Recombinant insulin Production

Recombinant insulin Production

Insulin, synthesized by the β-cells of the islets of Langerhans in the pancreas, controls the level of glucose in the blood.

An insulin deficiency manifests itself as diabetes mellitus, a complex of symptoms which may lead to death if untreated. 

Insulin used in this treatment was originally obtained from the pancreas of pigs and cows.

Problems of animal-derived Insulin

1. Side effects in some patients
2. Purification procedures are difficult
3. Potentially dangerous contaminants cannot always be completely removed

Features that facilitate its production by recombinant DNA techniques - 

1. Human protein is not modified after translation by the addition of sugar molecules
2. Size of the molecule is small, comprising two polypeptides, one of 21 amino acids (the A chain) and one of 30 amino acids (B chain)

Preproinsulin precursor which contains the A and B segments linked by a third chain (C) and preceded by a leader sequence.

Method -

1. The leader sequence is removed after translation and the C chain excised, leaving the A and B polypeptides linked to each other by two disulphide bonds.
2. Then the artificial gene was ligated to a lacZ′ reading frame present in a pBR322-type vector of E. coli.
3. The insulin genes were expressed as fusion proteins, consisting of the first few amino acids of β-galactosidase followed by the A or B polypeptides which were separated by a methionine residue.
4. Then insulin polypeptides cleaved from the β-galactosidase segments by treatment with cyanogen bromide.
5. Then purify the A chains & B chains then finally carry-out disulphide bond formation

Citric Acid & vinegar Production

Citric Acid Production

Microbes involved

Aspergillus niger (mainly)

Asp. wenti

Candida quillermondi

Candida lipolytica

Candida oleiphila.

Basis of the Production of Citric Acid

Citric acid is an intermediate in the citric acid cycle (TCA). The acid can accumulate by one of the following methods –

a.     By mutation – giving rise to mutant organisms which may only use part of a metabolic pathway, or regulatory mutants; that is using a mutant lacking an enzyme of the cycle.

b.     altering the environmental conditions - temperature, pH, medium composition

                           i.          isocitrate dehydrogenase, are inhibited by anaerobiosis, hence limited aeration is done on the fermentation so as to increase the yield of citric acid.

                         ii.          Low pH

                       iii.          Addition of small amount of citric acid

Conditions & Requirements

1.     Media –

C source –

            N source -  Organic & Inorganic (ammonium nitrate)

Salts & Minerals

Growth factors

Water

2.      Temperature - 30⁰ C

3.      pH – 3.5 (HCl is added to adjust the medium to low pH)

4.      Time – 5-14 days

5.      Trace elements -  Zn, Mn, Fe, Cu. use to initiate the reaction

6.      KH₂PO₄, MgSO₄

7.     As high aeration is deleterious to citric acid production

8.     mechanical agitation is not necessary and air may be bubbled through

Production

Fermentation can be either surface or submerged.

Surface fermentation:

Mainly do in Japan by using rice bran

culture-  Aspergillus niger

Temp. pH, Time, media – same

aeration - limited

Submerged fermentation:

culture-  Aspergillus niger (mainly)

Temp. pH, Time, media – same

2 salts are added.  MgSO₄, 7H₂O – 1%

                              KH₂PO₄ - 0.05-2%

Copper is used up to 500 ppm as an antagonist of the enzyme aconitase which requires iron

If beet molasses used as the substrate – Ferro-cyanide is added to reduce Fe percentage.

Harvest/ Recovery/Downstream process/Extraction (draw a flow chart)

The broth is filtered until clear & extract citric acid.

Add Ca(OH)₂ to precipitate Calcium citrate

Wash the precipitate & add Dil. H₂SO₄ ; it results CaSO₄ + citric acid

then, Dried to form crystals of citric acid.

Powder & package

Uses-

1.     Food industry

a.      as food acidulant in the production of jams, sweets, and soft drinks.

b.     as an artificial flavoring

c.      Production of processed cheese (Sodium citrate)

2.     medicine and pharmaceutical industry

a.      blood transfusion & prevention of blood clotting. (Sodium citrate)

b.     as a source of energy

3.     cosmetic industry

a.      In astringent lotions (e.g.- aftershave lotions)

b.     In hair sprays

4.     Other

a.      as a chelating agent; it chelates Fe³⁺ & help in oil recovery

b.     Production of Detergents.

Acetic acid/ Vinegar Production

Vinegar is a product resulting from the conversion of alcohol to acetic acid by acetic acid bacteria, Acetobacter spp

Types of Vinegar

1.     Cider vinegar, apple vinegar - from fermented apple justice

2.     Wine vinegar, grape vinegar – from Fermented grape juice

3.     Malt vinegar - from fermented barley malt

4.     Spirit vinegar - from distilled alcohol

5.     Flavored vinegar

Microbes involved

Acetobacter aceti

Gluconobacter oxydans

bacteria should have following characteristics –

1.     tolerate high concentrations of acetic acid

2.     require small amounts of nutrient

3.     not over oxidize the acetic acid formed

4.     should have high yield

.

CH₃CH₂OH + (O)    →                             CH₃CHO + H₂O

Ethyl alcohol oxygen                                                 Acetaldedyde Water

CH₃CHO + H₂O          →                             CH₃CH(OH)₂

Hydrated acetaldehyde

CH₃CH(OH)₂ + (O)       →                          CH₃COOH + H₂O

                                       Dehydrogenase              Acetic acid

1 gm of alcohol →yield 1.304 gm of acetic

Production

The 3 methods used for the production of vinegar

1.     Orleans Method (slow method)

2.     Trickling (or quick) Method

3.      Submerged Fermentation

Orleans Method (slow method) – now days not use (see pic in written book)

·       wine left in open vats/tubs became converted to vinegar by acetic acid bacteria entering it from the atmosphere.

·       wine was put in wooden vessels and left in the open & Add small amount of vinegar to initiate fermentation as an inoculum of acetic acid bacteria

·       then, a thick film (mother liquor film) of acetic acid bacteria formed on the wine and converted it in to vinegar in about 5 weeks

Limitations

1.     Slow process

2.     less efficient, yielding 75-85%

3.     contamination

The Trickling Generators (Quick) Method (see pic in written book)

·       The vessel is made out of wood or stainless steel.

·       broth + culture add only up to 4/5 of total volume.

·       alcohol-acetic acid mixture spray at the top.

·       wood pieces of the broth – provide surface for biofilm formation

·       cooling water jacket is used to regulate the temperature between 29°C - 35°C. this is determined by thermometers placed in vessel.

·       ethyl alcohol level must be maintained; not fall below 0.3-0.5%

·       Final acidity of the vinegar is about 12%

·       Have 80% of efficiency.

Advantages

1.     High yield

2.     Contamination decreased.

Submerged Fermentation

aeration is crucial due shortage of oxygen because of the highly acid conditions of submerged production

Can be carried out by –

a.      Fringes’ acetator

b.     The tower fermenter

Fringes’ acetator

·       It consists of a stainless steel tank fitted with internal cooling coils and a high speed agitator fitted through the bottom.

·       Temperature -  30°C

·       Air is sucked at supplied to the fermenter

·       Foaming is interrupted with an automatic foam breaker

·       operated batch wise, and 35hrs batch cycle produce 12% vinegar

Advantages

1.     High yield

2.     smaller space required.

3.     Automatic system; so easy in operation

4.     Can produce multiple types of vinegar

The tower fermenter

·       developed in the UK

·       The fermenter is 2 feet in diameter, 20 feet tall in the tubular section; expansion chamber 4 feet in diameter and 6 feet high.

·       working volume -  3,000 liters

Harvest/ Recovery

1.     Clarification by Filtration

             Submerged method much turbid than tricking method and so filtration is much

             important in Submerged method. Kieselguhr use as a filter aid in filtration process

2.     pasteurized at 60-65°C for 30 minutes

3.     concentrated by freezing