Role of some Role of some fermentation parameters fermentation parameters

on cyclosporin A on cyclosporin A production by a new production by a new isolate of isolate of Aspergillus Aspergillus

terreus.terreus.

Presented byPresented by

SEEMA RENUSEEMA RENU

Struture of Cyclosporin AStruture of Cyclosporin A

• Cyclosporin A is a cyclopeptide composed of 11 Cyclosporin A is a cyclopeptide composed of 11 amino acidsamino acids

• It was first derived from submerged culture of It was first derived from submerged culture of aerobic fungi identified as strains of aerobic fungi identified as strains of Trichoderma Trichoderma polysporumpolysporum. .

• Trichoderma polysporumTrichoderma polysporum currently identified as currently identified as Tolypocladium inflatum Tolypocladium inflatum also known as also known as Tolypocladium niveum. Tolypocladium niveum.

• T. inflatum T. inflatum also produces numerous other also produces numerous other cyclosporins all composed of 11 amino acids cyclosporins all composed of 11 amino acids differing from one another in only one amino acid. differing from one another in only one amino acid.

• For example the second amino acid in cyclosporine For example the second amino acid in cyclosporine A is alpha amino butyric acid whereas that in A is alpha amino butyric acid whereas that in cyclosporine B is alanine.cyclosporine B is alanine.

Discovery of Cyclosporin Discovery of Cyclosporin AA

• It was discovered in the early 1970 by research workers It was discovered in the early 1970 by research workers at Sandoz, Ltd in Basel, Switzerland during a search for at Sandoz, Ltd in Basel, Switzerland during a search for fungal metabolites with antibiotic properties. fungal metabolites with antibiotic properties.

• One of the metabolites released, known as 24-556 had One of the metabolites released, known as 24-556 had antifungal properties and very low toxicity toward the antifungal properties and very low toxicity toward the mammalian cells when tested at Sandoz. mammalian cells when tested at Sandoz.

• The two components of 24-556 were termed as The two components of 24-556 were termed as Cyclosporin A and Cyclosporin B where as Cycloporin A Cyclosporin A and Cyclosporin B where as Cycloporin A was much more active than Cyclosporine B. was much more active than Cyclosporine B.

• Human transplantation was first performed in 1978 Human transplantation was first performed in 1978 using cyclosporin A. Since then it has a major role in using cyclosporin A. Since then it has a major role in allogenic transplantation and autoimmune diseases.allogenic transplantation and autoimmune diseases.

Mechanism of actionMechanism of action• Cyclosporin A works by selectively affecting the Cyclosporin A works by selectively affecting the

production of T and B lymphocytes produced in the production of T and B lymphocytes produced in the bone marrow and thymus. bone marrow and thymus.

• Individuals with autoimmune diseases such as Individuals with autoimmune diseases such as rheumatoid arthritis produces excessive amount of rheumatoid arthritis produces excessive amount of toxic molecules such as reactive oxygen toxic molecules such as reactive oxygen intermediates that cause inflammation in the intermediates that cause inflammation in the joints. joints.

• Cy A reduces the inflammation by selectively Cy A reduces the inflammation by selectively acting against the production of these acting against the production of these lymphocytes. lymphocytes.

A macrophage engulfing a bacterium and releasing packets of toxic molecules (reactive oxygen intermediates) that breaks down and destroy the bacterium

Cyclosporin A and kidney Cyclosporin A and kidney damagedamage

• The widespread use of cyclosporin A The widespread use of cyclosporin A showed that it causes damage to the showed that it causes damage to the kidneys. kidneys.

• Studies proved that the damage can Studies proved that the damage can be avoided by using cyclosporin A in be avoided by using cyclosporin A in lower amounts and in conjunction lower amounts and in conjunction with a steroid called prednisone. with a steroid called prednisone.

Fermentation parameters Fermentation parameters and Cy A production by and Cy A production by A. A. TerreusTerreus

• The conditions affecting the production of Cy A by The conditions affecting the production of Cy A by A. TerreusA. Terreus included the composition of the included the composition of the cultivation medium, fermentation time course, cultivation medium, fermentation time course, inoculum nature, medium volume, agitation rate inoculum nature, medium volume, agitation rate and pH value.and pH value.

• Different fungi were investigated for Cy A Different fungi were investigated for Cy A production.production.

• – – sign indicated no production of Cy A, ++ showed sign indicated no production of Cy A, ++ showed a moderate amount of Cy A production, whereas a moderate amount of Cy A production, whereas three +++ means higher production of Cy A as three +++ means higher production of Cy A as judged by the darkness of the colony color.judged by the darkness of the colony color.

The yield of the Cy A increased as the fermentation time course increased. The highest yield of Cy A in mg/ml was on the day 10. The biomass yield of A. terreus (g/l) was kept on decreasing after day 10.

The best yield of Cy A was obtained by a 48-h-old culture. As the age increased production of Cy A start to decrease.

Maximum yield of Cy A was obtained when the volume of the inoculum size was 2ml/100ml.

The size of the medium also affected the production of Cy A. A 250 ml Erlenmeyer flask containing production medium equal to 100ml afforded the highest production of Cy A

The formation of Cy A by A.terreus was also affected by the agitation rate. The maximum production was at 200 rpm

The production was maximum in an acidic medium with a pH 5.3. Cy A production decreased with an increase in pH.

Buffer is a partially neutralised acid that resists changes in pH. The results showed that on using citrate buffer the maximum yield (84.22 mg/L) of Cy A was obtained. Increased pH also affected the texture of the fungal pellets by making it weaker.

RESULTSRESULTS

• The considerable amount of Cy A as The considerable amount of Cy A as a secondary metabolite, was a secondary metabolite, was produced under above described produced under above described conditions, proved conditions, proved A. terreus A. terreus as a as a potential source.potential source.

• Still no data on its use for Cy A Still no data on its use for Cy A production. production.

• It does used to extract lovastatin an It does used to extract lovastatin an agent used to lower cholesterol.agent used to lower cholesterol.