[精选]第九章抗生素的生产8238.pptx

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定义：是生物在其生命过程中产生的，并在低浓度下定义：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。物质。Microbial Products,effective at lowMicrobial Products,effective at low concentrations,which have the ability to kill or inhibit concentrations,which have the ability to kill or inhibit the growth of certain micro-organisms.the growth of certain micro-organisms.History:History:1919世纪世纪7070年代，法国年代，法国Pasteur,Pasteur,发现有些微生物可以抑制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。象来治疗一些感染性疾病。19281928年，年，概述 In 1928 Scottish bacteriologist Alexander In 1928 Scottish bacteriologist Alexander Fleming reported the antibacterial action of Fleming reported the antibacterial action of cultures of a cultures of a Penicillium Penicillium speciesspecies He observed that He observed that Penicillium notatumPenicillium notatum killed killed some of his some of his Staph.aureusStaph.aureus culture dishes culture dishes Discovery by accident?Culture dishes Discovery by accident?Culture dishes contaminated when left out for two weeks contaminated when left out for two weeks while Fleming was on holiday,instead of while Fleming was on holiday,instead of being incubated being incubatedFleming noticed that the substance secreted Fleming noticed that the substance secreted was an inhibitor of many bacterial species was an inhibitor of many bacterial speciesHe published a paper on the active ingredient He published a paper on the active ingredient which he called penicillin.This was the first which he called penicillin.This was the first demonstration that a substance produced by demonstration that a substance produced by micro-orgs could inhibit or kill other microbesmicro-orgs could inhibit or kill other microbesFleming could not isolate penicillin.10 yrs Fleming could not isolate penicillin.10 yrs later scientists later scientists Florey and ChainFlorey and Chain successfully successfully isolated penicillin.isolated penicillin.1943-19451943-1945，scale-up production of scale-up production of pencillium.pencillium.By 1945,enough penicillin was produced to treat 7 million per year.Discoverer of Discoverer of penicillinpenicillinBorn into a farming Born into a farming family in Scotland,family in Scotland,moved to London at moved to London at 1313Awarded Awarded Scholarship to study Scholarship to study medicinemedicine Alexander FlemingZone of InhibitionThis picture This picture illustrates the effect illustrates the effect of penicillin on a of penicillin on a fungus.Note the fungus.Note the clear ring of clear ring of inhibition.It was this inhibition.It was this observation which observation which initiated Flemings initiated Flemings further studies.further studies.Howard FloreyAustralian-bornAustralian-bornStudied Science&Studied Science&Medicine in AdelaideMedicine in AdelaideAwarded Awarded Scholarship at Scholarship at Oxford UniversityOxford UniversityReturned to England Returned to England to complete a PhDto complete a PhDErnst ChainOf Russian-German Of Russian-German descentdescentGraduated in Graduated in Chemistry and Chemistry and Physiology Physiology Left Berlin when Left Berlin when Nazis came to Nazis came to powerpowerHired by Florey to Hired by Florey to work on penicillin work on penicillin projectproject到目前为止，有到目前为止，有8000多种抗生素，多种抗生素，2/3是是由放线菌（由放线菌（strepomyces）产生的，并产生的，并通过化学结构的改造，总共有通过化学结构的改造，总共有3万多种半万多种半合成抗生素，它们的分子量为合成抗生素，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，普遍应用的包括半合成和盐类的抗生素有素有350多种，其中以青霉素、头孢霉素、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四环素类、氨基糖苷类和大环内脂类主要抗生素的使用量（主要抗生素的使用量（Harvey,1998)抗生素种类抗生素种类 用量用量 (kg/year)(kg/year)动物动物19961996人类人类1997/19981997/1998头孢类头孢类四环类四环类氨基糖类氨基糖类大环内酯类大环内酯类121,603121,603323,151323,15137,05837,05871,22271,222314,498314,49847,50047,5005,4095,40947,69647,696Some Clinically Important AntibioticsAntibioticsAntibioticsProducer Producer OrganismOrganismActivityActivitySite or mode Site or mode of actionof actionPenicillinPenicillinPenicillinPenicillinchrysogenumchrysogenumGram-positiveGram-positivebacteriabacteriaWall synthesisWall synthesisBacitracinBacitracinBacillusBacillussubtilissubtilisGram-positiveGram-positivebacteriabacteriaWall synthesisWall synthesisNeomycinNeomycin(新霉新霉素）素）StreptomycesStreptomycesfradiaefradiaeBroad Broad spectrumspectrumProtein Protein synthesissynthesisStreptomycinStreptomycinStreptomycesStreptomycesGriseusGriseusGram-negativeGram-negativebacteriabacteriaProtein Protein synthesissynthesisTetracyclineTetracyclineStreptomycesStreptomycesmediterraneimediterraneiBroad Broad spectrumspectrumProtein Protein synthesissynthesis抗生素的分类根据抗生素的来源分根据抗生素的来源分根据抗生素的来源分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放线菌产生的，占目前发现的抗生素的放线菌产生的，占目前发现的抗生素的放线菌产生的，占目前发现的抗生素的2/32/3。真菌产生的，主要包括青霉菌。真菌产生的，主要包括青霉菌。真菌产生的，主要包括青霉菌。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（菌素。动植物产生的，蒜中的蒜素，动物脏器中的鱼素（菌素。动植物产生的，蒜中的蒜素，动物脏器中的鱼素（菌素。动植物产生的，蒜中的蒜素，动物脏器中的鱼素（ekmolin).ekmolin).根据作用谱分根据作用谱分根据作用谱分根据作用谱分:广谱抗生素，如氨苄青霉素，可抑制广谱抗生素，如氨苄青霉素，可抑制广谱抗生素，如氨苄青霉素，可抑制广谱抗生素，如氨苄青霉素，可抑制G-&G+G-&G+细菌细菌细菌细菌抗抗抗抗G+G+菌的抗生素：如青霉素。菌的抗生素：如青霉素。菌的抗生素：如青霉素。菌的抗生素：如青霉素。抗抗抗抗G-G-菌的抗生素：如链霉素菌的抗生素：如链霉素菌的抗生素：如链霉素菌的抗生素：如链霉素抗真菌的抗生素：如制霉菌素抗真菌的抗生素：如制霉菌素抗真菌的抗生素：如制霉菌素抗真菌的抗生素：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（抗癌抗生素：如阿霉素（抗癌抗生素：如阿霉素（抗癌抗生素：如阿霉素（Adriamycin)Adriamycin)目前在抗病毒、抗癌、等方面还没有理想的抗生素。目前在抗病毒、抗癌、等方面还没有理想的抗生素。目前在抗病毒、抗癌、等方面还没有理想的抗生素。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根据抗生素的结构分类根据抗生素的结构分类根据抗生素的结构分类以以以以-内内内内酰酰胺胺胺胺类类抗生素：包括其青霉素、抗生素：包括其青霉素、抗生素：包括其青霉素、抗生素：包括其青霉素、头孢头孢菌素菌素菌素菌素类类等。它等。它等。它等。它们们含含含含有一个四元内有一个四元内有一个四元内有一个四元内酰酰胺胺胺胺环环，为为目前最受重目前最受重目前最受重目前最受重视视的一的一的一的一类类。Penicillin StructureB-内酰胺是抗生素具有抗菌活性所必需的，而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基团则赋予抗生素的稳定性、抗菌谱等的变化。性、抗菌谱等的变化。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环醇四环类抗生素：四环素类、土霉素以四个苯为母核。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。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。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。喹诺酮类抗生素，如环丙沙星、诺氟沙星。按照作用机制分类按照作用机制分类抑制细胞壁合成的抗生素抑制细胞壁合成的抗生素影响细胞膜功能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，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，以乙酸、丙酸为单位的衍生物，如红霉素等丙酸衍。生物如红霉素等丙酸衍。生物抗生素的应用对抗生素的评价：对抗生素的评价：有较大的差异毒力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。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致病菌对抗生素不易产生耐药性不引起过敏反应不引起过敏反应理化性质稳定，便于提取、制剂和贮藏。理化性质稳定，便于提取、制剂和贮藏。抗生素的计量单位抗生素的计量单位：效价单位：青霉素效价单位为能在效价单位：青霉素效价单位为能在50ml肉汤培养基中完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。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1667个单位1 mg链霉菌素碱相当于1000个单位Industrial evolution of penicillin productionDateDateYield(units/mL)Yield(units/mL)DevelopmentDevelopment192919292-202-20Wild-type Wild-type(P.notatum)(P.notatum)1941194140-8040-80WTWT1943194380-10080-100New WT New WT(P.chrysogenum)(P.chrysogenum)19441944100-200100-200Colony selectionColony selection19441944300-500300-500X-irradiationX-irradiation19451945800-1000800-1000UV-irradiationUV-irradiation194919491500-20001500-2000Chemical mutagenesisChemical mutagenesis1951195124002400Chemical mutagenesisChemical mutagenesis1953195327002700Strain selectionStrain selection1960196050005000Strain selectionStrain selection197019701000010000Strain selectionStrain selectionCommon antibiotics and their sourcesBacitracinBacitracinCephalosporin(s)Cephalosporin(s)（头孢（头孢菌素）菌素）ChloramphenicolChloramphenicol（氯霉（氯霉素）素）CycloheximideCycloheximide（放线菌（放线菌酮）酮）HygromycinHygromycinPenicillinPenicillinStreptomycinStreptomycinTetracycline(s)Tetracycline(s)VancomycinVancomycinBacillus subtilisBacillus subtilisCephalosporium Cephalosporium spsp.S.venezuelaeS.venezuelaeS.griseusS.griseusS.hygromycesS.hygromycesP.chrysogenumP.chrysogenumS.griseusS.griseusS.aurofaciensS.aurofaciensS.orientalisS.orientalisBeta-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。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，对耐药菌有效有效，便于口服，改善药理性质，提高生物有效性。，便于口服，改善药理性质，提高生物有效性。STRUTURE OF PENICILLINWhat 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 importanceMain VariantsAmpicillin（氨卡青霉素）MethicillinAmoxycillinMethicillin(甲氧霉素)19861986（13%13%，19981998，26%26%）Originally introduced Originally introduced to combat to combat S.S.AureusAureusB-Lactamase B-Lactamase resistantresistant1960s a semi-1960s a semi-synthetic broad synthetic broad spectrum compound spectrum compound producedproducedOver production of Over production of beta-lactamase beta-lactamase enzyme has been enzyme has been shown to contribute to shown to contribute to methicillin resistance.methicillin resistance.Ampicillin（氨卡青霉素）Discovered in 1963Discovered in 1963One of only few One of only few penicillins that can be penicillins that can be administered orallyadministered orallyHas improved acid Has improved acid stabilitystabilityBroad spectrum.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 BeechamsBenefits of semi-synthetic variantsCan 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 G6-APA（6-氨基青霉烷酸）First discovered in 1959 First industrial process of enzyme engineering Conversion of Pen G to 6-APAUsed in penicillin recovery processAction of 6-APAPGAPGA（青霉素酰化酶）（青霉素酰化酶）is the natural substrate is the natural substrate for Pen.Gfor Pen.GPenicillin G is hydrolysed in a reversible reaction Penicillin G is hydrolysed in a reversible reaction to 6-APA and phenylacetic acid.to 6-APA and phenylacetic acid.Vital intermediate for the production of semi-Vital intermediate for the production of semi-syntheic penicillinssyntheic penicillinsAcyl GroupNature of acyl group confers certain properties Nature of acyl group confers certain properties on the penicllin analogueon the penicllin analogueSelective hydrolysis occursSelective hydrolysis occurs by hydrolysis by by hydrolysis by lactamase-producing culture lactamase-producing culture lactamaseThese are the most common cause of bacterial resistance to beta lactam antibioticsMost important lactamase was produced by S.aureus.This was responsible for rise in resistance to penicillin in the 1950sBeta Lactam ring is crucial for penicillin to remain activeAction of lactamase lactamase destroys penicillin by hydrolyzing lactamase destroys penicillin by hydrolyzing a bond in this ringa bond in this ringSome Statistics10,985 tonnes of 6-APA were produced in 1998Of the 10,985,48%was used for ampicilllin production,and 27%for amoxicillin.ResistanceLinked 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，100%大肠杆菌抗链霉素ConclusionSemi-synthetic variants are now the favoured Semi-synthetic variants are now the favoured choice of antibitic for combating bacterial choice of antibitic for combating bacterial resistanceresistanceCan be orally administeredCan be orally administeredThey have a broadened range of specifityThey have a broadened range of specifityMore chemically stable than their native More chemically stable than their native counterpart.counterpart.抗生素的生产和提取工艺菌种菌种孢子孢子制备制备发酵发酵种子种子制备制备发酵液的预处理发酵液的预处理提取和精制提取和精制成品包装成品包装现代抗生素工业生产工艺现代抗生素工业生产工艺Configuration&ProductionReactors 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 designsBuilding a BioreactorThe most important design features to consider include:Construction MaterialCapacityImpellar DesignHeat ExchangerSpargerAnalytical InstrumentationThe Optimum Design1.1.Construction MaterialTypically stainless steel as its cheap,light,readily available&easy to sterilize2.2.Capacity 3.3.Industrial designs hold between 30-200m3 of fluid,must however consider impact on cost&environmental control3.3.Geometry4.4.Increased height to diameter ratio is favored as it aids agitation&aeration of viscous antibiotic broths4.4.Impellar5.5.Rushtons are standard,but axial designs also possible.Low rotational speeds minimize shear&increased impellar diameter compensates for reduced agitationAxial&Radial ImpellarsDifferent Impellar Designs5.5.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 brothsColdH2OInWarmH2OOut7.7.Analytical Instrumentation Probes for measurement of temp,pH glucose&DO conc.are fitted at various points around the vesselOptimum temp between 24-280CAlkaline pH must be reduced to neutralityGlucose must be monitored to prevent catabolite repressionDO must sustain cells but 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一般用溶媒体取法：利用抗生素在不同的phph条件下的化学条件下的化学状态（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。液相中（如有机溶剂），已达到浓酸和提纯的目的。Downstream ProcessingSteps in Downstream Processing:Downstream Processing Removal of Insolubles-Filtration Isolation of product-Solvent ExtractionPurification-Precipitation,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 fermentation broth are recovered by extraction into an appropriate solvent:Liquid-Liquid extractionSolvent 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 7.5Crystallisation and Lyophilisation 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 coat