Penicillin Production Process - Report Example

Penicillin Production Process Penicillin Production Process The 20th century saw various fascinating and serendipitous scientific inventions, and one of these great discoveries was the invention of penicillin by Sir Alexander Fleming (Schmidt & Moyer, 2006). The discovery and advanced development of penicillin contributed to the way of treating infectious diseases , which has since seen to the saving of lives of millions of people globally. Moyer and Schmidt (2006) affirm that penicillin is the first antibiotic to be used in healthcare to treat bacterial infections. In 1945, the greatest contributors for the invention and isolation of penicillin, Alexander Fleming, Sir Howard Florey, and Ernst B. Chain, were awarded the greatest Nobel Prize for the invention and isolation of penicillin. The antibiotic has been proven to possess vital therapeutic potential. The immune system of human beings can work to destroy harmful and toxic materials in the body, thanks to the white blood cells that attack these toxic materials before they can multiply. Our immune systems actively fight various infections when they detect any presence of harmful materials. However, some incidences occur that our immune system cannot function independently because the level of toxic materials has increased that the white blood cells cannot fight (Schmidt & Moyer, 2006). That is when antibiotics are necessary. Bacteria constantly rebuild their cell walls in a process known as peptidoglycan process. This process helps the bacterial to protect and maintain their cell structure. Penicillin operates by penetrating and damaging the cell walls of these bacteria, and kills the cells of the bacteria. However, over the years, bacteria has developed and become resistance. The bacteria generate β-lactamase that acts as the shied preventing the penetration and damage of their cell wall. The defense is neutralized by using β-lactamase incorporated in penicillin. Penicillin is majorly used to fight off infections by bacteria such as tonsillitis, penicillin helps in the protection of individuals with a weak immune system due to conditions or illness such as sickle cell anemia, and strengthen the immune systems of any sick individual that is receiving treatment and medical care and the disease can make him or her prone to infections. Most times penicillin is abbreviated as pen or PCN, and it is a member of antibiotics that is derived from penicillin V (oral use), benzathine penicillin, penicillium fungi, penicillin G, and benzathine penicillin (Sheehan & Logan, 2005). Various researches affirm that penicillin was the first antibiotic drug to be effectively used against various infections such as syphilis and illnesses caused by streptococci and staphylococci. Penicillin drug is globally used today, although most of the infections have become resistant to the drug. The entire groups of penicillin are β-lactam antibiotics, which are used in treating bacterial infections majorly caused by vulnerable organisms which, in most cases are caused by Gram positive (Jin, Wu, Lin, & Chen, 2008). There are three major processes to biosynthesis penicillin (benzypenicillin (Moyer & Coghill, 2005). The first step is through the condensation of three vital amino acids of L-valine, L-cysteine, and L-α-aminoadipic acid into tripeptite. The L-valine must undergo the epimerization process before condensing into tripeptide and becoming D-valine. The tripeptide that has been condensed is named as (L-α-aminoadipyl)-L-cysteine-D-valine (ACV). The epimerization and the condensation reactions are catalyzed by (L-α-aminoadipyl)-L-cysteine-D-valine synthetase (ACVS) enzyme which is a NRBS-nonribosomal peptide synthetase (Sarkar & Modak, 2006). The second step entails the biosynthesis and conversion through oxidation of linear ACV to bicyclic IPNS- intermediate isopenicillin N byisopenicillin N synthase that is encoded using the pcbC. The isopenicillin does not portray any strong antibiotic activity making it a weak intermediate. The last step entails the transamidation by the isopenicillin in which, th eaminodipyl side chain in the isopenicillin is removed, and it is exchanged for a side chain of phenylacetyl (Moyer & Coghill, 2005). The gene penDe encodes the reaction, and it makes it a unique process in the penicillin extraction process. Various enhanced penicillin families exist that aims at fighting the infectious bacteria. They include aminopenicillin, antistaphylococcal penicillin, and the strongest antipseudomonal penicillin. Scientists use the term “penam” to describe the basic skeleton structure of penicillin antibiotic. The skeleton has a basic molecular structure of R-C9H11N2O4S the R in the structural formula is a variable side chain. A normal penicillin antibiotic has a molar weight of 313-334g/mol (Jin et al., 2008). However, a penicillin type that has additional molecular groups that is attached has a molar mass of 500g/mol. For example dicloxacillin has a 492g/mol molar mass while cloxacillin has a 476g/mol molar mass. The core structure of penicillin is: The production of penicillin emerged due to the immediate result of the World War II. The production of penicillin was in large scale due to the demand that was created by the war. There were many jobs that were available in the United States at the home front (Jin et al., 2008). The penicillin production board was, therefore, formed to monitor, produce, and distribute the penicillin to the home front that saw the prosperity of the penicillin production. The War Production Board came up with strategy that would see the mass production of fine penicillin and distributing it to their allied troops that were fighting in Europe. In 1943, the production of penicillin was approximately 425 million units annually. However, in modern years, the production of penicillin has incorporated the use of biotechnology evolution that has led to the increased production and mutations of penicillin. These methods include ITCHY, DNA shuffling, strand overlap PCR, and PCR (Schmidt & Moyer, 2006). Penicillin is a metabolite fungus, “Penicillium” which is produced when stress inhibits the growth of fungus (Schmidt & Moyer, 2006). Penicillium cannot be achieved during an active growth. The feedback process in the pathway of synthesis of penicillin limits the production of penicillium. . AcCoA + α-ketoglutarate → homocitrate → L-α-aminoadipic acid → β-lactam + L-Lysine The L-Lysine is the by product and it inhibits the homocitrate production, therefore, scientists must avoid the existence of exogenous lysine in the penicillin production process. The Penicillium cells grow using the fed-batch technique (Moyer & Coghill, 2005). The process entails subjecting the cells to stress to produce large quantity of penicillin. Carbon sources in the production process are vital in the process, because glucose will inhibit the penicillin production process, but lactose does not interfere in the production process of penicillin. The pH levels of lysine, oxygen, phosphate, and nitrogen must be controlled automatically during the entire process. Though the exact or precise penicillin composition and production is difficult to determine or quote, by virtue of such facts is that the actual users term the information as “trade secrets.” A large proportion of the commonly used media comprises of ingredients such as lactose, calcium carbonate, glucose, cornsteep liquor solids, potassium dihydrogen phosphate (KH2PO4), penicillin precursor, and edible oil. Rhinehert and Li (2006) promulgated a typical and usel medium that have the following components: fermentable carbohydrates, lactose, organic nitrogen source, glucose, Phenyl acetic acid, KH2PO4, edible oils, Calcium Carbonate, and Organic Salts. The Calcium carbonate acts as the buffer in the process. The Organic salt maintains the salt balance in the experiment. The penicillin fermentation is carried in the fed-batch mode, and various experiments prove that glucose should not be put in high quantities in the penicillin production process because it inhibits penicillin production (Rhinehert & Li, 2006). In addition, Rhinehert and Li affirm that penicillin is a subordinate fungus, and the fed-batch mode provides the ideal conditions that are necessary for the penicillin production (2006). A typical fermentation temperature needs a temperature of 24oC and a pH of 6 to 6.5 is maintained to maximize the production of penicillin. The pressure that is found in the bioreactor is higher compared to the atmospheric pressure. This prevents the contamination such that external contamination cannot enter the bioreactor. Oxygen that is vital for the viability of the fermentation process is provided by the sparging of air bubbles. The sparging rate depend on the amount of the medium used, and for every 2 m3 of culture used be about 2.5 m3 of sparging bubbles are used. The impeller in the bioreactor is necessary for the mixing of culture medium. The fungal cells are hardy and they can handle a speed rotation of 200rpm (Schmidt & Moyer, 2006). After sterilization, the pH in the process is maintained at 5.5 to 6.0, and a high lactose level that ranges between 4 to 5% is necessary accompanied by an increased agitation environment and aeration maintained in the bioreactor. The medium used in the bioreactor is sterilized at high heat and pressure sterilized together or through the holding tube (Moyer & Coghill, 2005). The pressurized steam is usually used and the medium in the bioreactor is heated to a temperature of 121oC and at a pressure of 30psi, twice the atmospheric pressure. The high temperatures that are used in short time durations are applied to minimize or reduce the degradation level of various media components. The production media has both precursor and lactose that are not initially included in the inoculums media (Jin et al., 2008). Fig 1.0 Production and recovery of penicillin The general flow sheet diagram for the recovery and purification of penicillin is shown in the above figure (Fig 1.0). The process follows sequential steps that are outlined. 1. Once the procedure has undergone the fermentative process, that is, at the harvest stage, the complete fermentation of the penicillin culture is then subjected to filtration process. The heavy duty vacuums acts as a filter that gets rid of unwanted solid residues and mycelium (Moyer & Coghill, 2005). Filtration process is vital at this stage in the bioprocess flow. The bio separation is required to eliminate the biomass found in the culture such as impurities and fungus, away from the components that contain penicillin. Various researches has led to the discovery of different methods of filtrations, however, the Rotary filter is the major filtration method that is used in this process. It is employed in this experiment because it can be used in the penicillin production process in large scale. A Non-oxidizing agent such as phosphoric acid is introduced in the filtration stage. The acid has a pH of 8.5, and this is not conducive for production of penicillin, therefore, the pH should be lowered to a pH of 6.5 achieved at this stage. 2. The pH of the fermented broth is brought down to 2 to 2.5 by addition of sulphuric acid or phosphoric acid. The acids convert the penicillin that is formed to its ionic form (Halsall, Taylor, & Thibaul, 2008). 3. The fermented broth that is formed is extracted as soon as possible using pod bielniak countercurrent solvent extractor using a recommended organic solvent such as butyl acetate, amyl acetate, or methyl isobutyl ketone (Moyer & Coghill, 2005). To dissolve the penicillin that is present in the filtrate, butyl acetate or amyl acetate is used to dissolve the penicillin because they act better than water at the recommended pH. Penicillin is found in the resultant solution from the reactor and any other solid formed at this stage is considered as waste. Centrifugation is then performed to separate the aqueous penicillin and the solid waste formed in the process. A chamber or a tubular bowl is used and the supernatant is transferred down the downstream chambers to continue the extraction process. Penicillin will dissolve in the provided solvent and then subjected to a series of many extraction processes that aims at obtaining a purified penicillin product (Sarkar & Modak, 2006). The acetate solution is mixed with the phosphate buffer, and then followed by a solution of chloroform, again phosphate buffer is added to the solution and finally, an ether solution is mixed in the solution. The ether solution allows the penicillin concentration to be higher, and it allows the solution to be mixed with sodium bicarbonate solution that yields penicillin sodium salt. Sodium penicillin allows the penicillin to be in a stable condition that can be stored at room temperature. The product of sodium-penicillin is got from the liquid media by basket centrifugation process, in which the resultant solid products are as a result of the ease of removing solids (Halsall, 2008). Penicillin that is obtained is then extracted into an aqueous solution by the organic solvent by carefully adding a requisite sodium hydroxide or potassium hydroxide that leads to the formation of sodium or potassium salt of the resultant penicillin (Sarkar & Modak, 2006). 4. The resulting solution that contains the respective penicillin salts is acidified and extracted using the organic solvent methyl isobutyl ketone 5. The shifts that occur between the solvent and aqueous medium assist greatly in the purification process of penicillin (Sheehan & Logan, 2005). 6. The product that is formed is then subjected to scrupulous back-extraction with sodium hydroxide solution severally until the extraction process of penicillin is completed. The penicillin undergoes several processes that enable it to crystallize as either potassium or sodium penicillin (Rhinehert & Li, 2006). 7. The obtained crystalline penicillin is then washed, and dried under a vacuum. The final product must follow the specifications that are laid down by official Compendia. Drying the product is needed because it helps remove moisture that might be present in the penicillin. Hot gas is pumped through the base of the compartment that contains the powered salt in a process known as fluid bed drying. Moisture from the aqueous penicillin is removed and much drier penicillin is achieved. The penicillin salt achieved are stored in huge containers and then stored in a dry environment. It is then polished and packet in various types of products such as penicillin in pills or liquid penicillin (Jin et al., 2008). Clinical trials are performed on this drug to determine a specific dosage. Figure 1.0 shows a typical penicillin fermentation plant. In the real sense, the culture medium can be successfully batched and sterilized in the fermenter itself. Most of the fermenters get attached to the batching vessel and to the other sterilizers as indicated in the figure. The different feed vessels that are duly connected in the final stage and the fermenters are employed to supplement the precursors and the nutrients during the fermentative process. Importantly, the final stage and the seed fermenter must operate under strict and aseptic environments (Sheehan & Logan, 2005). The bioreactor is made up of SS that has a capacity that ranges between 30 to 300 m3 agitations by two thirds of flat-peddled impellers, the compressed sterile injections performs the aeration in the reactor. The employment of chilling water by the cooling coils maintains the temperature at a 26oC. The live-steam injections perform sterilization in the fermenter (Jin et al., 2008). Bibliography Halsall, W., Taylor, Y., & Thibaul, J. 2008, "Multicriteria optimization of gluconic acid production using net flow," Bioprocess and Biosystems Engineering, vol 2, no 2, 299-307. Jin, X., Wu, Q., Lin, X., & Chen, Q. 2008, "Immobilization of penicillin G acylase on a composite carrier with a biocompatible micro-environment of chitosan,"J Chem Technol Biotechnol, vol 6, no 5, 1710–1716. Moyer, A. J., & Coghill, A. D. 2005, "Penicillin: IX. The Laboratory Scale Production of Penicillin in Submerged Cultures by Penicillium notatum Westling (NRRL 832)," Bacteriology, vol 7, no 5,15-67. Rhinehert, K., & Li, H. 2006, "Heuristic random optimization," Computers & Chemical Engineering, vol 7, no 4, 427-444. Sarkar, D., & Modak, M. J. 2006, "Optimisation of fed-batch bioreactors using genetic algorithms," Chemical Engineering, vol 5, no 8, 2283-2298. Schmidt, W. H., & Moyer, A. 2006, "Penicillin: I. Methods of assay," vol 9, no 7, Bacteriology, 17-26. Sheehan, J. D., & Logan, H. D. 2005, "The Total Synthesis of Penicillin V," vol 4, no 8, American Chemical Society, 3089-3097. Read More