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Production of Benzyl penicillin
Introduction:
Penicillin is known as the first naturally discovered antibiotic. It is generally obtained from a
number of forms of Penicillium moulds. Penicillin is not a single molecule but a group of
narrowly correlated compounds, all of which having the same basic ring-like structure (βlactam). It is resulting from two amino acids (valine and cysteine) through a tripeptide
intermediate stage. The third amino acid of this tripeptide is substituted by an acyl group (R) and
the nature of this acyl group produces explicit properties on altered types of penicillin.
The finding of penicillin and its therapeutic usages was one of the most important scientific
discovery of the 20th century. The production of penicillin has transformed dramatically since its
beginning of production from late 1930’s and early 1940’s. Currently, with advancements of new
techniques different ways were found for more penicillin production with very viable and
efficient purification process. The increase resistance of bacteria with production of penicillinase
has therefore limited the effectiveness of penicillin as a clinical treatment. In order to fight this,
scientists have moved for production of semi-synthetic derivatives of penicillin in the
anticipation that these will resemble the properties essential to beat the resistance problem.
There are two different categories of penicillin.
Biosynthetic penicillin: these are class of natural penicillin are harvested from the mold. The
harvestation of penicillin is done through different fermentation techniques.
Semi-synthetic Penicillin: these are class of manmade synthesized penicillin. These are
produced by changing different using synthetic chemical pathways for penicillin production.
Some examples of semisynthetic derivatives of penicillin are Ampicillin, Penicillin V,
Carbenicillin, Oxacillin, Methicillin, etc. These compounds contain the basic Penicillin structure,
but have been decisively adapted chemically by eliminating the acyl group to leave 6aminopenicillanic acid and then addition of acyl groups that yield new properties. These recent
semi-synthetic penicillins have numerous precise properties such as resistance to intestinal acids
so that they can be taken orally.
General Process for production of Penicillin
Like all other industrial products, production of penicillin begins from the basic raw materials to
the downstream processes ensuing in the final product. It is important to note that in practical,
enterprises generally have their own explicit set of standards and hence alteration of the
procedure is necessary to meet their needs and also to optimize quality and quantity.
In any bioprocess facility, there has to be an upstream and after process (downstream Processing)
for final product recovery. The upstream processes in this case are referring to processes which
are uptake in the
fermenter prior to the product yield/formed, while the after processes
(downstream) refers to the processes that are done in fermenter after product yield.
Fermentation
To activate the fermentation procedure, a small quantity of these spores will be inoculate into a
(normally 250-500ml) conical flask. The flask will be incubated at prefixed temperature for
several days to achieve maximum production of penicillin. At this stage, spores growth will be
initiated with adaptation in utilizable carbon and nitrogen sources, such as starch and corn-steep
liquor. Temperature is commonly maintained at 23-28˚C and pH at 6.5, although there may be
some changes fabricated to facilitate optimum growth. Generally, fermenter is equipped with
baffles for achieving more oxygen levels.
Some important factors for Penicillin Production
Media Consideration and supplies:
For maximum production of benzyl penicillin, optimization of medium and media constituents
are important factors. The overall objective for media formulation is facilitation of synthesis of
cell materials and formation of anticipated components. At the same time, the media should
provide the most acceptable and favorable environment for the maximum production of benzyl
penicillin. Characteristically, all microorganisms require Carbon, Hydrogen, Oxygen, Sulphur
and Nitrogen for cell growth and cell maintenance. In many cases, microorganisms require lesser
quantities of trace elements such as Cu, Mn and Co (this will commonly rest on on the water
source as maximum water sources contain small amounts of the growth factors such as vitamins
or amino acids as well. Also, certain organisms such Penicillium chrysogenum produces
antibiotics, enzymes or other secondary metabolites which frequently require initiators like
purine/pyrimidine bases or organic acids to harvest said metabolites.
At this stage, it is all-important to accord abrupt reasons for the differences in primary and
secondary metabolism. Primary metabolism is the metabolism of activity production for the cells
and for its own biosynthesis. Typically, in aerobic bacilli (such as Penicillium chrysogenum), it
involves the about-face of sugars such as glucose to pyruvic acid and the assembly of activity via
the TCA cycle. Secondary metabolism commendations the assembly of metabolites that are not
acclimated in energy assembly for penicillin from Penicillium chrysogenum. In this case the
metabolite is getting activated as a defense apparatus adjoin added microorganisms in the
environment.
Generally, this Production of penicillin is done in stages. In the aboriginal stage, primary
metabolism will be emphasised. Media for this production will be focused on accomplishing best
advance and biomass production. At this stage, Glucose (Starch) will usually be the primary
antecedent of carbohydrate for the culture. This is because, lot of metabolites are acclimated for
activity metabolism and thus gives the best yields in agreement of growth. At this time, a
substrate is frequently added that will accord a readily accessible antecedent of Nitrogen as ablebodied such as corn steep liquor. Both these substrates have the ability for the maximum amount
of product (antibiotic) formation. This is because advance and antibacterial assembly are
mutually commutual and inversely proportionate. Readily accessible carbon and nitrogen sources
tend to arrest antibacterial assembly (this is accepted as Catabolite Repression, area an accessory
metabolite is inhibited by the attendance of an added readily accessible substrate).
Limiting the bulk of carbon and nitrogen accessibility typically does this. Hence, the bulk of
carbon antecedent that is added have to be carefully controlled. In adjustment to do this, lactose
is frequently acclimated as carbohydrate source in this allotment of the fermentation. It is as well
actual important that the nitrogen source is anxiously controlled, as balance nitrogen will
abundantly arrest antibacterial production. The solution to this is to use the fed-batch adjustment
to augment the culture. As declared above, this allows us to add the substrate to the reactor in
baby increments and to even change the substrate if we so desire.
This is the best way to change from one production date to another. There are as well several
added considerations for production of penicillin. First of all, as mentioned above, at this date
there is frequently a charge for the accession of precursor molecules (e.g. purine/pyrimidine) into
the medium.
Process considerations and Scale up
This part deals with the assorted accomplish in a beverage process, from the antecedent
activation of a preserved ability appropriate up to the all-encompassing automated assembly of
the antibiotic of interest. As can be imagined, there are abundant factors that have to considered,
with altered role at stages in the fermentation processes.
Almost all fermentations activate with the preserved culture. This is a demonstrative established
ability from which added cultures are grown. Frequently, this will be a stock that has been
adapted over time (genetically or not) to accord the best accessible yield of microbe. For this
reason, it is generally desirable to use as little of accessible to admit the beverage process.
Often the best way to use these cultures is in the spores stages. This is because spores are actual
aggressive structures that can survive heat, desiccation, low pH and automated armament that
would about annihilate an accustomed abundant cell. To access their longevity, these spores are
generally kept in a cyropreservative aqueous medium and are in frozen state.
To activate the fermentation process, a small amount of these spores will be added into a small
(normally 250-500ml) conical flask and will be incubated for several days. At this stage, with
spore is adapting to constant changes in fermentor environment. In such the spores will start
utilizing carbon and nitrogen sources, such as starch and corn-steep liquor. At this stage, the
spores will activate to animate and form abundant cells. Temperature is commonly maintained at
23-28˚ C and pH at ~6.5, although there may be some changes fabricated to facilitate optimum
growth. The spores will generally accept baffles in it and be on an afraid accoutrement to
advance oxygen diffusion in the flask.
Once the all-embracing optimum parameters are reached, it is an applicable that vegetative
ability to alive in the flask, will be transferred to a 1 or 2 liter bench top reactor. This reactor will
be adapted with an amount of instruments to acquiesce the culture to be bigger empiric than it
was in the agitate flask. Typical ambit observed cover pH, temperature, and stirrer acceleration
and attenuated oxygen concentration.
For example, there may not be abundant oxygen accepting to the ability and appropriately it will
be oxygen starved. Another archetype would be top shear-rate arch to cell damage. To resolve
these problems, modifications will be fabricated to the action and/or the reactor. The majority of
optimization of the action occurs actuality in the bench-top reactor as well as acquisition of
important advice about the action of the cells.
At this point, the spore should be assuming filamentous morphology, as this is adopted for
penicillin production. As before, cell advance is antecedence at this stage. This reactor is
generally beheld as a blazon of ancestor for the beyond fermentations that follow.
Once this has been acknowledged the action is scaled-up upto a pilot-scale bioreactor. This
reactor will be in architecture to the bench-top reactor except it will accept an admeasurement of
about 100-1000 litres. The aim actuality is to appraise the after effect of scale-up on the culture.
At this stage, hopefully advance will abide as before, however, there are generally abrupt
changes in performance. This can be due to changes in the analysis of the ability (remember
Penicillium chrysogenum is a filamentous fungi and appropriately pseudoplastic) that may or
may not be correctable.
If the pilot-plant date is acknowledged again plan can activate on an automated calibration
operation. This is now actual abundant an engineering problem. The reactor accept to be capable
of active aseptically and the architecture accept to reflect assurance of requirements. Different
Carbon and nitrogen sources will be added sparingly alongside forerunner molecules for
penicillin fed-batch style.
Therefore, it is generally accepted that an efficient adjustment for the abatement of this artefact
should be done in the reactor. Other systems, such as cooling supply can also be considered. If
all this goes to plan, maximum production of penicillin will be obtained.
Fermentor
An important allotment of any antibiotic production is the display and agreement of fermenter
getting acclimated for the fermentation. Generally, the purpose of a fermenter is to accommodate
a contained, controlled, akin environment in which the can antibiotic production is both safe and
practical and which optimises the accurate objectives of the fermentation.
Within these parameters, there is a huge ambit of flexibility. Adaptability is frequently a above
priority in any lab. Other primary factors cover cost, believability and safety. It should be
acclaimed about that this adaptability tends to abatement with scale. As reactors become beyond
advances there is less allowance for error.
When a reactor is getting made for a specific purpose there are important ambit that will
abundantly affect the reactors action performance.
1. Reactor Size: How ample does the reactor charge to be in adjustment to accomplish optimum
production?
2. Reactor Configuration: How will the reactor be configured? For archetype should impellers be
acclimated to apply automated agitation (stirred tank) or will an air-jet arrangement be
acclimated for bond (bubble column)?
3. Mode of operation: How will substrate be added? Will it be accumulation fed or continuously
fed?
4. Conditions central the reactor: What temperature and pH should the reactor be maintained at?
How will contamination be avoided? And how will these conditions be controlled?
Regardless of how the aloft ambit are approached, there are still are amount of common adapted
appearance that the requirements for a good reactor. Some of the lot are accepted and
abbreviated below:
Functional:
 High gas/liquid accumulation transfer.
 Reasonable heat-transfer.
 Prevention of accession after damaging microorganisms.
 Good bulk-flow and bond to anticipate the conception of asleep zones in the reactor.
 Good comestible transfer.
Economic:
 Easy to accomplish aseptically.
 Reasonably adjustable apropos action requirements.
 Low ability consumption.
 Stable beneath clashing conditions.
 Cheap, robust, simple and able-bodied accepted for scale-up.
Mass Alteration is apparent if there is bond two apparatus of capricious concentrations. Mass
Alteration processes are amenable for the movement of the dye through the baptize until calm is
reached. This is actual important if we are attempting to abound aerobic bacilli in media. If the
oxygen is bubbled through the fermenter tank, there will be top concentrations of oxygen apart of
the bubbles but low absorption everywhere abroad in the tank. Since oxygen absorption is such a
vital basic of aerobic metabolism, it is capital that there is acceptable alteration of oxygen
beyond the gas/liquid interface.
The Sparger delivers this oxygen. One botheration with gas/liquid interfaces is the accumulation
of foam. The impeller that serves to breach up the cream about relieves this. Heat Alteration is
important because metabolism as an action tends to accord off heat, as does the automated bond
by the impellers. If the reactor needs to be operated at a constant temperature of ambit of
temperatures, again it is analytical that there is some kind of arrangement to air-condition the
reactor. This is commonly accomplished through an aggregate of a cooling anorak and airconditioned baffles getting anesthetized about the tank.
Bulk Breeze and Bond are accomplished by the impellers. Most reactors use the Rushton Flatbladed disc agent apparent in the diagram. Fluid breeze in a reactor can be declared as
Newtonian or non-Newtonian. Newtonian fluids appearance connected bendability and beeline
shear amount adjoin microburst stress. However abounding fluids are non-Newtonian and are
described as Bingham or Pseudoplastic in nature.
Common Procedures:
Fermentation for penicillin is usually done in the fed-batch approach as glucose have to not be
added in top amounts at the level of advance which will after effect in low crop of penicillin.
Generally, pencillium grpwth arrest as in high level glucose. In accession to that, penicillin is an
accessory metabolite of the fungus, therefore, the fed-batch approach is ideal for such articles as
it allows the top assembly of penicillin. The archetypal fermentation altitude for the Penicllium
mold, usually requires temperatures at 20-24˚C while pH altitude are kept in amid 6.0 to 6.5. The
burden in the bioreactor is usually abundant college than the atmospheric pressure (1.02atm) this
is to anticipate contagion from occurring as it prevents alien contaminants from entering.
Sparging of air bubbles is all-important to accommodate acceptable oxygen the activity of the
fungus. Depending on the aggregate of medium, for 2 cubic metres of culture, the sparging
amount should be about 2.5 cubic metres per minute. The impeller is all-important to mix the
ability analogously throughout the ability medium, fungal beef are abundant able and they are
able to handle circling acceleration of about 200rpm.
Removal of biomass
Filtration is all-important at this point of the bioprocess flow, as bioseparation is appropriate to
abolish the biomass from the ability such as the bane and added algae abroad from the average
which contains the penicillin product. There are abounding types of filtration methods accessible
today, however, the Rotary exhaustion clarify is frequently active as it able to run in connected
approach in any ample calibration operations. Add this point non-oxidising acerbic such as
phosphoric acerbic are alien as pH will be as top as 8.5. In adjustment to anticipate accident of
action of penicillin, the pH of the abstraction should be maintained at 6.0-6.5.
Extraction
Penicillin liquefy in the solvent will now endure in a sequence of pulling out procedure to find
better purity of the penicillin product. The acetate secures is aboriginal alloyed with a phosphate
buffer, followed by a chloroform solution, and alloyed afresh with a phosphate absorber and
assuredly in an ether solution. Penicillin is present in top absorption in the ether secures and it
will be alloyed with a sodium bicarbonate to access the penicillin-sodium salt, which accept
penicillin to be stored in an abiding crumb anatomy at allowance temperature. The penicillinsodium alkali is acquired from the aqueous actual by bassinet centrifugation, in which debris are
gentlyremoved.