Penicillin Production

Tom OHare & Lynne White Abstract The discovery of penicillin and its medicinal uses was arguably the most important
scientific discover y o f the 20th centur y. From the observation of an accidental

inocu lation to the application o f knowledge and techno lo gy, the pro duction of penicillin is a process that has changed dramatically since its beginnings in the late 19 0!s and ear ly 19"0!s. #s new ways were found to ensure that more penicillin was being pr oduced and that the purification process was as effective as possible, the production could increase, allo wing the use of penicillin in times of war to save the lives o f sold iers who otherwise would have died due to infection of their wo unds. $ince then, the development of large scale production has allowed penicillin to be used whenever needed to kill off bacteria and prevent ser io us infection, however, this has also been its downfall. The ability of some bacteria to now produce penicillinase to break down and render penicillin co mp letely useless has co me abo ut due to the wide scale use of the drug and has therefore limited the effectiveness of penicillin as a clinical treatment. %n order to combat this, scientists have turned to semi&synthetic derivatives of penicillin in the hope that these will have the properties necessary to beat the resistance problem. Penicillin and its history 'enicillin was the first naturally occurring antibiotic discovered ('rontosil, the first chemical used to cure certain infectious diseases, had been disco vered in 19 but had serious side effects). There are now more than *0 antibiotics, which are su bstances that ar e produced by microbes and that fight bacteria, fungi and other micro bes harmful to humans & the word means against (anti) life (bio). 'enicillin is obtained in a number of forms from 'enicillium moulds.

Penicillin: C16H18 !O"#

#s shown in the above diagram, penicillin is not a single compound but a group of clo sely related compounds, all with the same basic ring& like structure (a +&lactam) der ived from two amino acids (valine and cysteine) via a tripeptide intermediate. The thir d amino acid of this tripeptid e is replaced by an acyl group (, in the diagram below) and the nature o f this acyl gr oup pro duces specific properties on different types of penicillin. -acter ia repr oduce by divid ing to produce two new cells. They enlarge to abo ut twice their si.e before the /0# chro mo some is cop ied. The two new chromosomes mo ve apart and a cell wall forms between them. -ut if penicillin is present, the new cell wall won1t be able to fo rm. %t doesn1t harm old bacterial cell walls, but it sto ps new ones forming. This means the bacteria can1t r eproduce, so the disease can1t spread. 'enicillin acts by blocking the activity of the en.yme transpeptidase, which cross co nnects long po lymers of sugars that fo rm the bacterial cell wall. The +&lactam ring on penicillin (see structure) irreversib ly blocks the activity of the en.yme by co valently bonding with the functional end o f the en.yme. #s a result, newly&fo rmed cell walls will be structurally weak in some areas, causing water to rush in and rupture the cell. There are two different categories of penicillin. -iosynthetic penicillin is natural penicillin that is harvested from the mould itself through fermentation. The other form of penicillin is known as semi&synthetic. There are all kinds of what are called semi& synthetic derivatives of penicillin & like #mpicillin, 'enicillin 2, 3arbenicillin, 45acillin, 6ethicillin, etc. These compounds consist of the basic 'enicillin structure, but have been purposefu lly mod ified chemically by remo ving the acyl group to leave *&aminopenicillanic acid and then adding acyl gr oups that produce new properties. These modern semi&synthetic penicillins have vario us specific pro perties such as resistance to stomach acid s so that they can be taken o rally, a degree of resistance to penicillinase (or +&lactamase) (a penicillin&destroying en.yme produced by so me bacter ia) and an e5tended range of activity against some 7ram&negative bacteria. 'enicillin 7 (or ben.ylpenicillin) is the most widely used form and the same one we get in a shot (hypodermic) form.

Penicillin $ 'enicillin 7 is not stable in the presence of acid (it is therefore said to be acid&labile). $ince our stomach has a lot of hydro chloric acid in it (the p8 can be around 2.0), if we were to ingest penicillin 7, the compound would be destroyed in our stomach before it could be absorbed into the bloodstream, and would therefo re not be any good to us as a treatment fo r infection somewhere in our body. %t is fo r this reason that penicillin 7 must be taken by intramuscular in9ection & to get the compound in our bloodstream, which is not acidic at all. 6any of the semi&synthetic penicillins can be taken orally. The history of penicillin is a fascinating reminder of how the most freak and seemingly rando m of occurrences can lead to very significant scientific disco veries. #lso seeing as how the discovery o f penicillin is arguably man!s most important med ical (if scientific) ad vance to date, some discussion of its o rigins are necessary. This histo ry clearly displays that discovery in itself is not enough, there must also be a drive (in this case the huge numbers of soldiers d ying fr om secondar y infections in the second world war) to utilise this new d isco very to its fu llest. %n 192:, while working in $t. 6ary!s 8ospital in ;ondon, bacter io logist #le5ander Fleming was conducting research on the flu. 8e had been searching for antibacterial agents, influenced by his wartime e5perience. 8e had witnessed the deaths of many soldier s that died, not from the wounds they receiv ed during co mbat, but from secondar y infections of those wounds. <hile he was on holidays, a bit of blue&green mould had fallen into a discarded culture plate containing $taphylococcus aureus, forming a clear patch in the surrounding area. From this he could conclude that the mould was pro ducing an antibiotic substance. 8e named the antibiotic penicillin, after the 'enicillium notatum mo uld that produced it and 1929, he published the resu lts of his investigations, noting that his disco very mig ht have therapeutic value if it cou ld be

produced in =uantity. >nfortunately it couldn!t, and it would be 10 years before another significant leap forward for penicillin would occur. %n 19 : /r. 8owar d <. Flo rey came across Fleming1s paper on penicillin (which oddly had languished in obscurity). <hile Fleming!s lab was poorly e=uipped with no staff support, Florey1s lab was well e=u ipped and staffed with a team of scientists at 45for d >niversity that included /r. ?rnst -. 3hain. %t was 3hain who began e5tracting penicillin into a purified and powerful antibiotic. #t first penicillin was made using old dairy e=uipment and after a great deal of effort, enough was e5tracted for e5per imentation to beg in. ?ig ht white mice were ino culated with deadly Streptococcus ger ms, fo llowed by in jection of penicillin in half the mice. All of the untreated mice died the ne5t day while the treated mice all recovered. 0o w it was time for the first human test. #lbert #le5ander , a ":&year& old ;ondon po liceman had developed septicaemia as a result of a small cut on his face. <hen treated with penicillin #le5ander began to recover within the day. 8owever Flor ey1s team didn1t have enough of the drug to see the patient through to a full reco very and he later re& lapsed and died. 8owever by 19"1, it was acknowledged that penicillin was indeed a worthwhile drug and could save thousands of lives. %n the same year Florey travelled to the >nited $tates (which at the time was still neutral) to co ntinue his work with penicillin. -ecau se the >nited $tates intended to enter into <orld <ar %% in another few months the penicillin pro9ect, which became declared a war pro9ect, was g iven to p priority (and fu nd ing). Florey and his team were able to use beer&brewing techno logy to pro duce the huge amo unts of the mouldy li=uo r needed fo r penicillin production. This underwent a slow purification process to produce the large amounts of clinically usable penicillin that became available for military use in early 19"01s. %n 'eoria, %llino is a blue&gr een mou ld was fo und gr owing o n a mouldy cantaloupe in a market. This mould was identified as 'enicillium chrysogenum and pro duced appro 5imately 200 times as much penicillin than what Florey!s team were working with ('enicillium notatum). $cientists began to tr y to increase the amount of penicillin produced by '. chrysogenum, by irradiating it with @&rays and >2 rays in order to induce mutations of this species. They succeeded and developed a mutant that produced 1000 times the amount o f penicillin than Fleming1s original culture. #t the same time scientists began to grow the mould in the first deep tank fermenters. "

-y late 19" , mass production of the drug had commenced and by the end of the war, many companies were manufacturing the drug, including the 6erck, $=uibb and 'fi.er . %n 19"A Fleming was awarded the 0o bel 'ri.e in 'hysio lo gy and 6edicine along with Florey and 3hain. %n the coming years, strains and techni=ues were improved upon and 'enicillin saved tens of thousands of lives. 8owever, it all began with a bit of blue&green mould falling onto a discarded culture plate. %ermenters #n important part of any fer mentatio n process (o f which penicillin is an e5amp le) is the type and configuration of fer menter being used fo r the fer mentatio n. 7enerally, the purpose of a fermenter is to provide a contained, controlled, homo genous enviro nment in which the fermentation can proceed in a manner that is bo th safe and practical and which optimises the particular o b9ectives o f the fermentation. <ithin these parameters, there is a huge range of variability and fle5ibility. Fle5ibility is fre=uently a ma9o r ob9ective in a lab. 4ther primar y factors include co st, reliability and safety. %t should be noted however that this fle5ibility tends to decrease with scale. #s reactors become larger and larger ther e is less and less room fo r error. <hen a reactor is being designed fo r a specific purpose there are a number of impo rtant parameters that will greatly affect the reacto rs pro cess perfo rmance. 1. ,eactor $i.eB 8ow large do es the r eactor need to be in o rder to achieve optimum rates of productionC 2. ,eactor 3onfigurationB 8ow will the r eactor be configuredC For e5ample sho uld impellers be used to e5ert mechanical agitatio n (stirred tank) or will an air& 9et system be used for mi5ing (bubble colu mn)C . 6ode of operationB 8ow will substrate be addedC <ill it be batch fed or co ntinuously fedC ". 3onditions inside the reactorB <hat temper atur e and p8 should the reactor be maintained atC 8ow will contamination be avoidedC #nd how will these co nd itions be controlledC ,egardless o f ho w the above parameters are approached, there are still are nu mber of co mmon desired features that are re=uirements (both functional and economic) fo r a good working reactor. $ome o f the mo st commo n are summari.ed belo wB %unctional: D 8igh gasEli=uid mass transfer. A