Penicillin: History, Structure, Production & Recovery

Content

• Introduction
• History
• Structure
• Penicillin molecule and its precussor
• Penicillinase
• 6- amino penicillanic acid
• Penicillin production

Introduction

• 1st antibiotic to be produced and still one of the best antibiotic available.
• Penicillin is a group of antibiotics, isolated from common molds known as Penicillium Molds.
• It is active against many gram-positive bacteria, Nocardia, Actinomycetes.
• It generally, interferes with cell wall synthesis of organisms which is sensitive and it is active only against vegetative cell.

History

• Sir, Alexander Fleming in 1929, first observed the antibiotic properties and possible therapeutics value of penicillin.

• He observed that an airborne contaminant, later shown to be Penicillium notatum inhibited the growth of a culture of Staphylococcus aureus on agar medium in the area surrounding the fungal activity.
• Fleming further showed that this inhibitory effect Caused by a soluble, diffusible mold product.
• Penicillin observed by Fleming was Penicillin-G.

Structure

• Several penicillins are closely related in structure.
• Common chemical nucleus and a side chain attached to this nucleus.

• Various compounds resembling the side chain can be added as precursor to fermentation medium & through microbial strain are directly incorporated into Penicillin molecule.
• Fungi: Aspergillus & Penicillium.
• Various penicillin differs as nature of their “R’’ side chain.

Penicillin molecule and its Precursor

• Whereas, in present day commercial terms penicillin is regarded as the Penicillin-G.
• The Penicillin nucleus consists of two amino acids, L-cysteine and D-valine.

Penicillinase

• Penicillinase is an extracellular enzyme adaptively produced by members of the coliform group of bacteria, by most Bacillus sp.
• Major factor for penicillin resistance during infection.
• This enzyme hydrolyses penicillin to penicilloic acid.

6-amino penicillanic acid

• Sensitive to Penicillinase enzyme.
• Can be recovered from fermentation broth.
• Chemically converted to penicillin by chemical addition of various R side chains. Yield of this material is low.
• Penicillin acylase enzyme cleaves side chains of penicillin so resulted in commercial production 6-Amino penicillanic acid.

Penicillin production

• Commercially, penicillin was first produced in stationary mat culture.
• Production of penicillin is previously achieved by surface process i.e. solid-state fermentations and surface liquid fermentation.

Penicillin production mainly consists of three steps:

1. 1. Inoculum preparation
   2. Production fermentation
   3. Product recovery

Inoculum preparation

• Inoculum: Penicillium chrysogenum.
• For inoculum production, spores from heavily sporulated working stocks (special agar sporulation) are suspended in water or in a dilute solution of a nontoxic wetting agent, such as  1:10,000 sodium lauryl sulfonate.
• These spores are then poured in the flask which contains wheat bran and nutrient solution for heavy sporulation.
• Incubate for at 24°C for 5-7 days to provide heavy sporulation.
• Resulting spores are directly inoculated into inoculation tanks.
• Incubated at 25 to 26°C for 3 to 5 days exact duration of fermentation depends on various factors.

Production fermentation

• Method: fed batch or batch.
• Media: (Jackson 1958)
  1. Corn steep liquor solids-3.5%
  2. Lactose: 3.5%
  3. Cellulose: 1%
  4. CaCO3: 1%
  5. K2HPO4: 0.4%
• Edible oil:  0.25%
• Fermenter: stirred tank or air lift tank.
• Temperature: 25-27°C.
• Aeration: 0.5 – 27 1 VVM.
• Anti-foam: edible oil
• pH: set at 5.5 to 6.0 which increased up to 7-7.5.
• Inoculum media is same as production media.
• Lactose precursor is not added, at start of fermentation.
• PH remain constant, glucose, corn steep liquor, Carbon compounds and ammonia are utilised.
• Carbon compounds depleted, pH rise to 7-7.5.
• At the end of fermentation pH rises to 8 higher because depletion of lactose brings an autolysis in mycelium.
• Penicillium is harvested before this.

Product Recovery

• Harvest broth from fermenter tank by filtration.
• Chill to 5-10°C.
• Acidify filtrate to pH 2.0 to 2.5 with H2SO4.
• Penicillin extracted from  aqueous filtrate into butyl acetate or amyl acetate.

• Discard aqueous fraction
• Add potassium acetate to the solvent extract in a crystallization tank to crystallize as potassium salt.
• Recover crystal from centrifuge.
• Further processing.
• Packaging.

Reference and Sources

• 1% – https://www.slideshare.net/guest08f61f/project-antibioticsbyharkiran-presentation
• 1% – https://quizlet.com/182638247/microbiology-iclicker-smartwork5-quiz-1-flash-cards/
• 1% – https://www.researchgate.net/publication/11010850_Optimization_of_a_fermentation_m
  edium_for_the_production_of_Penicillin_G_acylase_from_Bacillus_sp
• 1% – https://mafiadoc.com/download/practical-fermentation-technology_59da452a1723dd5e
  e2b54f83.html

Also read:

• Ethanol : production, recovery and uses
• Microbiology Disciplines: Bacteria, Viruses, Fungi, Archaea and Protists
• Antimicrobial Chemotherapy: History, Characteristics and Tests
• Citric acid: Introduction, Fermentation, Recovery and Uses
• Streptomycin : chemical structure, production , recovery and uses.

Penicillin: History, Structure, Production & Recovery