纳他霉素高产菌株的链霉素抗性选育及其发酵工艺的优化

用紫外线对纳他霉素(Natamycin)产生菌褐黄孢链霉菌(Streptomyces gilvosporeus)ATCC13326菌株孢子进行诱变处理后，利用链霉素抗性突变选育得122株链霉素抗性突变株。其中纳他霉素产量高于出发菌株的有13株。突变株SG-56的生产能力达到出发菌株的146％，研究了其发酵性能，优化了培养基组成和发酵工艺条件，纳他霉素产量为2．81g／L，较出发菌株产量提高了170％。     

尼莫克汀高产菌株诱变选育研究

目的 为了提高尼莫克汀产量以满足生产需求。方法 本试验以蓝灰链霉菌N20-69为出发菌株,探索了紫外(UV)诱变、常压室温等离子体(ARTP)诱变、亚硝基胍诱变、UV氯化锂复合诱变与ARTP缬氨酸氧肟酸复合诱变育种。结果 筛选到了11株高产突变株,均将出发菌株提高了20%以上,其中突变株NAX-96遗传性稳定,最适种龄为19 h。在500 L发酵罐发酵实验中,突变株NAX-96在生产能力上优势明显,尼莫克汀的产量达到3593μg/mL,比出发菌株N20-69(2477μg/mL)提高了45%。结论 研究结果对实际生产有重要指导作用。     

新型常压室温等离子体-紫外复合诱变选育埃莎霉素I高产菌株#br#

目的 通过对埃莎霉素Ⅰ产生菌WSJ-IA进行诱变选育研究,以期获得埃莎霉素Ⅰ高产菌株。方法 使用多功能等离子体诱变系统(multifunctional plasma mutagenesis system, MPMS)对出发菌株的孢子进行等离子体和紫外复合诱变,设定不同的诱变时间处理孢子悬液,通过致死率确定合适的诱变条件,利用突变株摇瓶发酵效价筛选出正突变菌株。结果 在MPMS射频功率为100W,处理距离5mm,气体流量12.5SLM,等离子体-紫外辐射时间为50s时,菌株致死率为96.08%。在此诱变条件下,以突变株的初筛效价为指标的突变率、正突变率分别达到63.96%和22.52%,复筛效价是出发菌株1.5倍以上的有5株,占复筛菌株的9%。最终筛选出一株发酵单位比出发菌株提高221%、埃莎霉素Ⅰ组分含量提高192%的正突变株IA-425。42L自动发酵罐发酵结果表明,该菌株埃莎霉素Ⅰ产量达到(2000±200)μg/mL左右。结论 新型等离子体复合紫外诱变方式,可有效提高菌株的埃莎霉素Ⅰ发酵产量和组分含量。这为埃莎霉素Ⅰ的大规模发酵和临床前研究奠定了良好基础。     

达托霉素生产菌(ATCC31568)的紫外诱变和抗生素抗性筛选

目的 建立一种提高达托霉素生产水平的方法.方法 以玫瑰孢链霉菌(ATCC31568)为出发菌株,利用紫外诱变结合抗生素抗性筛选以提升达托霉素生产水平.结果 通过对致死率和正突变率的考察,确定紫外诱变时间为45s,利福平和庆大霉素的合适筛选浓度分别为1.4μg/mL、1.0μg/mL,得到的突变株经摇瓶发酵后,采用高效液相色谱仪(HPLC)对最终发酵液进行检测.筛选到的突变株Ge-27最高产量达到79.44 mg/L,比原始菌株提高了42.2％.结论 采用紫外诱变结合抗生素抗性筛选得到了达托霉素合成水平提高的菌株.     

低能离子注入诱变达托霉素高产菌株及其发酵的研究(英文)

通过离子注入技术对达托霉素产生菌玫瑰孢链霉菌(Streptomyces roseosporus)Z0327进行诱变选育,使用N+作为离子源,注入能量为20keV,并研究了不同离子注入参数对菌株存活率的影响以及正负突变率的关系,初步探讨N+离子注入对达托霉素生产菌所产生的生物学效应。通过癸酸钠抗性筛选法获得多株遗传稳定性较好的高产突变株,其中突变株M1208发酵单位比出发菌株提高了206%,经发酵工艺研究,100L发酵终效价达到1720mg/L。     

60Co诱变选育平阳霉素高产株的研究

目的 筛选平阳霉素高产菌株。方法 以平阳霉素产生菌-平阳链霉菌为出发菌株,采用60C0诱变处理并通过遥瓶试验。结果 获得一株高产突变株,其产素能力达到出发菌株效价的1.86倍。传代试验表明该突变株的高产遗传特性稳定。结论     

紫杉醇产生菌美丽镰刀霉K1781的诱变育种

采用紫外线与亚硝酸对美丽镰刀霉(Fusarium mairei)K178的孢子进行复合诱变,于含40μg/ml制霉菌素的平板上进行筛选,获得31株突变株,其中5株的紫杉醇摇瓶发酵产量高于出发菌株,突变株UH23的紫杉醇产量为259.8μg/L,进一步优化其发酵工艺条件,紫杉醇产量达286.4pg/L,较出发菌株提高了29.9%.     

普那霉素产生菌的原生质体诱变育种

普那霉素产生菌始旋链霉菌（Streptomyces pristinaespiralis ）11．2在含0．5％甘氨酸的种子培养基中培养到对数生长期，收集菌丝体，经2mg／ml溶菌酶在30℃下作用90min可获得大量的原生质体，其再生率为5．1％。始旋链霉菌11.2原生质体经UV诱变并在含普那霉素的再生平板上筛选普那霉素抗性菌株，从中获得一高产：突变株始旋链霉菌ZP-07，普那霉素产量达到1．59g／L，比出发菌株提高101．3％。     

组合抗性筛选法选育达托霉素高产菌株

目的利用组合抗性筛选法选育达托霉素高产菌株。方法以玫瑰孢链霉菌(Streptomyces roseosporus ATCC 11379)为出发菌株,通过在不同浓度梯度的达托霉素和链霉素复合抗性平板上进行抗性筛选。结果筛选到一株高产达托霉素的突变株D1000-S3-2,经摇瓶发酵验证达托霉素发酵单位可达59mg/L,比出发菌株提高了63.8%。结论实验证明组合抗性筛选是一种简单高效筛选方法。     

纳他霉素产生菌的诱变育种及其发酵条件的优化

目的 以纳塔尔链霉菌(Streptomyces natalensis)SIPI-NHF-09为出发菌株进行诱变选育和培养条件优化,以提高其纳他霉素的产量.方法 采用紫外和微波结合对纳塔尔链霉菌SIPI-NHF-09进行诱变选育,并对获得的高产突变株SIPI-UW-10-22的发酵条件进行了初步优化.结果 经过连续3轮的紫外和微波诱变,获得高产突变株SIPI-UW-10-22,其纳他霉素(Natamycin)产量达到3.15g/L,是出发菌株的3.5倍,且该菌株经多次传代,遗传性能稳定.对SIPI-UW-10-22发酵条件优化后获得其发酵条件为:以5.5％玉米淀粉加0.5％葡萄糖为碳源,2％黄豆饼粉加1％酵母粉为氮源,种龄46h,初始pH7.0,接种量8％,摇床转速220r/min,28℃培养126h.此条件下,纳他霉素的产量达到4.07g/L,较优化前提高了1.29倍.结论 紫外与微波诱变相结合的方法简便有效,所获得高产菌株遗传性状稳定.     

普那霉素产生菌的推理选育

根据普那霉素的生物合成途径对普那霉索产生菌始旋链霉菌进行推理选育，原始出发菌株Streptomyces pristinaespiralis ATCC25486经过12次单菌落分离，其间经历5次UV诱变并分别筛选0．1％AA^r突变株、0．3％AA^r突变株、0．1％VH^r突变株、200u／ml KTM^R突变株、0．1％DOG^r突变株，获得了突变株S．pristinaespiralis 12-55，其生产能力较原始出发菌株S．pristinaespiralis ATCC25486提高了100倍，达到3000u／ml。传代试验表明普那霉素高产突变株S.pristinaespiralis 12-55的高产性能遗传特性稳定。     

螺旋霉素产生菌螺旋霉素链霉菌遗传育种

本文是利用抗生素抗性与产量之间的相关性来进行螺旋霉素产生菌螺旋霉素链霉菌的遗传育种,旨在获得高效价菌株,从而提高螺旋霉素的生产水平。首先用正交试验法确定出发菌株最佳发酵培养基,并在此基础上通过紫外线诱变,和在螺旋霉素浓度梯度平板上筛选螺旋霉素抗性菌株。按上述方法理性化筛选得到的菌株多为正突变株。从本实验得到一株螺旋霉素抗性菌株SPM~r-248,其抗性较出发菌株提高300％,生产能力较出发菌株提高81.3％。菌株SPM~r-248的高效价生产性能遗传特性稳定。在7m~3罐做发酵放大,与出发菌株相比,发酵效价提高31.4％,发酵指数提高18.8％,具有一定的学术意义和经济价值。     

埃坡霉素高产菌株的选育及发酵培养基的优化

以埃坡霉素产生菌粘细菌纤维堆囊菌为出发菌株,经亚硝基胍诱变处理后,在含有前体丙酸盐的平板中筛选抗性突变株,得到一株高产突变株N-19,埃坡霉素产量为18.5 mg/L,比出发菌株提高了69.7.通过优化发酵培养基的碳、氮源组成,埃坡霉素产量达36.1 mg/L,此时埃坡霉素B和埃坡霉素A的比值为0.4:在摇瓶发酵过程中补加前体丙酸盐,埃坡霉素B和埃坡霉素A的比值增至2.1.     

Characterisation of in vitro-selected mutants of Ureaplasma parvum resistant to macrolides and related antibiotics

Resistant mutants of Ureaplasma parvum were selected by serial passages of a susceptible strain in subinhibitory concentrations of different macrolides and related antibiotics (erythromycin, azithromycin, josamycin, quinupristin, quinupristin/dalfopristin, pristinamycin and telithromycin). Mechanisms of resistance were characterised by sequencing portions of genes encoding 23S rRNA and ribosomal proteins L4 and L22. Mutants with significantly increased minimum inhibitory concentrations could be selected with all the selector antibiotics, except quinupristin and pristinamycin. Mutants harboured mutations in domain V of the 23S rRNA gene at nucleotides G2056, G2057 or A2058 (Escherichia coli numbering) and in conserved portions of ribosomal proteins L4 and L22. Most of the mutations were associated with complete loss of macrolide and ketolide activity, whereas streptogramin combinations were less affected.     

Avermectin产生菌异亮氨酸诱导变种的选育

用紫外线处理ａｖｅｒｍｅｃｔｉｎ（ＡＶＭ）产生菌ＳｔｒｅｐｔｏｍｙｃｅｓａｖｅｒｍｉｔｉｌｉｓＸＣ１－２５，并在含有Ｌ－异亮氨酸（Ｌ－Ｉｌｅ）的平板培养基上筛选Ｌ－Ｉｌｅ诱导变种。结果表明：Ｉｌｅｉ变株发酵产生的ＡＶＭＢ１ａ效价高于自然分离株与紫外线诱变株，其中Ｉｌｅｉ变株ＸＣ２－２６的ＡＶＭＢ１ａ效价较亲株提高２２％。该变株经自然分离获得菌株ＸＣ３－８，其ＡＶＭＢ１ａ效价比出发菌株提高５０％以上，传代试验表明菌株ＸＣ３－８的形态和高产性能稳定，它在７ｍ３发酵罐生产试验，产生ＡＶＭＢ１ａ效价与发酵指数均比出发菌株提高５６％。     

利福霉素B产生菌─—地中海诺卡氏菌的推理选育

利福霉素Ｂ的生物合成受芳香族氨基酸（Ｔｒｐ、Ｔｙｒ、Ｐｈｅ）的反馈抑制。用紫外线处理Ｎ．ｍｅｄｉｔｅｒｒａｎｅｉＸＣ－１０２菌丝悬浮液后，并在含０．１％芳香族氨基酸的琼脂平板上分离芳香族氨基酸抗性变种。结果表明，芳香族氨基酸抗性变种的发酵效价高于自然分离株和紫外线诱变株；色氨酸抗性变种（Ｔｒｐｒ）的发酵效价又高于酪氨酸抗性变种（Ｔｙｒｒ）和苯丙氨酸抗性变种（Ｐｈｅｒ）；０．５％Ｔｒｐｒ变种比０．１％Ｔｒｐｒ变种更高产。其中０．５％Ｔｒｐｒ变种ＸＣ－５４０的生产能力较出发菌株ＸＣ－１０２增加４２．１６％。传代试验表明ＸＣ－５４０的遗传特性稳定，在７ｍ３罐做发酵放大，与出发菌株相比，发酵效价和发酵指数分别提高５１．１１％和５１．０２％。     

Mutagenesis of the rapamycin producer Streptomyces hygroscopicus FC904

Rapamycin (RPM) is produced by Streptomyces hygroscopicus FC904 isolated from soil in Fuzhou, China. It is a triene macrolide antibiotic with potential application as an immunosuppressant and drug for human gene therapy. In an attempt to improve rapamycin production, mutation and screening of the parent culture have been carried out. Thousands of survivors were obtained after mutagenesis by NTG (3 mg/ml) and UV (30 W, 15 cm, 30 seconds) of spore suspensions. None showed improved production of RPM. We determined the susceptibility to antibiotics of S. hygroscopicus FC904 by two fold dilutions of antibiotics in oatmeal agar plates. It was found that the strain was resistant to penicillin, erythromycin, RPM, tetracycline and chloramphenicol, but susceptible to mitomycin C (MIC, 10 microg/ml) and aminoglycosides such as gentamicin (MIC, 0.1 microg/ml), kanamycin (MIC, 0.1 microg/ml) and streptomycin (MIC, 0.3 microg/ml). Protoplasts of strain FC904 were prepared after finding the best conditions for their formation. They were treated with gentamicin, erythromycin, mitomycin C and NTG. Surprisingly, gentamicin was especially effective for obtaining higher RPM-producing mutants. Mutant C14 was selected by exposing the protoplasts of the parent strain FC904 to 1 microg/ml of gentamicin at 28 degrees C for 2 hours. A higher RPM-producing mutant (C14-1) was obtained from the protoplasts of mutant C14 treated with gentamicin, and its titer was 60% higher than that of the parent strain FC904 by HPLC analysis. Another improved mutant (C14-2) was obtained from the spores of mutant C 14 treated with 1 microg/ml of gentamicin plus 2 mg/ml of NTG at 28 degrees C for 2 hours. Mutant C14-2 had a titer 124% higher than FC904. The possible mechanism for the effect of gentamicin by using protoplasts or spore suspensions will be discussed, i.e. the possibility of gentamicin being a mutagen or a selective agent.     

多杀菌素产生菌复合诱变选育及发酵培养基优化

目的 利用诱变结合抗性筛选方法选育多杀菌素高产菌株,并通过发酵培养基优化进一步提高多杀菌素产量。方法 分别确定链霉素、安普霉素和鼠李糖3种抗性的最小抑菌浓度(minimal inhibitory concentration, MIC),然后以S.s1-4为出发菌株,通过紫外(UV)结合链霉素、安普霉素、鼠李糖抗性因子诱变选育,在此基础上利用亚硝基胍(NTG)结合上述抗性因子诱变选育,并利用响应面实验设计对发酵培养基中葡萄糖、糊精、棉籽蛋白3种成分进行优化。结果 出发菌株经过紫外照射30s,涂布于抗性平板上,筛选得到S.s2-21,S.s2-21再用NTG处理30min,涂布于抗性平板上,最终获得1株遗传性状稳定的菌株S.s3-37,产量为78.26mg/L,提高了45.71%;发酵培养基优化后,其产量达83.00mg/L。结论 利用紫外和NTG结合抗性复合诱变选育获得多杀菌素高产菌株是有效的,通过发酵培养基优化,其产量较出发菌株提高了54.55%,获得良好的效果。     

Comparative effects of melatonin, zolpidem and diazepam on sleep, body temperature, blood pressure and heart rate measured by radiotelemetry in Wistar rats

RATIONALE: The role of melatonin (MLT) in mediating the sleep-wake cycle has been previously suspected of indicating that this substance could be a candidate for a new generation of hypnotics. OBJECTIVES: We investigated whether MLT acted as a sleep promoter or a modulator of sleep temporal timing related to cardiovascular and body temperature (Tb) adaptations to sleep induction. The pharmacological effects of MLT on sleep were compared with zolpidem (ZP) and diazepam (DZ). METHODS: The radiotelemetry system was used to record the electrocorticogram [slow wave sleep (SWS), paradoxical sleep (PS)], Tb, blood pressure and heart rate in six Wistar rats. DZ (3 mg/kg and 6 mg/kg), ZP (1, 3, 5 and 10 mg/kg) and MLT (2.5 and 5 mg/kg) were delivered intraperitoneally during light (L) and dark (D) periods. RESULTS: MLT increased the number of sleep cycles (L: 30%, D: 110%) and total duration (P<0.05) of PS (L: 70%, D: 150%). In return, ZP (10 mg/kg) presented no effect during L but increased total (40%) and mean duration (37%) of SWS during the D period. DZ modified mean duration of SWS (L: -27%, D: +26%) and increased total duration of SWS (+47%). ZP and DZ induced a more pronounced decrease in Tb than MLT but only DZ induced tachycardia and hypertension. CONCLUSIONS: We showed that MLT could not promote sleep and its cardiovascular adaptations despite hypothermia, but modulated the period of ultradian sleep cycles. DZ and ZP promoted sleep and induced hypothermia during the D period. Only DZ disrupted sleep architecture and induced adverse effects on cardiovascular parameters.