微生物转化左旋四氢巴马汀产生左旋紫堇达明高产菌株的紫外诱变选育

紫堇达明为中药元胡中高效、微量活性成分,灰色链霉菌(Streptomyces griseus ATCC 13273)具有转化左旋四氢巴马汀产生左旋紫堇达明的生物转化活性。本研究采用紫外诱变法对原始菌株进行诱变处理,以期获得左旋紫堇达明高产突变菌株。正交试验结果显示:孢子浓度为107个/mL,照射距离为30 cm,照射时间为30 s为最佳诱变条件。筛选得到的突变株UV-056对底物的转化率达到62.58%,左旋紫堇达明转化产率达到33.12%,分别较出发菌株提高了51.00%和67.63%。检测表明,突变株经连续传代后仍可保持较好的遗传稳定性。     

HPLC法测定盐酸左旋咪唑糖浆的含量

目的建立高效液相色谱法测定盐酸左旋咪唑糖浆含量的方法。方法色谱柱为Agilent-C18（250mm×4．6mm,5μm）;流动相为0．025mol／L枸橼酸（用三乙胺调节pH值为4．5）-乙腈（85：15）;检测波长为277nm;流速为1．0ml／min。结果盐酸左旋咪唑在5—100μg／ml范围内有良好的线性关系（r=0．9998）,平均回收率（n=9）为100．9％（RSD=1．4％）。结论本法简单、快速、准确,可用于盐酸左旋咪唑糖浆的定量分析。     

HPLC-ECD法测定复方甲哌卡因注射液中左旋异肾上腺素的含量

目的：建立HPLC—ECD法测定复方甲哌卡因注射液中左旋异肾上腺素含量的方法。方法：采用Venusil XBP C18色谱柱（150mm×4．6mm,5μm）,流动相为醋酸盐缓冲液（取冰醋酸50ml,庚烷磺酸钠1．2g,乙二胺四醋酸二钠0．04g,加水930ml,用2mol·L^-1的氢氧化钠溶液调节pH至3．4,加水稀释至1000ml）-甲醇（90：10）;柱温20～25℃;流速为1．0ml·min^-1;电化学检测器检测。结果：左旋异肾上腺素在0．2～10μg·ml^-1范围内线性关系良好,r=1．0000（n=6）;平均回收率为100．9％,RSD0．6％（n=9）。结论：方法快速,准确,灵敏,可作为注射液中微量肾上腺素类药物的测定方法。     

甲磺酸左旋氧氟沙星治疗泌尿道感染临床疗效及安全性

目的：研究甲磺酸左旋氧氟沙星对泌尿道感染的治疗作用及安全性。方案：对30例细菌性泌尿道感染患者静脉滴注甲磺酸左旋氧氟沙星400mg/d,疗程5－7d；对70例泌尿道手术患者术后预防性静脉滴注甲磺酸左旋氧氟沙星400mg/d，疗程5d。结果：治愈率86．7％，有效率93．3％，只有2例（2％）出现了轻度的皮疹；2例（2％）出现肝功能轻度升高反应。结论：甲磺酸左旋氧氟沙星不仅适用于泌尿道感染，而且安全，不良反应少。     

阿霉素产生菌突变株H6125转化研究

本文探讨了不同培养条件下,菌龄和再生温度对链霉菌质粒转化阿霉素突变株H6125的影响。结果证明,利用改良的R_2YE培养基在适宜的条件下可使pH702和PH922的转化率分别达到5×10~5/μg DNA和0.6×10~5/μg DNA。转入质粒的H6125遗传表型发生改变,其发生机理尚不清楚。     

反相高效液相色谱法测定左旋氧氟沙星血药浓度

本文建立了反相高效液相色谱法测定Levofloxacin（左旋氧氟沙星）的血药浓度方法，样品经液－液提取后，C(18)柱上分析，流动相为甲醇：0．01M磷酸盐缓冲液：0．5M四丁基溴化铵，紫外检测波长294nm，线性范围0．5～4．0μg／ml，平均回收率为97．17％，日内误差＜7％，日间误差＜8％，本法具有简便、灵敏、快速、准确的优点，已用于药代动力学的研究。     

左旋氧氟沙星治疗尿路感染82例

目的：为观察左旋氧氟沙星对尿路感染的疗效,选择146例患者,均有尿频、尿急、尿病等膀胱刺激症状,尿常规检查WBC＞10个／HP,清洁尿中断尿培养均阳性,菌落计数＞10CFU／ml。方法：随机分两组,治疗组82例,给予左旋氧氟沙星0．2g／次,po,bid,严重感染或绿脓杆菌等所致感染者,剂量增至0．3g／次;对照组64例,给予氧氟沙星0．6g／d,Po,tid,两组疗程均7～14d。结果：治疗组和对照组总有效率分别为93．9％（77／82）和90．6％（58／64）;痊愈率为84．1％（69／82）和85．9％（55／64）;膀胱刺激症状好转或消失时间为（2．5±1．1）和（2．8±1．0）d。经Radii检验差异不显著（U=0．14,P＞0．05）。细菌清除率分别为92．6％（76／82）和90．6％（58／64）,经X检验亦差异不显著（X=0,P＞O．05）。两组均未见严重不良反应发生。结论：主旋氧氟沙星对尿路感染有效,安全可靠。     

HPLC法测定盐酸左旋咪唑糖浆的含量

目的：采用高效液相色谱法测定盐酸左旋咪唑糖浆的含量。方法：采用Agilent TC—C18色谱柱（4．6mm×150mm，5μm）；以0．05mol·L^-1磷酸二氢钾溶液（二乙胺调节pH7．0）-乙腈（60：40）为流动相；流速1．0mL·min^-1；检测波长为215nm；柱温：室温。结果：盐酸左旋咪唑线性范围为5．14～25．70μg·mL^-1（r=0．9998），平均回收率（n=6）为98．3％，RSD为0．56％。结论：本方法简便、准确、重现性好，可用于测定盐酸左旋咪唑糖浆的含量。     

左旋泮托拉唑钠肠溶片的制备及其稳定性考察

目的：制备左旋泮托拉唑钠肠溶片并考察其稳定性。方法：以3因素3水平（碳酸钠的含量0,5％，10％；黏合剂的种类MC，HPMC和PVP；浓度2％，5％，8％）的正交试验设计进行片芯处方筛选，并参考中国药典2000年版附录考察了制剂的稳定性。结果：确定了以聚乙烯吡咯烷酮（PVP）为黏合剂、10％碳酸钠为稳定剂的处方，恒温加速试验及长期留样试验6个月，含量及有关物质未见明显改变。结论：本法制备肠溶片的处方工艺简便，易于控制和操作。     

高效液相色谱法测定复方枣仁胶囊中左旋延胡索乙素含量

目的建立测定复方枣仁胶囊中左旋延胡索乙素含量的高效液相色谱（HPLC）法。方法采用Discovery C18（150mm×4．6mm,5／xm）,以甲醇-0．1％磷酸（63：37,pH=6．0）为流动相,检测波长为281nm,流速为1．0mL／min,柱温为30℃。结果左旋延胡索乙素质量浓度在63．52～635．2μg／mL范围内与峰面积线性关系良好,r=0．99998（n=7）,平均回收率为99．93％,RSD为0．7％（n=6）。结论HPLC法灵敏、快捷、准确,可用于复方枣仁胶囊中左旋延胡索乙素含量的测定。     

Streptomyces griseus ATCC 13273对去氢骆驼蓬碱的转化研究

利用微生物转化的方法,选取灰色链霉菌(Streptomyces griseus ATCC 13273)为转化菌株,对去氢骆驼蓬碱的转化进行研究。该菌株可以将去氢骆驼蓬碱转化为两种转化产物(记为H1和H2),通过MS、1H-NMR和13C-NMR分析,转化产物分别鉴定为哈尔酚和N-羟基去氢骆驼蓬碱。并对转化条件进行初步优化,优化后的转化条件为:初始pH 8.0,两步活化法以4%的接种量转接,48 h后加入底物(以N,N-二甲基甲酰胺溶解),发酵液中去氢骆驼蓬碱终浓度为0.25 mmoL/L,发酵时间为5 d。转化条件优化后H1和H2的产率分别达到8.70%和12.76%;该研究丰富了S.griseus ATCC 13273的作用底物范围,为哈尔酚和N-羟基去氢骆驼蓬碱的合成提供了一条新途径。     

Microbial transformations and 13C-NMR analysis of colchicine

Several microorganisms were screened for their ability to biotransform colchicine, and two were selected for preparative scale fermentations. Streptomyces spectabilis and Streptomyces griseus both produced O2-demethylcolchicine and O3-demethylcolchicine but in different amounts. The 13C-NMR assignments of colchicine, O10-demethylcolchicine, and trimethylcolchicinic acid are reported and were used to help identify the structures of the metabolites.     

北极放线菌BF-1发酵条件优化及代谢产物抑菌活性研究

目的优化发酵工艺以提高北极放线菌BF-1发酵液中抑菌活性物质的产量;测定BF-1次级代谢产物的体外抑菌活性。方法以发酵液抑菌活性为指标,采用单因素实验和正交实验对放线菌BF-1发酵培养基和发酵条件进行优化;琼脂稀释法测定BF-1发酵液最低抑菌浓度。结果最佳发酵培养基:淀粉5g.L-1,NH4Cl 5g.L-1,黄豆15g.L-1,MgSO40.25g.L-1,海水晶30g.L-1;最佳发酵条件:28℃,起始pH7,接种量5%;BF-1发酵液对绿脓杆菌的最低抑菌浓度(MIC)为640μg.mL-1。结论北极放线菌BF-1发酵液中次级代谢产物具有显著的体外抑菌活性,优化后BF-1发酵液的抑菌活性与优化前相比提高了约2.6倍。     

原生质体诱变及高通量筛选选育链霉素高产菌株

目的 通过诱变和筛选方法的研究，提高灰色链霉菌(Streptomyces griseus)生产链霉素的水平。方法 优化灰色链霉 菌的原生质体的生成和再生条件，并对得到的原生质体进行紫外诱变，然后利用微孔板高通量方式对获得菌株进行筛选。结果 经过诱变选育获得一株菌NP-11703，其链霉素产量在100L罐上比出发菌株提高了21.8%。结论 用紫外诱变原生质体，可以有 效提高灰色链霉菌产链霉素的能力。结合高通量筛选模型的应用，可大大加快高产菌株的筛选效率。     

Production and optimization of oxytetracycline by a new isolate Streptomyces rimosus using response surface methodology

A new microbial strain showing broad spectrum antibacterial and antifungal activity was isolate from soil of Chhattisgarh and characterized as Streptomyces rimosus MTCC 10792 (gene sequence similarity 99.52%). The antibacterial compound was produced by the isolate purified by silica gel chromatography and chemically characterized as oxytetracycline and production of the antibiotic was statistically optimized using response surface methodology. The three independent variables, namely concentrations of glucose (10?g/l), soybean meal (10?g/l), and calcium carbonate (1?g/l) were found to be the most important for production antibiotic by a one-factor-at-a-time study. For optimization, the individual and interaction effects of the studied variables were evaluated by response surface methodology (RSM) using central composite design (CCD). Antibiotic production was increased nearly ten times (470?mg/l) as compared with the normal unoptimized production medium (47?mg/l) by applying statistical design.     

Microbial specific carboxylation of (–)-huperzine A by Streptomyces griseus

In a scale-up microbial transformation of (-)-huperzine A (1) by Streptomyces griseus CACC 200300, a specific carboxylated derivative (2)was yielded together with two known hydroxylated metabolites. The structure of 2 was characterized as 16-carboxyl huperzine A on the basis of IR, HRMS and NMR spectroscopic data analysis.     

New antibiotic-producing streptomycetes, selected by antibiotic resistance as a marker. I. New antibiotic production generated by protoplast fusion treatment between Streptomyces griseus and S. tenjimariensis

A novel antibiotic was found after performing an interspecific fusion treatment between Streptomyces griseus and S. tenjimariensis by the selection of clones with a unique antibiotic resistance. Nonantibiotic-producing mutants of streptomycin (SM)-producing S. griseus SS-1198 with resistance to SM and istamycin (IS)-producing S. tenjimariensis SS-939 with resistance to kanamycin (KM) were protoplasted, mixed with polyethyleneglycol and regenerated. Resistant clones to both SM and KM were found among spores of the regenerated culture at a frequency of 10(-6). Their growth appearance was identical with that of S. griseus. Antibiotic productivity was found only in clones resistant to both 20 approximately 50 micrograms/ml of KM and 400 micrograms/ml of SM. The antibiotic produced by a selected strain, SK2-52, proved to be different from SM and IS.     

New antibiotic-producing streptomycetes, selected by antibiotic resistance as a marker. II. Features of a new antibiotic-producing clone obtained after fusion treatment

A new antibiotic-producing Streptomyces strain SK2-52 obtained by a protoplast fusion treatment between Streptomyces griseus NP1-1 and S. tenjimariensis NM16 showed taxonomical features identical with those of S. griseus. The strain resistant to wider range of aminoglycoside antibiotics than the parental strains. This multiple resistance corresponded to the activities of streptomycin kinase and acetyltransferase which were probably derived from S. griseus NP1-1. Clones with fast-growth and reduced antibiotic productivity frequently segregated from strain SK2-52, while their antibiotic resistance was stable. The results suggest that the fusion treatment caused a genetic change in S. griseus which enhanced the expression of genes for unique multiple resistance to aminoglycoside antibiotics and also induced new antibiotic production.     

Structures of grixazone A and B, A-factor-dependent yellow pigments produced under phosphate depletion by Streptomyces griseus

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) acts as a microbial hormone that induces morphological development and secondary metabolism in Streptomyces griseus. A diffusible yellow pigment is produced by S. griseus in an A-factor-dependent manner under phosphate depletion. Detailed analysis of the pigment production by S. griseus cultivated in minimal liquid medium containing different concentrations of phosphate showed that the pigment was actively produced in the presence of low concentrations of phosphate and the production of the pigment was completely repressed in the presence of 2.5 mM KH2PO4. HPLC analysis of the culture supernatant showed that the pigment consisted of two major, structurally related compounds and they were produced at different ratios depending on the concentration of phosphate in the medium. The structures of the two major compounds, designated as grixazone A and B, were determined by spectroscopic analyses as 1-[[2-(acetylamino)-2-carboxyethyl]thio]-2-amino-3-oxo-8-formyl-3H-phenoxiazine and 1-[[2-(acetylamino)-2-carboxyethyl]thio]-2-amino-3-oxo-8-carboxyl-3H-phenoxiazine, respectively. Grixazone A was a novel compound, although grixazone B was reported in a patent as a parasiticide produced by Streptomyces sp. DSM3813.