1、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,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: 19世纪70年代，法国Pasteur,发现有些微生物可以抑制炭疽杆菌
2、，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，,概述,In 1928 Scottish bacteriologist Alexander Fleming reported the antibacterial action of cultures of a Penicillium speciesHe observed that Penicillium notatum killed some of his Staph. aureus culture dishesDiscovery by accident? Culture dishes contaminated when
3、 left out for two weeks while Fleming was on holiday, instead of being incubated,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
4、 by micro-orgs 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.,Discoverer of penicill
5、in Born into a farming family in Scotland, moved to London at 13 Awarded Scholarship to study medicine,Alexander Fleming,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 Flemings further studi
6、es.,Howard Florey,Australian-born Studied Science & Medicine in Adelaide Awarded Scholarship at Oxford University Returned to England to complete a PhD,Ernst Chain,Of Russian- German descent Graduated in Chemistry and Physiology Left Berlin when Nazis came to power Hired by Florey to work on penicil
7、lin project,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,主要抗生素的使用量（Harvey, 1998),Some Clinically Important Antibiotics,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
8、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-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。,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
9、谱等的变化。,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，,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。
10、生物,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。,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1
11、667个单位 1 mg链霉菌素碱相当于1000个单位,Industrial evolution of penicillin production,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. venezuelaeS. g
12、riseus S. hygromyces P. chrysogenum S. griseus S. aurofaciens S. orientalis,Beta-Lactam Structure,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。,STRUTURE OF PENICILLIN,What is a semi-synthetic variant of penicillin?, Discovered that na
13、tural 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,Main Variants, A
14、mpicillin（氨卡青霉素） Methicillin Amoxycillin,Methicillin(甲氧霉素) 1986（13%，1998，26%）,Originally introduced to combat S. Aureus B-Lactamase resistant 1960s a semi-synthetic broad spectrum compound produced Over production of beta-lactamase enzyme has been shown to contribute to methicillin resistance.,Ampic
15、illin（氨卡青霉素）,Discovered in 1963One of only few penicillins that can be administered orally Has improved acid stability Broad spectrum.,Amoxycillin（羟氨卡青霉素）,The preffered choice of drug for oral use. The highest absorbance levels in the bodyFirst marketed in 1998 by Smitkline Beechams,Benefits of semi
16、-synthetic variants,Can be administered orally as they are resistant to stomach acidThey have a broader spectrum of activityThe semi-synthetic variant of penicillin is chemically more stable than the volatility of the B-Lactam ring, in the penicillin G,6-APA（6-氨基青霉烷酸）, First discovered in 1959 First
17、 industrial process of enzyme engineering Conversion of Pen G to 6-APA Used in penicillin recovery process,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
18、-syntheic penicillins,Acyl Group,Nature of acyl group confers certain properties on the penicllin analogue Selective hydrolysis occurs by hydrolysis by lactamase-producing culture, lactamase,These are the most common cause of bacterial resistance to beta lactam antibiotics Most important lactamase w
19、as produced by S. aureus. This was responsible for rise in resistance to penicillin in the 1950s Beta Lactam ring is crucial for penicillin to remain active,Action of lactamase, lactamase destroys penicillin by hydrolyzing a bond in this ring,Some Statistics,10,985 tonnes of 6-APA were produced in 1
20、998Of the 10,985, 48% was used for ampicilllin production, and 27% for amoxicillin.,Resistance,Linked to overuse of a specific agent or to the introductiob of a new class of lactamBy the late 1970s 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，10
21、0%大肠杆菌抗链霉素,Conclusion,Semi-synthetic variants are now the favoured choice of antibitic for combating bacterial resistanceCan be orally administeredThey have a broadened range of specifity More chemically stable than their native counterpart.,抗生素的生产和提取工艺,菌种,孢子制备,发酵,种子制备,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
22、,Configuration & Production,Reactors must be correctly designed if the fermenting organisms potential is to be achievedAn understanding of the impact of configuration on production can help highlight suitable designs,Building a Bioreactor,The most important design features to consider include:Constr
23、uction Material Capacity Impellar Design Heat Exchanger Sparger Analytical Instrumentation,The Optimum Design,Construction MaterialTypically stainless steel as its cheap, light, readily available & easy to sterilizeCapacity Industrial designs hold between 30-200m3 of fluid, must however consider imp
24、act on cost & environmental control,GeometryIncreased height to diameter ratio is favored as it aids agitation & aeration of viscous antibiotic brothsImpellarRushtons are standard, but axial designs also possible. Low rotational speeds minimize shear & increased impellar diameter compensates for red
25、uced agitation,Axial & Radial Impellars,Different Impellar Designs,Heat ExchangerHeat generated by biological reactions & agitation is removed by cooling jackets. Limpet, or half pipe, is common choiceSpargerC-shaped preferable to single pipe, as they improve O2 transfer in the viscous broths,Cold H
26、2OIn,Warm H2O Out,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 bu
27、t promote fermentation,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。,Downstream Processing,Steps in Downstream Processing:,Downstream Processing,Removal of Insolubles,-Filtration,Isolation of product,- Solvent Extraction,Purification,- Pre
28、cipitation, Electrophoresis,Polishing,- crystallisation, drying,Penicillin G is in solution exocellularly,Filtration,Remove cells by filtration,Sterile conditions must be utilised to avoid contamination of filtrate with B-lactamase producing micro-organisms.,Components dissolved in aqueous fermentat
29、ion broth are recovered by extraction into an appropriate solvent: Liquid-Liquid extraction,Solvent Extraction,Solvents: amyl acetate（醋酸戊酯）, butyl acetate（醋酸丁酯） and methyl isobutyl ketone（甲基异丁酮） at pH 2-2.5.,Penicillin then extracted back into an aqueous buffer at pH 7.5,Crystallisation and Lyophili
30、sation,An excess of potassium or sodium acetate is added in a mixing tank to induce crystallization. A centrifuge or drum filtration unit then collects the crystals which are washed and dried to produce the final product.,Commercial scale lyophilisation (freeze-drying) is used to dry and preserve the sensitive biological product.,For parenteral use, the antibiotic is packed in sterile vials as a powder or suspension.,For oral use, the antibiotic is tabletted with a film coating.,Crystal Structure,
