南昌霉素高产菌株的链霉素抗性基因突变诱变筛选研究

通过对链霉素对南昌霉素（Nanchangmycin)产生菌NS－41－80菌株孢子的致死浓度测定基础上,采用诱变剂甲基磺酸乙酯（EMS）的不同诱发剂量对菌株孢子进行诱变处理,诱变处理的孢子涂布在含链霉素（10ug/mL)致死浓度的高氏平板上,获得了大量的链霉素抗性基因（str)突变株。然后从3,000株链霉素抗性基因（str)突变株中通过初筛获得比诱变出发菌株产素能力提高20％以上的菌株202株,再进一步通过摇瓶复筛,获得比出发菌株产素能力分别提高100％,200％,300％高产菌株为48株,7株和1株,分别为复筛菌和初筛菌株的23．76％和1．60％,3．46％和0．23％,0．5％和0．03％,将产素能力提高240％以上5个菌株连同出发菌株连续3批次进行摇瓶发酵结果,5个突变株的产素能力均比出发菌株的产素能力提高57％－96．4％,其中突变株80－5．3－165菌株摇瓶发酵单位达6,000ug/mL以上,3批次摇瓶平均发酵单位达5,855ug/mL,建立了南昌霉素高产菌株的链霉素抗性基因突变诱变快速高效的筛选方法。     

妥布霉素产生菌诱变育种的研究

本实验以妥布霉素产生菌黑暗链霉菌（Ｓ．ｔｅｎｅｂｒａｒｉｕｓ）ＡＴＣＣ１７９２０为出发菌株,经紫外线处理,获得一株产量较高且稳定的菌株ＵＶ－５９,其抗生素效价比原株提高９２．３％对ＵＶ＿５９菌株进行高温处理,得到一株Ｔ－５４１菌株,所产抗生素只有两个组分,比原出发株减少一个组份,不再含有出发株产生的氨甲酸卡那霉素;再对Ｔ－５４１菌株进行原生质体制备,分别用紫外线和紫外线加氯化锂以及亚硝基胍诱变处理原生质体,获得四株高产菌株,效价比原株提高１１５～１５０％,且稳定。此外还研究了变异菌株的形态与产量的关系。     

羊肚菌原生质体诱变筛选高生物量高氨基酸含量菌株

研究影响尖顶羊肚菌ＣＧＡＣ－９５０６原生质体制备、再生因素基础上,首次诱变羊肚菌原生质体,进行高生物量高氨基酸含量菌株选育。原生质体诱变再生株Ｍ．ｃｏｎｉｃａ ＣＧＡＣ－９５０６３７生物量比出发株提高７．３％,总氨基酸比出发株提高３８％。     

羊肚菌原生质体诱变筛选高生物量高氨基酸含量菌株

研究影响尖顶羊肚菌（MOrchellaconicaPers、）CGAC－9506原生质体制备、再生因素基础上,首次诱变羊肚菌原生质体,进行高生物量高氨基酸含量菌株选育。原生质体诱变再生株MconicaCGAC－950637生物量比出发株提高7．3％,总氨基酸比出发株提高38％。     

产胞外青霉素酰化酶菌株的选育

以短小杆菌（B．pumilus）B－97为出发菌株,经过连续两次紫外线诱变处理,分离得一突变株B－U－29。其酶活力为4．56U／ml,较出发菌株酶活力提高113．3％。对B－U－29菌株进行连续两次亚硝酸处理,分离得一正变稳定株B—H－29,酶活力为4．93u／ml,较出发菌株酶活力提高了20％。     

啤酒酵母胞外多糖发酵条件的研究

以啤酒酵母S－12为出发菌株,用紫外线＋氯化锂作为复合诱变剂,获得一株产胞外多糖量较高的变株S－12－4,比出发菌株提高33．3％,同时对变株进行了最佳培养条件的研究,结果表明：最适碳源和氮源分别为大米糖3％、酵母粉0．37％及NH4Cl0．32％,最适发酵条件为起始PH6．0、培养温度26℃,发酵周期为30h,在此基础上进行培养,变株S－12－4产胞外多糖最高可达38.2mg/100mL,比初始条件提高了54％。     

紫外线诱变选育聚丁二酸丁二醇酯（PBS）高降解活性菌株

以PBS降解菌HJ03（Alternariasp．）为出发菌株,通过紫外诱变,透明圈初筛及PBS薄膜复筛,获得一株降解能力增强且对温度和pH耐受力均得到提高的突变株HJ10。与出发菌株相比,HJ10在培养初期气生菌丝少,而培养7d时菌落致密且生长速度较快。经过连续继代培养7代后发现,突变株的降解活力保持了良好的遗传稳定性,其降解率较出发菌株提高百分比达14．4％以上;在最适降解温度范围内（25℃～30℃）和不适宜降解的温度条件下突变菌株Ⅻ10的降解率均高于出发菌株;各pH条件下,突变株对PBS的降解能力明显优于出发菌株,尤其在pH5．0时降解率提高了22．80％。     

拟除虫菊酯类农药降解菌的紫外线诱变

拟除虫菊醑类农药降解菌阴沟肠杆菌(Enterobacter cloacap)w10j15菌株经紫外线诱变后,筛选出突变株55株。分别在30℃,转速180rpm条件下培养3d,测降解力,获得正突变菌株6株,经加药普通斜面传种10代,2株(UW19,UW2)降解力保持稳定。降解力较强的UW19对联苯菊醑、甲氰菊醑、氯氰菊醑的降解率分别达70．40％、84．04％、70．87％,比出发菌株降解率提高了近20％;UW2也比出发菌株提高了约10％的降解率。     

木聚糖酶高产菌株的诱变*

出发菌株Aspergillus niger M1经过紫外线诱变得到一株木聚糖酶活力提高30％的突变株A．niger J506。木聚糖酶谱带检测发现,突变株成熟发酵液中有3种类型的木聚糖酶,而出发菌株中只有两种。经过正交试验得出突变株产酶的最佳发酵条件为：主碳源浓度4％、麸皮与玉米芯的比例为5：5、葡萄糖浓度0．1％、草酸铵浓度2．0％,培养基初始pH为5．0,250mL三角瓶的装液量为100mL。     

深黄被孢霉高产脂变株的选育及其发酵的研究

以深黄被孢霉（Mortierellaisabellina）AS3．3410为出发菌株,经紫外线,硫酸二乙酯和亚硝基胍复合诱变处理,选育成功高产脂深黄被抱霉M018变株,其摇瓶培养菌体油脂含量达65．6％,比出发菌株提高133％。60m³罐三级发酵培养菌体油脂含量高达79．2％,生物量达37．8g／L．气相色谱分析表明变株M018r-亚麻酸的含量比出发菌株提高了53％。连续传代试验表明M018是一稳定的变株。该变株油脂合成的最佳碳源为葡萄糖,最佳氮源为酵母膏,最佳C／N为     

4103例泌尿生殖道支原体感染及药敏分析

目的通过分析泌尿生殖道支原体感染及药敏情况,为临床提供用药指导。方法采用支原体检测试剂盒进行支原体属培养和药敏试验。结果4103例患者标本中,检出支原体属1336株,总阳性率为32．56％;其中单一解脲脲原体（Uu）感染1227例,阳性率为91．84％;单纯人型支原体（Mh）感染15例,阳性率为1．12％;Uu＋Mh混合合感染94例,阳性率为7．04％。解脲脲原体对阿奇霉素、红霉素、交沙霉素、罗红霉素、米诺环素、强力霉素敏感性高;单纯人型支原体对交沙霉素、米诺环素、强力霉素敏感。结论泌尿生殖道支原体感染以Uu为主。支原体大多具有多重耐药性,临床治疗需根据药敏结果选药物。     

A Rapid Microbiological Assay for Nystatin Using Rt+ Enriched Yeast Cells

A simple, rapid (30 min) microbiological assay for nystatin is discussed. It is based on the efflux of Rb⁺ ions from nystatin treated yeast cells which had been grown in a medium enriched with this element. Results obtained with this method and the conventional agar diffusion method for nystatin in raw materials and finished products compare favourably both in accuracy and reproducibility. For simplicity and reproducibility, a cryogenically-stored inoculum is advocated; its use gives a confidence interval ( P = 0·05) on six results of < 5%.     

Biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The polyene macrolide antibiotic nystatin, produced commercially by the bacterium Streptomyces noursei, is an important antifungal agent used in human therapy for treatment of certain types of mycoses. Early studies on nystatin biosynthesis in S. noursei provided important information regarding the precursors utilised in nystatin biosynthesis and factors affecting antibiotic yield. New insights into the enzymology of nystatin synthesis became available after the gene cluster governing nystatin biosynthesis in S. noursei was cloned and analysed. Six large polyketide synthase proteins were implicated in the formation of the nystatin macrolactone ring, while other enzymes, such as P450 monooxygenases and glycosyltransferase, were assumed responsible for ring decoration. The latter data, supported by analysis of the polyene mixture synthesised by the nystatin producer, helped elucidate the complete nystatin biosynthetic pathway. This information has proved useful for engineered biosynthesis of novel nystatin analogues, suggesting a plausible route for the generation of potentially safer and more efficient antifungal drugs.     

Interaction of nystatin with nystatin-resistantCandida tropicalis

Nystatin-resistant yeast Candida tropicalis was obtained after UV illumination and plating on nystatin-containing media. The mutant contained no ergosterol in the plasma membrane but bound nystatin to a degree similar to that of the wild strain (1.2 vs. 1.5 nmol per mg dry solid). Respiration of the mutant on glucose was reduced by 36% in the presence of 25 microM nystatin. This corresponded to a 25-43% decrease of the uptake of monosaccharides. Transport of amino acids was reduced by nystatin in the mutant by 44-86%, as compared with a 84-95% reduction in the wild strain. The intracellular ATP content was reduced by nystatin equally in the wild strain and in the mutant (by 43 and 47%). Nystatin appears to affect specifically membrane transport processes of nonelectrolytes while both the H+-extruding ATPase and the membrane potential are unaffected.     

Structural analysis and biosynthetic engineering of a solubility-improved and less-hemolytic nystatin-like polyene in Pseudonocardia autotrophica

Polyene antibiotics such as nystatin are a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Previously, using a polyene cytochrome P450 hydroxylase-specific genome screening strategy, Pseudonocardia autotrophica KCTC9441 was determined to contain an approximately 125.7-kb region of contiguous DNA with a total of 23 open reading frames, which are involved in the biosynthesis and regulation of a structurally unique polyene natural product named NPP. Here, we report the complete structure of NPP, which contains an aglycone identical to nystatin and harbors a unique di-sugar moiety, mycosaminyl-(α1-4)-N-acetyl-glucosamine. A mutant generated by inactivation of a sole glycosyltransferase gene (nppDI) within the npp gene cluster can be complemented in trans either by nppDI-encoded protein or by its nystatin counterpart, NysDI, suggesting that the two sugars might be attached by two different glycosyltransferases. Compared with nystatin (which bears a single sugar moiety), the di-sugar containing NPP exhibits approximately 300-fold higher water solubility and 10-fold reduced hemolytic activity, while retaining about 50% antifungal activity against Candida albicans. These characteristics reveal NPP as a promising candidate for further development into a pharmacokinetically improved, less-cytotoxic polyene antifungal antibiotic.     

Characterization of the P450 monooxygenase NysL, responsible for C-10 hydroxylation during biosynthesis of the polyene macrolide antibiotic nystatin in Streptomyces noursei

The nysL gene, encoding a putative P450 monooxygenase, was identified in the nystatin biosynthetic gene cluster of Streptomyces noursei. Although it has been proposed that NysL is responsible for hydroxylation of the nystatin precursor, experimental evidence for this activity was lacking. The nysL gene was inactivated in S. noursei by gene replacement, and the resulting mutant was shown to produce 10-deoxynystatin. Purification and an in vitro activity assay for 10-deoxynystatin demonstrated its antifungal activity being equal to that of nystatin. The NysL protein was expressed heterologously in Escherichia coli as a His-tagged protein and used in an enzyme assay with 10-deoxynystatin as a substrate. The results obtained clearly demonstrated that NysL is a hydroxylase responsible for the post-polyketide synthase modification of 10-deoxynystatin at position C-10. Kinetic studies with the purified recombinant enzyme allowed determination of K(m) and k(cat) and revealed no inhibition of recombinant NysL by either the substrate or the product. These studies open the possibility for in vitro evolution of NysL aimed at changing its specificity, thereby providing new opportunities for engineered biosynthesis of novel nystatin analogues hydroxylated at alternative positions of the macrolactone ring.     

An unexpected role for the putative 4'-phosphopantetheinyl transferase-encoding gene nysF in the regulation of nystatin biosynthesis in Streptomyces noursei ATCC 11455

The nysF gene encoding a putative 4'-phosphopantetheinyl transferase (PPTase) is located at the 5' border of the nystatin biosynthesis gene cluster in Streptomyces noursei. PPTases carry out post-translational modification of the acyl carrier protein domains on the polyketide synthases (PKS) required for their full functionality, and hence NysF was assumed to be involved in similar modification of the nystatin PKS. At the same time, DNA sequence analysis of the genomic region adjacent to the nysF gene revealed a gene cluster for a putative lantibiotic biosynthesis. This finding created some uncertainty regarding which gene cluster nysF functionally belongs to. To resolve this ambiguity, nysF was inactivated by both insertion of a kanamycin (Km) resistance marker into its coding region, and by in-frame deletion. Surprisingly, the nystatin production in both the nysF::Km(R) and DeltanysF mutants increased by ca. 60% compared to the wild-type, suggesting a negative role of nysF in the nystatin biosynthesis. The expression of xylE reporter gene under control of different promoters from the nystatin gene cluster in the DeltanysF mutant was studied. The data obtained clearly show enhanced expression of xylE from the promoters of several structural and regulatory genes in the DeltanysF mutant, implying that NysF negatively regulates the nystatin biosynthesis.     

Susceptibilities of Candida albicans Mouth Isolates to Antifungal Agents, Essentials Oils and Mouth Rinses

Forty Candida albicans strains isolated from patient's mouth with fixed orthodontic appliances were analyzed to their susceptibilities to antifungal agents, mouth rinses and essential oils. Susceptibility to fluconazole, econazole, miconazole and ketoconazole, amphotericin B and nystatin was assessed by the disk diffusion (DD) method based on the Clinical and Laboratory Standards Institute M44-A protocol, and by Etest (fluconazole and amphotericin B). The susceptibilities to mouth rinses and essential oils were also determined by the DD technique. All isolates tested were susceptible (S) to amphotericin B, nystatin and fluconazole. The overall concordance between the DD and the Etest was 100% for amphotericin and fluconazole. One isolate was resistant to econazole (2.5%) and the other to ketoconazole (2.5%). Econazole and ketoconazole had the highest percentages of susceptible dose dependent (SDD), 55 and 95%, respectively. Regarding to the susceptibility isolates profile, seven phenotypes were detected, and the 3 more represented (90% of the isolates) of them were SDD to one, two or three azoles. The study of mouth rinses showed a high variability of efficacy against C. albicans. The results showed that the isolates susceptibility to essential oils differed (P < 0.05). The profile activity was: cinnamon > laurel > mint > eucalyptus > rosemary > lemon > myrrh > tangerine. The main finding was that the susceptibility to cinnamon and laurel varied among the three more representative antifungal phenotypes (P < 0.05). The susceptibility of econazole-SDD isolates to cinnamon and lemon was higher than those of the econazole-S yeasts (P < 0.05). In contrast, econazole-SDD isolates were less affected by laurel than econazole-S counterparts (P < 0.05).     

Effects of the pore-forming agent nystatin on giant phospholipid vesicles

The effects of the polyene pore-forming agent nystatin were investigated on individual giant unilamellar phospholipid vesicles (GUVs), made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), in different methanol-water solutions using phase-contrast optical microscopy. Three characteristic effects were detected in three different nystatin concentration ranges: vesicle shape changes (between 150 and 250μM); transient, nonspecific, tension pores (between 250 and 400μM); and vesicle ruptures (above 400μM). Both the appearance of the transient tension pores and the vesicle ruptures were explained as being a consequence of the formation of size-selective nystatin channels, whose membrane area density increases with the increasing nystatin concentrations. Our results also show that nystatin is able to form pores in the absence of sterols. In addition, study of the cross-interactions between nystatin and methanol revealed mutually antagonizing effects on the vesicle behavior for methanol volume fractions higher than 10%.     

High-performance liquid chromatographic determination of liposomal nystatin in plasma and tissues for pharmacokinetic and tissue distribution studies

A reliable reversed-phase high-performance liquid chromatographic method was developed for the determination of liposomal nystatin in plasma. Nystatin is extracted by 1:2 (v/v) liquid–liquid extraction with methanol. Separation is achieved by HPLC after direct injection on a μBondapak™ C₁₈ analytical column with a mobile phase composed of 10 mM sodium phosphate, 1 mM EDTA, 30% methanol and 30% acetonitrile adjusted to pH 6. Detection is by ultraviolet absorbance at 305 nm. Quantitation is based on the sum of the peak area concentration of the two major isomers of nystatin, which elute at 7.5–8.5 and 9.5–10.5 min. The assay was linear over the concentration range of 0.05 to 50 μg/ml. The lower limit of quantitation was 0.05 μg/ml, sufficient for investigating the plasma pharmacokinetics of liposomal nystatin in preclinical studies. Accuracies and intra- and inter-day precision showed good reproducibility. With minor modifications, this method also was used for assaying nystatin in various non-plasma body fluids and tissues.