个化合物，利用波谱方法将其鉴定为：金毛壳菌素A（10）、金毛壳菌素B（11）、Eugenetin（12）、Eugenitol（13）、Chaetoquadrin A（14）、Chaetoquadrin B（15）、Chaetoquadrin G（16）、Chaetoquadrin H（17）、Chaetochromin A（18）、Sterigmatocystin（19）、O-methylsterigmatocystin（20）、3β-羟基-麦角甾-5，7，22-三烯（21）和过氧麦角甾醇（22）。 4．综述了聚醚类抗生素的结构、生物合成、生物活性及作用机理。 The genus Streptomyces (Actinomycetes) is an important group of microbe. Most antibiotics known nowdays are discovered from species of Streptomyces. The fungi of the genus Chaetomium have attracted much attention because various kinds of secondary metabolites with diverse bioactivities have been found from them. Thus, the bioactive compounds from two strains of Streptomyces and the secondary metabolites of Chaetomium aureus were investigated. 1. Three compounds were isolated from the ethyl acetate extract of the fermentation broth of Streptomyces hygroscopicus. They are identified to be elaiophylin (1), nigericin (2), and antibiotic RK955A (3) on the basis of their spectroscopic data. Compounds 1-3 possess antibacterial activities against Staphyloccocus aureus. 2. It was found that the extract of the fermented broth of a strain of Actinomycetes could inhibit some tumor cel lines. Separation of the bioactive fraction led to the isolation of six compounds. They were characterized to be phenylacetamide (4), phenylpropylamide (5), trans-cinnamamide (6), 3- (N- (formylmethyl) acetamide) indole (7), guanylicacid (8), and guanosine (9). 3. From the fermented broth of Chaetomium aureus, 13 compounds were isolated for the first time. They were determined to be chaetomiumycin A (10), chaetomiumycin B (11), eugenetin (12), eugenitol (13), chaetoquadrin A (14), chaetoquadrin B (15), chaetoquadrin G (16), chaetoquadrin H (17), chaetochromin A (18), sterigmatocystin (19), O-methylsterigmatocystin (20), 3β-hydroxyergosta-5, 7, 22-triene (21) and peroxy-ergosterol (22). Compounds 10 and 11 are new ones. 4. Structure, biosynthesis, biological activity, and mechanisms of polyether antibiotics were reviewed.

链霉菌S227抗耐药菌活性成分的研究

本论文以从四川峨嵋山森林土壤中分离筛选获得的一株产抗耐药性活性化合物的链霉菌S227为材料，对发酵液中活性物质的分离纯化及抗耐药性活性进行了研究。 建立了抗耐药性活性的定性、定量检测方法。建立的管蝶法活性定量检测的标准回归方程为：D=4.8229Ln(C)+3.6326 R=0.9972 ；纸片法活性定量检测的标准回归方程为：D=5.5Ln(C)-12.794 R=0.999。 根据建立的样品活性的检测方法，测定了发酵液的初始活性。实验证明活性物质的温度、pH稳定性好。 通过活性的定性、定量追踪方法，分别利用等体积的石油醚、乙酸乙酯、正丁醇在不同的pH梯度下萃取，确定了pH3条件下正丁醇能最大程度的萃取活性物质，说明活性物质极性很大。对正丁醇萃取相经过两次硅胶柱层析及薄层层析分离得到具有抗耐药菌活性的纯化样品S227-4。 经过核磁共振氢谱、碳谱数据分析初步确定S227-4为四聚糖，通过糖的水解实验初步确定S227-4由葡萄糖和半乳糖组成。 纸片法活性检测表明S227-4具有抗耐药菌活性。采用MIC测定法对该样品抗耐药活性进行研究。在证明该样品本身不具有抗菌活性的基础上，以临床分离的耐药性金黄色葡萄球菌为指示菌，考察了该样品与抗生素联合使用时对耐药菌抗生素MIC（最小抑菌浓度）值的影响，结果表明在不影响菌体生长的浓度条件下，该样品能明显降低多株耐药菌对多种抗生素的MIC值，不同程度地恢复所测试耐药菌对相应抗生素的敏感性。如S227-4与青霉素钠联用可以使S. aureus 12352的MIC降低8倍，而与红霉素联用可以使S. aureus 12334的MIC降低128倍。 The purification process and the activity of the anti bacterial drug resistance compounds produced by Streptomyces S227 isolated from the forest soil sample of the Mountain E’MEI in Sichuan Province were studied in this thesis. Quantitative and qualitative activity assay methods of the active compounds were established. The regression equation of the tube method was D=4.8229Ln(C)+3.6326, R=0.9972. The regression equation of the paper method was D=5.5Ln(C)-12.794, R=0.999. According to the established activity assay method, the incipient activity of the broth was evaluated. And it was proved that the stability of the active compounds was good. By quantitative and qualitative activity tracing method, petroleum ether, ethyl acetate and butanol were used to extract the active compounds at different pH. The result showed that butanol was the most effective agent for active component recovery at pH3. From the butanol extraction a purified sample, S227-4, was isolated by silica gel column chromatography and thin-layer chromatography . S227-4 was proved to be a tetra- saccharide by 1H-NMR and 13C-NMR. And its monosaccharides include glucose and galactose by hydrolysate analysis. The anti-drug resistant activity of S227-4 was tested in vitro by MICs assay using different drug resistant Staphylococcus aureus strains isolated clinically. The sample itself showed no anti-microbial activity in growth inhibitory experiment, but when it was used together with different antibiotics, it could remarkably decrease the MICs of different clinically isolated drug-resistant bacterial strains to these antibiotics. For example, when S227-4 was used with penicillin, the MIC of S. aureus 12352 decreased 8 times compared with that when penicillin was used alone. Meanwhile when it was used with erythromycin the MIC of S. aureus 12334 deceased 128 times compared with erythromycin alone.

克拉维酸产生菌的诱变育种与发酵工艺优化研究

以克拉维酸产生菌棒状链霉菌Streptomyces clavuligerus CCRC11518（ATCC 27064）III50为出发菌株, 首先比较各种物理和化学诱变剂处理对其克拉维酸生物合成的影响, 确定了亚硝基胍为棒状链霉菌诱变育种的诱变剂及其处理剂量: 2mg/ml、40min. 经浓度为2mg/ml的亚硝基胍处理40min后, 采用新颖理性化筛选方法, 通过逐步筛选自身代谢产物抗性突变株、克拉维酸抗性突变株和链霉素抗性突变株, 最终得到一株克拉维酸高产菌VI118(效价633μg/ml), 其克拉维酸效价是出发菌株(效价377μg/ml)的167.9%. 该高产突变株在琼脂斜面培养基上连续传接10代, 克拉维酸效价保持稳定. 通过单因子和多因子摇瓶正交试验, 对高产菌株VI118的发酵条件进行了研究, 确定最佳发酵条件: 甘油60g, 水解植物蛋白 60g, KH2PO4 0.5 g, 玉米浆 7.5g, MnSO4•H2O 0.34g, MgSO4•7H2O 0.99g, FeSO4•7H2O 0.56g, 蒸馏1000ml, pH 7.0, 发酵培养基装量20ml/250ml三角瓶, 接种量10％, 培养温度28ºC, 220r/min摇床培养72h后测定效价. 在最佳发酵条件下克拉维酸效价达到651μg/ml, 同时把初始发酵培养基的昂贵成分替换为廉价的工业原料. 通过摇瓶分批补料试验, 得到最佳补料物质和补料方式：在上述最佳发酵条件下, 分别在发酵培养48h、56h、64h、72h时补加4ml无菌水, 80h发酵结束, 克拉维酸效价达到905μg/ml. 在不增加原料成本的情况下通过摇瓶补料方式克拉维酸效价为未补料的139.0％, 总产量为未补料的264％. By a novel rational screening method, mutant Streptomyces clavuligerus CCRC11518（ATCC 27064）III50（titres 377μg/ml）, as the clavulanic acid-producing parent strain, was treated by NTG (2mg/ml) for 40min, and the self-generated metabolites resistant mark, the clavulanic acid resistant mark and the streptomycin resistant mark were added step by step. Finally, the mutant VI118（titres 633μg/ml）with the three marks was obtained. The clavulanic acid productivity of this mutant was increased by 167.9% compared with the parent strain. After reproducing 10 generations on the agar medium slant, the productivity of this mutant was stable. The optimum fermentation conditions were established as followings: glycerol 60g, acid hydrolyzed vegetable protein 60g, KH2PO4 0.5g, corn steep liquor 7.5g, MnSO4•H2O 0.34g, MgSO4•7H2O 0.99g, FeSO4•7H2O 0.56g, distilled water 1 liter, pH 7.0, 20ml in 250ml shake-flask, inoculation 10%(v/v), fermentation temperature 28ºC, rotation speed 220 r/min, time 72h. The clavulanic acid productivity was 651μg/ml, while used the low-priced industrial raw materials. After studying on fed-batch in the shake-flask, the optimum fed-batch manner was obtained: under optimum fermentation conditions, at 48h, 56h, 64h and 72h, adding 4ml distilled water into each flask, fermentation ending at 80h. The clavulanic acid productivity was increased by 139% compared with no fed-batch, meanwhile the total yield was increased by 264%.

石油污染土壤生物修复过程中微生物生态研究

The analysis on microbiological ecology for four types of oil contaminates soils showed that the bacteria utilizing the oil as carbon sources increase,wheras the fugi become less .Zooloea and Bacillu are the dominant bacteria ; Mocor and Cunninghamella ,and Fursarium are the dominant fungi streptomyces take the superiority among the actinomyces.The anaiysis on esterase activity showed that the microbes above mentioned have abilies of degrading esters. The biodeg radationrates are 55.45%,56.74%,38.37% and 45.19%respectively,after 53 days,the biodegradation rate can be increased by 12.6% when the dominant microbes are added.

Chandrananimycins A-C: Production of novel anticancer antibiotics from a marine Actinomadura sp isolate M048 by variation of medium composition and growth conditions

In our screening of marine actinomycetes for bioactive principles, three novel antibiotics designated as chandrananimycin A (3c), B (3d) and C (4) were isolated from the culture broth of a marine Actinomadura sp. isolate M045. The structures of the new antibiotics were determined by detailed interpretation of mass, 1 D and 2 D NMR spectra.