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第九章：抗生素的生产

第九章：抗生素的生产. 王雪青 2003.12.15. 抗生素的生产. 概述 抗生素的分类 抗生素的应用 半合成抗生素 青霉素的生产和提取工艺. 抗生素的生产. 概述. 定义：是生物在其生命过程中产生的，并在低浓度下有选择性地抑制或杀灭其它微生物或肿瘤细胞的有机物质。 Microbial Products,effective at low concentrations,which have the ability to kill or inhibit the growth of certain micro-organisms. History:

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第九章：抗生素的生产

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  1. 第九章：抗生素的生产 王雪青 2003.12.15

  2. 抗生素的生产 • 概述 • 抗生素的分类 • 抗生素的应用 • 半合成抗生素 • 青霉素的生产和提取工艺

  3. 抗生素的生产 概述 • 定义：是生物在其生命过程中产生的，并在低浓度下有选择性地抑制或杀灭其它微生物或肿瘤细胞的有机物质。Microbial Products,effective at lowconcentrations,which have the ability to kill or inhibit the growth of certain micro-organisms. • History: 19世纪70年代，法国Pasteur,发现有些微生物可以抑制炭疽杆菌，提出了利用微生物抑制另一种微生物的现象来治疗一些感染性疾病。1928年，

  4. In 1928 Scottish bacteriologist Alexander Fleming reported the antibacterial action of cultures of a Penicillium species • He observed that Penicillium notatum killed some of his Staph. aureus culture dishes • Discovery by accident? Culture dishes contaminated when left out for two weeks while Fleming was on holiday, instead of being incubated

  5. Fleming noticed that the substance secreted was an inhibitor of many bacterial species • He published a paper on the active ingredient which he called ‘penicillin’. This was the first demonstration that a substance produced by micro-org’s could inhibit or kill other microbes • Fleming could not isolate penicillin. 10 yrs later scientists Florey and Chain successfully isolated penicillin. • 1943-1945，scale-up production of pencillium. By 1945,enough penicillin was produced to treat 7 million per year.

  6. Alexander Fleming • Discoverer of penicillin • Born into a farming family in Scotland, moved to London at 13 • Awarded Scholarship to study medicine

  7. Zone of Inhibition • This picture illustrates the effect of penicillin on a fungus. Note the clear ring of inhibition. It was this observation which initiated Fleming’s further studies.

  8. Howard Florey • Australian-born • Studied Science & Medicine in Adelaide • Awarded Scholarship at Oxford University • Returned to England to complete a PhD

  9. Ernst Chain • Of Russian- German descent • Graduated in Chemistry and Physiology • Left Berlin when Nazi’s came to power • Hired by Florey to work on penicillin project

  10. 到目前为止，有8000多种抗生素，2/3是由放线菌（strepomyces）产生的，并通过化学结构的改造，总共有3万多种半合成抗生素，它们的分子量为100-1200，普遍应用的包括半合成和盐类的抗生素有350多种，其中以青霉素、头孢霉素、四环素类、氨基糖苷类和大环内脂类到目前为止，有8000多种抗生素，2/3是由放线菌（strepomyces）产生的，并通过化学结构的改造，总共有3万多种半合成抗生素，它们的分子量为100-1200，普遍应用的包括半合成和盐类的抗生素有350多种，其中以青霉素、头孢霉素、四环素类、氨基糖苷类和大环内脂类

  11. 主要抗生素的使用量（Harvey, 1998)

  12. Some Clinically Important Antibiotics

  13. 抗生素的分类 根据抗生素的来源分 放线菌产生的，占目前发现的抗生素的2/3。真菌产生的，主要包括青霉菌和头孢菌素。细菌产生的，多粘杆菌，枯草杆菌，芽孢杆菌产生的粘多菌素。动植物产生的，蒜中的蒜素，动物脏器中的鱼素（ekmolin). 根据作用谱分: 广谱抗生素，如氨苄青霉素，可抑制G-&G+细菌 抗G+菌的抗生素：如青霉素。 抗G-菌的抗生素：如链霉素 抗真菌的抗生素：如制霉菌素 抗病毒的抗生素：如四环类抗生素对立克次体积较大病毒有一定的作用。 抗癌抗生素：如阿霉素（Adriamycin) 目前在抗病毒、抗癌、等方面还没有理想的抗生素。 根据抗生素的结构分类 以β-内酰胺类抗生素：包括其青霉素、头孢菌素类等。它们含有一个四元内酰胺环，为目前最受重视的一类。

  14. Penicillin Structure B-内酰胺是抗生素具有抗菌活性所必需的，而R基团则赋予抗生素的稳定性、抗菌谱等的变化。

  15. 氨基糖苷类抗生素：包括链霉素、庆大霉素含有氨基糖苷类或氨基环醇氨基糖苷类抗生素：包括链霉素、庆大霉素含有氨基糖苷类或氨基环醇 四环类抗生素：四环素类、土霉素以四个苯为母核。 多肽类抗生素：多粘菌素、杆菌肽由产孢子杆菌产生。 蒽环类抗生素：阿霉素、柔红霉素属于抗癌类抗生素。 喹诺酮类抗生素，如环丙沙星、诺氟沙星。 按照作用机制分类 抑制细胞壁合成的抗生素 影响细胞膜功能的抗生素 抑制病原菌蛋白质合成的抗生素，如四环素 抑制核酸合成的抗生素如丝裂霉素C 抑制生物功能作用的抗生素，

  16. 根据抗生素的生物合成途径分类 氨基酸、肽类衍生物，如青霉素、头孢霉素等寡肽抗生素 糖类衍生物，如链霉素等糖苷类抗生素 以乙酸、丙酸为单位的衍生物， 如红霉素等丙酸衍。生物

  17. 抗生素的应用 对抗生素的评价：有较大的差异毒力 在体内发挥其抗生效能，而不被血液、脑肾液等破坏，不大量与血清蛋白质产生不可逆结合。 给药后吸收快，并在被感染器官或组织中分布 致病菌对抗生素不易产生耐药性 不引起过敏反应 理化性质稳定，便于提取、制剂和贮藏。 抗生素的计量单位： 效价单位：青霉素效价单位为能在50ml肉汤培养基中完全抑制金黄色葡萄球菌菌株发育的最小计量。

  18. 一个链霉菌效价单位为能在1ml肉汤培养基中完全抑制大肠杆菌标准菌株所需的最小链霉菌素计量。一个链霉菌效价单位为能在1ml肉汤培养基中完全抑制大肠杆菌标准菌株所需的最小链霉菌素计量。 除了用效价单位之外，当物质被制成纯净的化学物质时，就可以用质量表示，而且与效价单位之间有一定的换算关系 如1mg青霉素钠盐相当于1667个单位 1 mg链霉菌素碱相当于1000个单位

  19. Industrial evolution of penicillin production

  20. Common antibiotics and their sources • Bacitracin • Cephalosporin(s)（头孢菌素） • Chloramphenicol（氯霉素） • Cycloheximide（放线菌酮） • Hygromycin • Penicillin • Streptomycin • Tetracycline(s) • Vancomycin • Bacillus subtilis • Cephalosporium sp. • S. venezuelae • S. griseus • S. hygromyces • P. chrysogenum • S. griseus • S. aurofaciens • S. orientalis

  21. Beta-Lactam Structure

  22. 半合成抗生素 用化学或生物化学的方法改变一直抗生素的化学结构或引入特定的功能基团而获得的新抗生素品种或其衍生物的总称。 其目的：减低毒副作用，增强抗菌力，扩大抗菌谱，对耐药菌有效 ，便于口服，改善药理性质，提高生物有效性。

  23. STRUTURE OF PENICILLIN

  24. What is a semi-synthetic variant of penicillin??? • Discovered that natural penicillins can be chemically modified by removing the R group and adding a new one that confers new properties • It is the variation in the R group that leads to the semi-synthetic forms of penicillin. • Over 20,000 semi-synthetic variants • 3 main variants of industrial importance

  25. Main Variants  Ampicillin（氨卡青霉素）  Methicillin  Amoxycillin

  26. Methicillin(甲氧霉素)1986（13%，1998，26%） • Originally introduced to combat S.Aureus • B-Lactamase resistant • 1960’s a semi-synthetic broad spectrum compound produced • Over production of beta-lactamase enzyme has been shown to contribute to methicillin resistance.

  27. Ampicillin（氨卡青霉素） • Discovered in 1963 • One of only few penicillins that can be administered orally • Has improved acid stability • Broad spectrum.

  28. Amoxycillin（羟氨卡青霉素） • The preffered choice of drug for oral use. • The highest absorbance levels in the body • First marketed in 1998 by Smitkline Beechams™

  29. Benefits of semi-synthetic variants • Can be administered orally as they are resistant to stomach acid • They have a broader spectrum of activity • The semi-synthetic variant of penicillin is chemically more stable than the volatility of the B-Lactam ring, in the penicillin G

  30. 6-APA（6-氨基青霉烷酸） •First discovered in 1959 • First industrial process of enzyme engineering •Conversion of Pen G to 6-APA • Used in penicillin recovery process

  31. Action of 6-APA • PGA（青霉素酰化酶） is the natural substrate for Pen. G • Penicillin G is hydrolysed in a reversible reaction to 6-APA and phenylacetic acid. • Vital intermediate for the production of semi-syntheic penicillins

  32. Acyl Group • Nature of acyl group confers certain properties on the penicllin analogue • Selective hydrolysis occurs by hydrolysis by lactamase-producing culture

  33.  lactamase • These are the most common cause of bacterial resistance to beta lactam antibiotics • Most important  lactamase was produced by S. aureus. This was responsible for rise in resistance to penicillin in the 1950’s • Beta Lactam ring is crucial for penicillin to remain active

  34. Action of  lactamase •  lactamase destroys penicillin by hydrolyzing a bond in this ring

  35. Some Statistics • 10,985 tonnes of 6-APA were produced in 1998 • Of the 10,985, 48% was used for ampicilllin production, and 27% for amoxicillin.

  36. Resistance • Linked to overuse of a specific agent or to the introductiob of a new class of  lactam • By the late 1970’s fewer than 10% of Staphlococcus infections could be cured by penicillin，到1999年，WHO报道95%的金黄色葡萄球菌抗青霉素，100%大肠杆菌抗链霉素

  37. Conclusion • Semi-synthetic variants are now the favoured choice of antibitic for combating bacterial resistance • Can be orally administered • They have a broadened range of specifity • More chemically stable than their native counterpart.

  38. 抗生素的生产和提取工艺 孢子制备 菌种 种子制备 发酵 发酵液的预处理 提取和精制 成品包装 现代抗生素工业生产工艺

  39. Configuration & Production • Reactors must be correctly designed if the fermenting organisms potential is to be achieved • An understanding of the impact of configuration on production can help highlight suitable designs

  40. Building a Bioreactor The most important design features to consider include: • Construction Material • Capacity • Impellar Design • Heat Exchanger • Sparger • Analytical Instrumentation

  41. The Optimum Design • Construction Material Typically stainless steel as it’s cheap, light, readily available & easy to sterilize • Capacity Industrial designs hold between 30-200m3 offluid,must however consider impact on cost & environmental control

  42. Geometry Increased height to diameter ratio is favored as it aids agitation & aeration of viscous antibiotic broths • Impellar Rushtons are standard, but axial designs also possible. Low rotational speeds minimize shear & increased impellar diameter compensates for reduced agitation

  43. Axial & Radial Impellars

  44. Different Impellar Designs

  45. Cold H2O In • Heat Exchanger Heat generated by biological reactions & agitation is removed by cooling jackets. Limpet, or half pipe, is common choice • Sparger C-shaped preferable to single pipe, as they improve O2 transfer in the viscous broths Warm H2O Out

  46. Analytical Instrumentation Probes for measurement of temp, pH glucose & DO conc. are fitted at various points around the vessel • Optimum temp between 24-280C • Alkaline pH must be reduced to neutrality • Glucose must be monitored to prevent catabolite repression • DO must sustain cells but promote fermentation

  47. 抗生素的提取 • 一般用溶媒体取法：利用抗生素在不同的ph条件下的化学状态（游离酸，碱或盐）存在时，在水及与水互不相容的溶媒中溶解度不同的特性，使抗生素从一液相转移到另一液相中（如有机溶剂），已达到浓酸和提纯的目的。

  48. Downstream Processing

  49. Downstream Processing Steps in Downstream Processing: • Removal of Insolubles -Filtration • Isolation of product - Solvent Extraction • Purification - Precipitation, Electrophoresis • Polishing - crystallisation, drying

  50. Filtration • Penicillin G is in solution exocellularly • Remove cells by filtration • Sterile conditions must be utilised to avoid contamination of filtrate with B-lactamase producing micro-organisms.

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