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This presentation is prepared by the coordination of a worthy group

Zahida Yaseen Mamoona Irshad Sahrish Khan

Health- care productsAntibiotics

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Antibiotics are prepared by industrial

microorganisms. Antibiotics are not produced in the greatest

quantity nor they are economically valuable. The other health care products are• Alkaloids• Steroids• Toxins• Vaccines , Vitamins and Enzymes

Introduction

Through genetic engineering we can produced variety of products likeMammalians proteins and peptides that have therapeutic properties

Medically important and established markets includes

•Insulin, Interferon ,Human growth hormones and monoclonal antibodies •All of these are extensively used to test various health and disease states.

Genetic Engineering Techniques

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Antibiotics are secondary metabolites produced by•Filamentous fungi and bacteria•Particularly actinomycetesApproximately 4000 antibiotics isolated from various organisms but 50 used as antimicrobial chemotherapyMedically important antibiotics are •Betalactams,penicilllines,cephloporins,aminoglycosides,(streptomycin), tetracycline

Antibiotics

Antibiotics(low market value)

Some antibiotics are fail to fulfill certain important criteria particularly•Lack of selectivity•Exhibiting toxicity to humans and animals•High production costsSome antibiotics are acts as antitumor agents e,g;Actinomycin and mytomycin(streptomyces peucetius and S.caeptiosus)

Some antibiotics are used to control microbial diseases in plantsSeveral antibiotics area also added to animal feed as growth promotersWith respect to resistance the antibiotics used for humans or may be withdrawn from used in animal feed. For exampleThe EU Commission voted to ban the application of•Bacitracin,spiromycin,tylomycin and virginomycin as growth promoters after january 1999

Tools(biochemistry and molecular

biology)

β-Lactam antibiotics are a broad class of antibiotics

This includes penicillin derivatives (penams), cephalosporins (cephems), monobactams, and carbapenems Most β-lactam antibiotics work by inhibiting cell wall biosynthesis in the bacterial organismmore than half of all commercially available antibiotics in use were β-lactam compounds

Beta-lactams

Bacteria often develop resistance to β-lactam

antibiotics by synthesizing a β-lactamase, an enzyme that attacks the β-lactam ring.

To overcome this resistance, β-lactam antibiotics are often given with β-lactamase inhibitors such as clavulanic acid.

β-lactam antibiotics were mainly active only against Gram-positive bacteria

yet the recent development of broad-spectrum β-lactam antibiotics active against various Gram-negative organisms has increased their usefulness

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Mmaoona Irshad Discovery of Penicllin Production Structure of Penicillin Commercial production Recovery methods

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Penicillin The first antibiotic discovered by Flemming Historically significiant

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Flemming

Discovery of Penicillin Late 1930’s Florey Chain and Heatly

characterized the inhibitory compound responsible to produce Penicillin

Developed a protocol to produce it in pure form Major advancements in both medicine and

fermentation technology Penicillin exhibits the properties of a typical

secondary metabolite P.notatum generated litlle more than 1 mg/L from

the surface cultures initially used for penicillin production

Corn steep liquor by product of maize

Corn steep liquor 20-25 fold increase in yieldContains various nitrogen sources, Growth factors and side chain precursors and remains as a major ingredient of penicillin production media.

Pencilllin chrysogenumIsolated from mouldy cantaloupmelon, results greater penicillin yields.Further increases in yield when production went over to submerged fermentation

Since 1940,s penicillin yield and fermentation has been vastly improved by extensive mutation and selection of producer strains.

Traditional approach involved random mutation and selection of higher producing strains.

Resulting mutants were grown on liquid medium and culture filtrates were assayed fro penicillin

This was a slow and painstaking as large number of strains had to be tested

Such methods were the key to the dramatically increased yields since the discovery of penicillin

Penicillin fermentations now produce yields in excess of 50g/L,

A 50,000 fold increase from the levels first produce by Fleming’s original isolation

Structure of PenicillinBasic structure if penicillin is 6-aminopenicillanic acid (6-APA)Composed of thiazolidine ring fused with beta lactam ring whose 6 amino position carries a variety of acyl subtituentsThis beta lactam thiazolidine, structure synthesized from

Alpha aminoadipate L-cystine L-valine

Side-chain precursors fermentation medium different biosynthetic penicillins

Natural penicillins can be modified chemically to produce compounds with improved characteristics

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General structure of Penicillin

Most penicillins are now semisynthetic obtained by chemical modification of natural penicillinModification is achieved by removing their natural acyl group, leaving 6-APA, to which other acyl groups can be added to confer new properties

Semisynthetic penicillins such as Methicillin, carbenicillin and ampicillin various imrovements includinga)Resistance to stomach acids to allow oral administrationb)A degree of resistance to penicillinasec)Extended range of activity to Gram negative bacteria

Chemical production of Penicillin

Fed batch process carried out aseptically in stirred tank fermenters of 40,000- 200,000 L capacity

Oxygen level is very important, must be maintained in 25-60mmol/L/h

Oxygen transfer rate viscosity, which increases as fermentation progresses

Maintained at 25-27 c and pH 6.5-7.7 Specific conditions depends on P.chrysogenum

strain used

Carbon sources used Various carbon sources used for penicillin

production, including glucose, lactose, sucrose, ethanol and vegetable oil

i. 65% is metabolized for cellular maintenanceii. 25% growthiii. 10% penicillin production Mixture of glucose and lactose used in past for

penicillin production Glucose good growth, poor penicillin

yield

Lactose poor growth, good yield Mode of feeding of carbon source is viatlly

important Corn steep liquor still used Acidic nature creates an environment for calcium

carbonate and phosphate buffer to neutralize the medium

Ammonia, mineral salts and specific side cahin precursors, e.g. phenyl acetic acid may also be added

Inoculum development initiated by adding lyophilized spores to a small fermenter

Fungal mycellium may then be grown further to one or two stages

Initially vegetative growth phase devoted to the development of biomass, which doubles every 6h

This high growth phase maintained for the first 2 days

Mycellium must develop as loose pellets rather than compact forms to ensure optimum yield

Carbon source is fed at low rate an dpenicillin production increases

Continues for 6-8 days Penicillin excreted into the medium is

recovered at the end of fermentation

Whole broth extraction can be performed but can be lead to downstream processing problems

Penicillin recovery follows removal mycellium using rotatory vacuum filters

Recovered mycellium is then washed to remove residual penicillin, prior to its use as animal feed or fertilizer

Recovery of antibioticTwo methods mostly used

1.Solvent extraction of the cell free medium2. ion pair extraction

Solvents extarction It gives yields of 90% It involves reducing the Ph of the filtered

medium to 2-2.5 by adding sulphuric or phosphoric acid

Followed by a rapid two stage continuous counter current at 03 c using acetone or ketones

Ion pair extraction It may be used at 5-7 ph, in which range penicillin

is stable Pigments or impurities are removed by treating

with activated charcoal Penicillin is then retrieved from the solvent by

adding sodium or potassium acetate Reduces solubility of penicillin and precipitates as

sodium potassium salt Penicillin crystals are separated by rotary vacuum

filtartion

Penicillin crystals are mixed with a volatile solvent as anhydrous ethanol, butanol or iso propanol to remove further impurities

Crystals are collected by filtration and air dried

At this stage penicillin is 99.5% pure This product may be further processed

to form a pharmaceutical grade product

Production of semisynthetic PenicillinsPenicillin (sometimes abbreviated

PCN or pen) is a group of antibiotics derived from Penicillium fungi.

the naturally occurring penicillins (those formed during the process of mold fermentation) and the semisynthetic penicillins (those in which the structure of a chemical substance—6-aminopenicillanic acid—found in all penicillins is altered in various ways)

To generate compounds with improved properties e.g acid stability,resistance to enzymic degradation ,broader spectrum of activity etc.

Remove the side chain of the base Penicillin to form 6-APA by penicillin Acylase from E.Coli

6-APA then acylated with an appropriate side chain to produce a semisynthetic penicillin.

Production of Cephalosporin Yields of Cephalosporins from fermentation

are much lower than those for penicillins 6-APA is used as a starting material Penicillin is converted by ring expansion to

6-APA & then to 7-ADCA A suitable side-Chain can also be attached

Emergence of Antibiotic resistanceAntibiotic resistance was recognized

soon after the natural penicillins were introduced

The use of antibiotics creats selection pressure favouring the growth of antibiotic resistant mutants, by the misuse and overuse of drugs

The emergence of pathogenic bacterial strains, show multiple resistance to a broad range of antibiotics

Many of the antibiotic-resistance genes of staphylococci carried on plasmids that can be exchanged with species of Bacillus and Streptococcus, providing the means for additional genes and gene combinations.

Some resistance genes are carried on transposons,that are segments of DNA that can exist either in the chromosomes or within plasmids

Uses of Antibiotics in Medicine

Cell wall synthesis inhibitors usually stop bacteria from forming their cell walls. They kill bacteria and not human cells because human cells do not form cell walls,e.g beta lactums, semisynthetic penicillins

Cell membrane inhibitors kill bacterial cells by disorganizing the outer membranes of bacteria,e.g polymyxin.

Protein synthesis inhibitors interfere with the process of translation in protein synthesis,e.g tetracyclines, chloramphenicol, macrolides,

Chemotheraputic agents affecting the synthesis of nucleic acids block the division and growth of cells by inhibiting synthesis of DNA and RNA. Most of these agents affect both animal and bacteria cells, so they cannot be used as an antibiotic.

Competitive inhibitors are mostly synthetic. These drugs work by disrupting the metabolic rate of bacteria. Some examples include sulfonamides, isoniazid, paraaminosalicylic acid, and ethambutol

Production of semisynthetic PenicillinsPenicillin (sometimes abbreviated

PCN or pen) is a group of antibiotics derived from Penicillium fungi.

the naturally occurring penicillins (those formed during the process of mold fermentation) and the semisynthetic penicillins (those in which the structure of a chemical substance—6-aminopenicillanic acid—found in all penicillins is altered in various ways)

To generate compounds with improved properties e.g acid stability,resistance to enzymic degradation ,broader spectrum of activity etc.

Remove the side chain of the base Penicillin to form 6-APA by penicillin Acylase from E.Coli

6-APA then acylated with an appropriate side chain to produce a semisynthetic penicillin.

Production of Cephalosporin Yields of Cephalosporins from fermentation

are much lower than those for penicillins 6-APA is used as a starting material Penicillin is converted by ring expansion to

6-APA & then to 7-ADCA A suitable side-Chain can also be attached

Emergence of Antibiotic resistanceAntibiotic resistance was recognized

soon after the natural penicillins were introduced

The use of antibiotics creats selection pressure favouring the growth of antibiotic resistant mutants, by the misuse and overuse of drugs

The emergence of pathogenic bacterial strains, show multiple resistance to a broad range of antibiotics

Many of the antibiotic-resistance genes of staphylococci carried on plasmids that can be exchanged with species of Bacillus and Streptococcus, providing the means for additional genes and gene combinations.

Some resistance genes are carried on transposons,that are segments of DNA that can exist either in the chromosomes or within plasmids

Uses of Antibiotics in Medicine

Cell wall synthesis inhibitors usually stop bacteria from forming their cell walls. They kill bacteria and not human cells because human cells do not form cell walls,e.g beta lactums, semisynthetic penicillins

Cell membrane inhibitors kill bacterial cells by disorganizing the outer membranes of bacteria,e.g polymyxin.

Protein synthesis inhibitors interfere with the process of translation in protein synthesis,e.g tetracyclines, chloramphenicol, macrolides,

Chemotheraputic agents affecting the synthesis of nucleic acids block the division and growth of cells by inhibiting synthesis of DNA and RNA. Most of these agents affect both animal and bacteria cells, so they cannot be used as an antibiotic.

Competitive inhibitors are mostly synthetic. These drugs work by disrupting the metabolic rate of bacteria. Some examples include sulfonamides, isoniazid, paraaminosalicylic acid, and ethambutol

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