抗肿瘤活性海洋放线菌的筛选及菌株HGF26的初步鉴定

对采自连云港海域的海泥样品进行放线菌选择性分离,用其发酵液进行抗肿瘤活性筛选,并对活性较好的菌株HGF26进行了初步鉴定。从海泥样品中共分离得到放线菌78株,以人肝癌细胞HepG2为靶标,活性筛选得到细胞毒活性达60％以上的阳性菌株3株,以其他5株肿瘤细胞为靶标的复筛表明,菌株HGF26的发酵液对多种肿瘤细胞具有显著的细胞毒活性,其中对胃癌细胞BGC823的细胞毒活性为79％,活性产物具有较好的酸碱和热稳定性。通过对菌株的培养特征、形态特征、生理生化特征和细胞壁成分分析,将菌株HGF26初步鉴定为微白黄链霉菌的海洋变种。     

链霉菌Z314的分类鉴定及其转化产物普伐他汀的纯化研究

从土壤中筛选得到1株可转化美伐他汀(compactin)生产普伐他汀(pravastatin)的放线菌Z314,根据形态观察、培养特征、生理生化鉴定以及16S rRNA序列分析,初步判定该菌株为链霉菌属中的黄质产色链霉菌(Streptomyces xanthochromogenes).经发酵条件优化,该菌株转化美伐他汀生产普伐他汀的产量可达1580mg/L,转化率为49.45%.发酵液经中空纤维素柱过滤、大孔树脂吸附和HPLC纯化,普伐他汀的回收率为45.06%,纯度为99.02%,为进一步产业化开发奠定了基础.     

链霉菌ZG0429的分类鉴定与链霉亲和素的分离纯化研究

从土壤中筛选得到1株高产链霉亲和素的放线菌ZG0429,根据形态观察、培养特征、生理生化鉴定以及16S rRNA序列分析,初步判定该菌株为链霉菌属中的淡紫灰链霉菌(Streptomyces lavendulae)。经发酵,ZG0429的链霉亲和素产量可达201.0mg/L。进一步采用硫铵沉淀和凝胶过滤层析纯化,链霉亲和素的回收率为76.87%,纯度可以达到97.03%。该方法简单易行,成本低廉,可得到高产量、高纯度、高活性的目的蛋白,为链霉亲和素发酵产品的大规模纯化提供了依据。     

Large scale production and semi-purification of kedarcidin in a 1000-L fermentor

Summary Actinomycete strain ATCC 53650 was grown in a 1000-L fermentor containing 680 L of medium and the production of kedarcidin was monitored by HPLC. The titers of kedarcidin in the fermentor cultures were 0.49–0.53 mg ml^–1. A quick and efficient purification method involving the use of anion exchange resin DE23 (batch adsorption-desorption) and an ultrafiltration system yielded high recovery (65% yield) of kedarcidin from the fermentor culture. Over 200 grams of lyophilized kedarcidin of 70% purity was recovered from each of two 1000-L fermentor cultures using this process.     

核甙类抗生素产生菌的筛选及其活性组分研究

从成都郊区土壤中筛选出一株能产生抗生素的放线菌 ,经过形态及培养特征观察、生理生化特性试验、细胞壁化学组成分析 ,确定其分类学归属为链霉菌属 ( Strep-tomyces)。从产生菌的固体培养物中分离得到活性物质、理化性质研究表明 ,该物质为胞嘧啶核苷衍生物。抗菌和抗肿瘤活性试验结果显示 ,此物质不仅具有抑菌活性 ,而且具有较强的抗肿瘤活性。     

重组水蛭素的纯化和鉴定

本文报导了化学合成的水蛭素基因在酵母细胞中得到表达,井能分泌水蛭素到胞外。将该菌株培养物的上清液经硫酸铵沉淀和Sephadex G-50过滤后,用DEAE-SephadexA-25进行阴离子交换层析,进而用HPLC反相层析,得到表达产物重组水蛭素。经SDS-PAGE,氨基酸序列分析,抗凝血酶活力分析及血浆滴定实验等方法鉴定,证明该基因表达产物与天然水蛭素HV_2相同。     

枯草芽孢杆菌B2菌株产生的表面活性素变异体的纯化和鉴定

利用6mol/L HCI沉淀枯草芽孢杆菌B2菌株的去细胞培养液,甲醇抽提获得脂肽类抗生素粗提物,过Sephadex LH-20层析柱获得粗纯化物,经MALDI-TOF-MS检测表明B2菌株仅含有表面活性素一种脂肽类抗生素。利用HPLC SMART SYSTEM,将粗纯化物过μPRC C2／C18层析柱对表面活性素变异体进行分离后获得纯化物。经MALDI-TOF-PSD—MS对纯化物的结构分析表明,B2菌株的表面活性素变异体由13、14和15个碳原子的脂肪酸链以及L-Glu-L-Leu—D—Leu—L-Val-L-Asp-D—Leu-L-Leu七环肽组成。     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Taxonomy and chemical characterization of new antibiotics produced by Saccharothrix SA198 isolated from a Saharan soil

Actinomycete strain SA198, isolated from a Saharan soil sample of Algeria, exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria, and phytopathogenic and toxinogenic fungi. The morphological and chemotaxonomic characteristics of the strain were consistent with those of the genus Saccharothrix. Analysis of the 16S rRNA gene sequence of strain SA198 showed a similarity level ranging between 97.2 and 98.8% within Saccharothrix species, S. australiensis being the most closely related. Two new active products were isolated by reverse HPLC using a C18 column. The ultraviolet–visible (UV–VIS), infrared (IR), mass, and ¹H and ¹⁴C nuclear magnetic resonance (NMR) spectra showed that these products were new bioactive compounds. The minimum inhibitory concentrations of these antibiotics showed a strong activity against fungi and moderate activities against Gram-positive and Gram-negative bacteria.     

<Emphasis Type="Italic">Streptomyces sanglieri</Emphasis> which colonised and enhanced the growth of <Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq. seedlings was antagonistic to <Emphasis Type="Italic">Ganoderma boninense</Emphasis> in in vitro studies

Actinomycete strain AUM 00500 was 99.5 % similar to Streptomyces sanglieri NBRC 100784^T and was evaluated for antagonistic activity towards Ganoderma boninense, the causative fungus of basal stem rot of oil palm. The strain showed strong antifungal activity towards G. boninense in in vitro and SEM analysis showed various modes of inhibition of the fungus. Ethyl acetate extracts of single culture and inhibition zone of cross-plug culture by HPLC indicated that strain AUM 00500 produced two different antibiotics of the glutarimide group namely cycloheximide and actiphenol. In greenhouse trials, oil palm seed treated with spores of S. sanglieri strain AUM 00500 at 10⁹ cfu/ml showed significant (P < 0.05) increase in oil palm seedlings growth when compared to the control. Streptomyces sanglieri strain AUM 00500 successfully colonised the epidermal surface of the roots of treated oil palm seedlings and it was recovered from root fragments plated on starch casein agar.     

深海放线菌08A4的鉴定及其抗真菌活性产物研究

从南海深海分离得到1株放线菌08A4,其发酵产物具有抗植物病原真菌活性,分离纯化得到3个化合物,通过1H-NMR初步鉴定为抗霉素类物质。结合形态学鉴定方法与16S rDNA序列分析方法,鉴定该菌株为微白黄链霉菌(Streptomyces albidoflavus)。     

Localization and Characterization of the Carbon Tetrachloride Transformation Activity of Pseudomonas sp. Strain KC

Previous research has established that Pseudomonas sp. strain KC rapidly transforms carbon tetrachloride (CT) to carbon dioxide (45 to 55%), a nonvolatile fraction (45 to 55%), and a cell-associated fraction ((equiv)5%) under denitrifying, iron-limited conditions. The present study provides additional characterization of the nonvolatile fraction, demonstrates that electron transfer plays a role in the transformation, and establishes the importance of both extracellular and intracellular factors. Experiments with (sup14)C-labeled CT indicate that more than one nonvolatile product is produced during CT transformation by strain KC. One of these products, accounting for about 20% of the [(sup14)C]CT transformed, was identified as formate on the basis of its elution time from an ion-exchange column, its boiling point, and its conversion to (sup14)CO(inf2) when incubated with formate dehydrogenase. Production of formate requires transfer of two electrons to the CT molecule. The role of electron transfer was also supported by experiments demonstrating that stationary-phase cells that do not transform CT can be stimulated to transform CT when supplemented with acetate (electron donor), nitrate (electron acceptor), or a protonophore (carbonyl cyanide m-chlorophenylhydrazone). The location of transformation activity was also evaluated. By themselves, washed cells did not transform CT to a significant degree. Occasionally, CT transformation was observed by cell-free culture supernatant, but this activity was not reliable. Rapid and reliable CT transformation was only obtained when washed whole cells were reconstituted with culture supernatant, indicating that both extracellular and intracellular factors are normally required for CT transformation. Fractionation of culture supernatant by ultrafiltration established that the extracellular factor or factors are small, with an apparent molecular mass of less than 500 Da. The extracellular factor or factors were stable after lyophilization to powder and were extractable with acetone. Addition of micromolar levels of iron inhibited CT transformation in whole cultures, but the level of iron needed to inhibit CT transformation was over 100-fold higher for washed cells reconstituted with a 10,000-Da supernatant filtrate. Thus, the inhibitory effects of iron are exacerbated by a supernatant factor or factors with a molecular mass greater than 10,000 Da.     

Purification and characterization of a novel beta-agarase from Vibrio sp. AP-2

beta-Agarase was purified from the culture fluid of a porphyran-decomposing marine bacterium (strain AP-2) by ammonium sulfate precipitation, successive column chromatography and DNase and RNase treatment. The final enzyme preparation appeared to be homogeneous on polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 20 kDa, a pH optimum of 5.5, and was stable in the pH region 4.0-9.0 and at temperatures below 45 degrees C. The beta-agarase was a novel endo-type enzyme which hydrolyzed neoagarotetraose, larger neoagarooligosaccharides and agar to give neoagarobiose [3,6-anhydro-alpha-L-galactopyranosyl-(1----3)-D-galactose] as the predominant product. The enzyme did not act on kappa-carrageenan. According to the criteria of Bergey's Manual of Systematic Bacteriology, the strain was assigned to the genus Vibrio.     

Preliminary screening for antibacterial and antimycobacterial activity of actinomycetes from less explored ecosystems

Actinomycetes from less explored ecosystems were screened for antibacterial and antimycobacterial activity. Crude bioactive compounds were produced by growing these strains by shake flask fermentation using soybean meal medium. Culture supernatant and mycelia were extracted with ethyl acetate and methanol, respectively. Antibacterial activity of crude extracts was tested by disc diffusion method against gram positive and gram negative bacteria. Actinomycete strains D10, D5, NEK5, ANS2, M104 and R2 showed prominent activity. Culture filtrates and crude extracts were tested against standard strain Mycobacterium tuberculosis H₃₇Rv and drug sensitive and drug resistant clinical isolates of M. tuberculosis by luciferase reporter phage (LRP) assay. Considerable variation was observed in antimycobacterial activity between actinomycete culture filtrates and solvent extracts. Actinomycete strains viz., D10, D5 (desert), CSA14 (forest), CA33 (alkaline soil), NEK5 (Neem plant), MSU, ANS2, R2 and M104 (marine) screened in the present study were found to be highly potent showing good antibacterial and antimycobacterial activity. Five of them such as A3, CSA1, EE9, ANS5 and R9 were exclusively active against M. tuberculosis. Secretary products of actinomycetes of rare ecosystems are meant to antagonize organisms in their respective environments. These are likely to be novel antimycobacterial compounds as they unknown to human pathogens.     

具抗菌活性放线菌菌株JSM 20.1的分离和鉴定

从药用植物银杏(Ginkgo biloba)根际土壤中分离到1株放线菌菌株JSM20.1,其发酵产物具有广谱抗菌活性。该菌株在多数培养基上生长良好,具有链霉菌属的典型形态特征。菌丝多分枝,不断裂;基内菌丝黄白色至棕褐色,气生菌丝黄白色至棕红色;孢子链直且长,偶有波曲,孢子柱状、光滑、不运动。在察氏、马铃薯浸汁、葡萄糖-天门冬酰胺、酵母膏-麦芽膏(ISP2)、燕麦(ISP3)、无机盐淀粉(ISP4)和甘油-天门冬酰胺(ISP5)琼脂上均产生可溶性色素,而在营养琼脂上不产生可溶性色素。生长温度范围为7～40℃,最适生长温度28℃;生长pH范围为5.0～10.0,最适pH7.0;能在含0～3%(质量与体积比)NaCl的培养基上生长,而在无NaCl的ISP2培养基生长最佳。根据其形态学特征、生理生化特征、细胞化学分类特征和基于16SrRNA基因序列的系统发育分析结果,菌株JSM20.1被鉴定为链霉菌属的有效发表种Streptomyces cyaneofuscatus的1个菌株。     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Kosinostatin, a major secondary metabolite isolated from the culture filtrate of Streptomyces violaceusniger strain HAL64

During a screening program, an actinomycete strain isolated from the Egyptian soil was investigated for its potential to show antimicrobial activity. The identification of this isolate was performed according to spore morphology and cell wall chemo-type, which suggested that this strain is a streptomycete. Further cultural, physiological characteristics and the analysis of the nucleotide sequence of the 16S rRNA gene (1480 bp) of this isolate indicated that this strain is identical to Streptomyces violaceusniger (accession number EF063682) and then designated S. violaceusniger strain HAL64. In its culture supernatant, this organism could produce one major compound strongly inhibits the growth of Gram-positive but the inhibition of Gram-negative indicator bacteria was lower. The antibiotic was separated by silica gel column chromatography and then purified on a sephadex LH-20 column and finally the purity was checked by HPLC. The chemical structure of the purified compound was determined using spectroscopic analyses (molecular formula of C33H32N2O10 and molecular weight of 617.21) and found to be identical to the kosinostatin, a quinocycline antibiotic which is known to be produced by Micromonspora sp. TP-A0468 (Igarashi et al., 2002) and to quinocycline B isolated from Streptomyces aureofaciens (Celmer et al., 1958). Although the antibiotic is known, the newly isolated strain was able to produce the antibiotic as a major product providing an important biotechnological downstream advantage.     

Purification and Genetic Determination of Bacteriocin Production in Enterobacter cloacae

Enterobacter cloacae (strain DF13) was found to produce a bacteriocin which could be induced by mitomycin C. In the supernatant fluid of the induced culture phagelike particles were found. The bacteriocin was partially purified from induced cultures by ammonium sulfate precipitation and gel-filtration on Sephadex G-150. Ultraviolet-absorbing material was eluted from the Sephadex column in three fractions. The biological activity was mainly present in the second fraction and is associated with a protein with a molecular weight of about 61,000. The phagelike particles were found in the first fraction and show no biological activity. Upon conjugation of E. cloacae strain DF13 with another strain of the same species and with Escherichia coli K-12S, the ability to produce bacteriocin was transferred. The new bacteriocinogenic strain produced bacteriocin, which could not be distinguished from that produced by E. cloacae strain DF13. Although transfer of the bacteriocinogenic factor often occurred together with transfer of the ability to produce phagelike particles, it was shown that these two factors are two separate genetic entities. In addition to a bacteriocinogenic factor, E. cloacae strain DF13 was found to carry two other transferable plasmids: one determining resistance against streptomycin and sulfanilamide and another determining resistance against penicillin.     

A new method for purification of staphylocoagulase by a bovine prothrombin-Sepharose column

Staphylocoagulase was isolated from a culture filtrate of Staphylococcus aureus, strain st-213, by a two step purification procedure of chromatography on a bovine prothrombin-Sepharose 4B affinity column and gel filtration on Sephadex G-25. The yield of the coagulase activity ranged from 75--83% and the purified preparation gave a single precipitin line in immunodiffusion tests against anti-crude and anti-purified staphylocoagulase sera. However, the final product was shown to contain one major and two minor components by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chemical analysis of the material indicated that it does not contain any cystine residues and that its NH2-terminal residue is a single isoleucine.